METHODS FOR DETERMINING THE NUCLEOTIDE SEQUENCE OF A NUCLEIC ACID OF INTEREST AND FOR DETERMINING TH

专利摘要:
pcr sequencing method and its use in hla genotyping the invention provides a pcr sequencing method, where the combination of indexing primers, an incomplete DNA shear strategy and a second generation sequencing technique (sequencing technique end in pairs) can make the length of pcr products that can be sequenced by a sequencer greater than the maximum sequencing length of the sequencer while making full use of the characteristics of the second generation sequencing technique such as high throughput and low cost, thereby greatly expanding its applicable scope. in addition, the present invention also provides indexing primers for the pcr sequencing method and the use of the method in genotyping, particularly in the analysis of hla, and also provides the pcr primers used, particularly the pcr primers for the hla genes. -a,b, hla-c and hla-dqb1.
公开号:BR112012032586B1
申请号:R112012032586-8
申请日:2011-06-30
公开日:2021-08-17
发明作者:Jian Li；Shiping Chen；Xiandong Zhang；Ying Liu；Caifen Zhang；Tao Liu；Meiru Zhao
申请人:Bgi Genomics Co., Ltd；
IPC主号:

专利说明:

Relevant Orders
[001] The present application claims the priority right of Chinese Patent Applications Nos. 201010213717.6, 201010213719.5, and 201010213721.2, as filed on June 30, 2010 and the right of priority of International Order Nos. PCT/CN2010/002150 and PCT/CN2010/002149 as filed on December 24, 2010, the contents of which are incorporated by reference in their entirety. TECHNICAL FIELD
[002] The present invention relates to the technical field of nucleic acid sequencing, in particular, the technical field of PCR sequencing. Furthermore, the present invention also relates to the DNA barcode technique and an incomplete DNA shear strategy. The method of the present invention is particularly applicable to second generation sequencing technique, especially to paired end sequencing technique, and is also applicable to HLA genotyping, in particular, a method for HLA-A, HLA-B genotyping , HLA-C and HLA-DQB1, and also provides the primer pairs for the PCR amplification used in the method. BACKGROUND
[003] The PCR sequencing method refers to a technique where the DNA fragments of a gene of interest are obtained by the PCR method, and the DNA fragments obtained from the gene of interest are subjected to DNA sequencing to obtain the formation of the DNA sequence of the gene of interest. PCR sequencing methods are widely applied in fields such as detecting a gene mutation and long-term genotyping.
[004] The DNA sequencing technique is mainly classified into the first-generation DNA sequencing technique represented by the Sanger sequencing method and the second-generation DNA sequencing technique represented by Illumina GA, Roche 454, ABI Solid, and the like . The DNA Sanger sequencing technique is characterized by simple experimental operations, visual and accurate results, and short experimental period, and thus it is widely applied in fields such as clinical gene mutation detection and genotyping, where a fast response time is highly required as for detection results. However, due to characteristics such as low yield and high cost, its application in fields where genotyping is carried out on a large scale is limited.
[005] When compared to the first generation DNA sequencing technique, the second generation DNA sequencing technique has the characteristics such as high sequencing throughput, low cost, high level of automation, and single molecule sequencing. Taking single-molecule Illumina GA sequencing as an example, a single sequencing operation generates data of 50G (about 50 billion) bases, 5 billion base data per day on average, and the average sequencing cost for the basis is less than 1/1000 of the sequencing cost in the Sanger method. Furthermore, the analysis of results can be directly performed by a computer. Thus, the second-generation DNA sequencing technique is an almost compatible technique for large-scale sequencing projects. However, the length of a continuous sequencing is generally short in the second generation DNA sequencing technique. Currently, the maximum bidirectional sequencing length is 200bp for Illumina GA; although the maximum bidirectional sequencing length can be up to about 500bp for the Roche 454 GS-FLX, the sequencing cost is higher and throughput is low. When a PCR amplification is of a length greater than the maximum sequencing length in a sequencer, complete sequencing of the entire amplification cannot be performed directly by sequencing, and the entire DNA sequence information of the amplification cannot be obtained. Due to the short maximum sequencing length, the application of the second generation DNA sequencing technique in the PCR sequencing method is limited. Furthermore, a gradual improvement of the sequencing technique to obtain a greater maximum sequencing length, is an urgent need to develop a new technique to overcome the deficiency of a maximum sequencing length of the second generation DNA sequencer in the field of PCR sequencing application.
[006] A human leukocyte antigen (HLA) is one of the most polymorphic gene systems found so far. It is a primary gene system to modulate a specific immune response in human bodies and determine an individual difference in disease susceptibility, and is closely associated with a rejection of a halogen organ transplant. It has been found in studies that the greater the degree of matching of genes such as HLA-A, B, C, DRB1 and DQB1, as well as the resolution is in a donor and in a recipient, the longer a transplant survives. a regular test item submits a potential donor and recipient to high-resolution HLA genotyping prior to hematopoietic stem cell transplantation.
[007] The current international standard high resolution HLA genotyping technique is a PCR sequencing method based on the Sanger sequencing technique, which comprises the PCR amplification of the corresponding HLA gene regions, sequencing of the amplified products , submit the sequencing result to genotyping with a professional genotyping software, and finally obtain the HLA genotyping information from the sample. It is characterized by visual results, high resolution and ability to detect new alleles. However, due to the characteristics of Sanger sequencing, such as high cost and low yield, this application in institutes such as the hematopoietic stem cell volunteer registry database (Bone Marrow Bank), in which the detection of HLA genotyping in large scale is required, is limited.
[008] It has been reported that a PCR sequencing method based on GS-FLX Roche 454 was used in HLA genotyping. However, since the cost for sequencing was relatively high, it was not significantly superior over the HLA genotyping technique based on Sanger sequencing in terms of sequencing throughput and sequencing costs. When compared to Roche 454 GS-FLX, Illumina GA has a sequencing length, but has obvious advantages in terms of sequencing throughput and sequencing cost. If the short-length maximal sequencing defect of Illumina GA can be overcome, its application in HLA genotyping will make up for the paucity of the current HLA genotyping method. CONTENTS OF THE INVENTION
[009] When conducting a sequencing analysis simultaneously for sequences associated with a specific gene, the PCR sequencing strategy is generally employed, where the combination of the indexing primer and the second generation sequencing technique are directly employed. When the maximum sequencing length of the sequencer can cover the length of the entire PCR product, the above strategy meets the requirements. When the maximum sequencing length of the sequencer does not cover the length of the entire PCR product, the Illumina GA needs to be replaced by the second generation sequencer having a longer maximum sequencing length (such as Roche 454 GS-FLX). If the maximum sequencing length still does not meet the requirements, a first generation sequencer has to be employed with cost and throughput scarification.
[010] The current situation is that Illumina GA has a super high sequencing throughput, but its maximum sequencing length is only 200bp; although the maximum sequencing length of the Roche 454 GS-FLX can reach 500bp, the cost for sequencing is relatively higher and the throughput is relatively below 1000bp, its throughput and cost are not comparable to those of second generation sequencers.
[011] Is there a technique capable of increasing the length of PCR products that can be fully sequenced by a sequencer without the cost and throughput scarification The combination of the first indices, the incomplete DNA shear strategy, and the second-generation sequencing technique in the present application can make the length of the PCR products that can be sequenced by the sequencer greater than the length of the sequence. - maximum sequencing of the sequencer while making full use of the characteristics of the second generation sequencing technique such as high throughput and low cost, thereby greatly expanding its applicable scope. The second generation sequencing technique employed in the present invention includes, among the second generation sequencing techniques, a paired end sequencing technique, and a PCR sequencing technique that has a DNA reference sequence for the PCR template.
[012] The present invention provides methods for PCR sequencing, whereby the resulting limitation of the maximum sequencing length is alleviated and the application of a second second generation DNA sequencing technique in the field of application of PRC sequencing is amplified. For example, when performing sequencing with the second generation sequencing technique, indexing primers having an indexing primer added to the 5' end are used, amplified PCR products are sheared, sheared products are terminally repaired and have deoxyadene - signal (A) attached at their 3' ends, and then attached to different free PCR adapters.
[013] A PCR sequencing method, based on the DNA barcode technique and incomplete DNA shear strategy, can greatly increase the number of specifically labeled samples without increasing the number of indexing primers (figure 5). In the present invention, the currently sequenced length of PCR products exceeds the maximum sequencing length of the sequencer by adding the indexing primers to the forward and reverse PCR primers, in combination, in combination with an incomplete DNA shear strategy and applying a second-generation sequencing technique.
[014] The addition of an indexing sequence at the front end of a primary amplification is intended to perform a simultaneous sequencing of a plurality of samples. Concretely speaking, a single indexing primer is added to each sample during PCR using a barcode/index-PCR technique in combination synthesizing an indexing primer by adding an indexing primer to the 5' end of a PCR primer . As such, during sequencing by the second-generation sequencing technique, samples have to be processed one by one only in the PCR step, and can be mixed together and processed simultaneously in the remaining experimental steps, and the final result for each sample can be traced due to its unique indexing initiator.
[015] The "Adapter" or "library adapter" indexing technique refers to a library indexing technique comprising adding different library adapters to multiple sequencing libraries (the different library adapters consist of different sequences, and the different part between sequences is called adapter index), building indexed sequencing libraries, then sequencing multiple different indexed sequencing libraries in a pool, where the final sequencing result for each sequencing library indexed is distinguishable. The term “free PCR library adapter” refers to a segment designed from the bases, whose main role lies in the auxiliary attachment of a DNA molecule to the sequencing chip and lies in providing the binding sites for the primary sequencing. universals, where the free PCR adapter can be directly attached to both ends of the DNA fragments in the sequencing library. Since no PCR is involved in inserting the adapter, the adapter is called a free PCR library adapter. For example, the free PCR library adapters used in the Examples of the present invention are from ILLUMIN.
[016] A method of building a free PCR library, where a library adapter index technique is used, refers to direct ligation of the library adapter to the two ends of the DNA fragment of the sequencing library. Since no PCR is involved in introducing the library adapter, it is called free PCR library construction. A DNA ligase can be used for ligation in the introduction process. Since no PCR is involved in the library construction process, an inaccuracy of the final results of the PCR slope results is involved during the construction of a library comprising the high sequence similarity PCR products.
[017] DNA amplification methods, DNA extraction methods, DNA purification methods and DNA sequence alignment methods as involved in the present invention may be any methods available in the art. Such methods can be selected by a person skilled in the art according to practical situations. As for the DNA sequencing methods, one skilled in the art can carry them out according to the conventional methods or following the instructions of the sequencer.
[018] The design of indexing initiators varies depending on the experimental platform applied. In view of the characteristics of the Illumina GA sequencing platform, the following factors are first considered when designing the indexing primers in the present invention: 1: a mononucleotide repeat sequence comprising 3 or more bases is avoided in the indexing primer sequences, 2: the total amount of base A and base C at the same location of all indexing primer counts for 30%-70% of the amount of all bases, 3: the GC content of the primary index sequence itself is between 40 and 60%, 4: indexing primers differentiate from each other by at least 4 bases, 5: sequences having a high sequence similarity to early Illumina GA sequencers are avoided in indexing primer sequences, and 6: the circumstance where the addition of the primary sequences to the primary results of PCR in dimer and serious clamp, are reduced.
[019] In the present invention, the two indexing primers (which are both identical and different) are added to two termini of a PCR product, respectively, so that the indexing primer in each PCR product can specifically label the information of the PCR product sample. The resulting PCR product undergoes incomplete shear. The so-called "incomplete shear" refers to the circumstance where the products comprise intact unsheared PCR products and partially sheared PCR products. Shear methods include, but are not limited to, chemical shear methods (such as enzymatic digestion) and physical shear methods. Physical shear methods include ultrasonic shear methods or mechanical shear methods. The sheared DNA is subjected to a 2% agarose electrophoresis, and all DNA strands between the maximum sequencing length and the maximum applicable DNA length of the sequencer are purified and coated by cutting the gel (the longest DNA applicable to the Illumina GA sequencer is 700bp, and the length refers to the length of the original DNA, which does not comprise the length of the library adapter sequence). Methods for purification and recovery include, but are not limited to, electrophoresis and gel slice recovery, and magnetic bead recovery. The coated DNA fragments are subjected to construction of sequencing libraries according to the procedures for the construction of sequencing libraries for the second generation sequencer, and then submitted to sequencing. Preferably, the sequencing libraries are constructed according to free PCR procedures for constructing the sequencing libraries, and the pair end method is used as the sequencing method. Free PCR construction of sequencing libraries is performed according to methods known to one skilled in the art. In the obtained sequencing data, sequence information for all test samples can be obtained by virtue of the indexing primer sequences. The sequence reads are aligned to the corresponding DNA reference sequences of the PCR products by BMA, and the complete sequence is assembled by overlap w linkage between the sequence reads (Figure 1). Bonding here refers to a paired end bonding relationship due to the paired end sequencing characteristics.
[020] In Illumina GA sequencing (Illumina Inc. Genome Analyzer Sequencer, referred to as Illumina GA for short), DNA sequence analysis is performed based on the principles of sequencing by synthesis. It can be applied to an aplotypic phase, and the data obtained finally refers to a series of base sequences and can be applied directly to alignment with reference sequences in the HLA database. Since it does not have the defect of a peaking error present in traditional typing software, it is advantageous for the automation of the typing software. Illumina GA has a high sequencing throughput. Currently, a single sequencing operation generates 50G (50 billion) of base data, 5 billion base data per day on average. Due to the high data throughput, a high sequencing depth can be achieved for each sequence, thus ensuring the reliability of the sequencing results.
[021] There are still no studies on the application of Illumina GA in the field of HLA typing. The present invention applies Illumina GA sequencing to the HLA typing field for the first time, and performs HLA typing with low cost, high throughput, high precision and high resolution using a PCR sequencing technique, based on the code technique DNA bars, incomplete DNA shear and free PCR library preparation.
[022] In the present invention, using a PCR sequencing technique that is based on the DNA barcode technique, incomplete DNA shear and free PCR library preparation, the samples to be analyzed are pooled; samples from each group are subjected to amplification of a fragment of interest from HLA genes with primers labeled by bidirectional indexing primers (maximum length of PCR products depends on the maximum length of DNA that can be applied in a sequencer; the length of Applicable maximum DNA is 700bp in current Illumina GA, and length is the original DNA length, which does not comprise library adapter sequence length); PCR products are pooled together by the same amount, then subjected to incomplete shear and an indexed free PCR DNA sequencing library preparation. Different indexed sequencing libraries, as obtained from various groups of samples, are mixed in an equal mole, all DNA fragments of a length greater than the maximum sequencing length of the sequencer are selectively overlayed and sequenced by an Illumina GA sequencer. DNA sequence readouts for each sample can be obtained by tracing the sequence information from adapter indices, indexing primers and PCR Primers into the total sequencing data. The resulting DNA sequences after assembly are aligned with the corresponding data in a professional IMGT HLA database, thus finally determining the HLA genotype of the sample.
[023] In the methods described above, after shearing such DNA, the DNA from samples from different groups are ligated to different library adapters during the preparation of the free PCR library, and therefore, in the following typing steps, the data from resulting sequencing can be plotted on the samples one by one based on the indexing primers and adapter indices used in each sample. The sequences from each sample are aligned to the known DNA reference sequence corresponding to the PCR product by software. Based on the overlapping sequence and the binding relationship, an intact sequence for the PCR product is assembled from the sheared DNA sequences.
[024] The present invention provides high resolution HLA genotyping methods based on Illumina GA sequencing techniques, thus performing a haplotypic sequencing and typing automation software, increasing the yield of HLA genotyping and reducing costs.
[025] Due to the requirement in the length of the DNA template in current sequencing techniques and the short read length in current sequencing techniques, the original PCR Primers for the HLA-SBT methods are no longer applicable to the new methods of high resolution HLA typing based on sequencing technique. The present invention designs new PCR Primers with good specificity and conservation, which amplifies Exons 2, 3, 4 of HLA-A, gene B independently, and whose PCR products have a length of no more than 700bp and are particularly applicable to Illumina GA (maximum DNA length applicable to current Illumina GA is 700bp). A set of PCR Primers as provided in the present invention is applicable to HLA genotyping for subjects (humans in particular) in a large scale, high throughput and low cost.
[026] In the technical solutions employed in the present invention, all the latest HLA-A/B gene sequences are downloaded from an IMGT/HLA internet website (http://www.ebi.ac.uk/imgt/hla /), and then written to the local disk as the HLA-A dataset; however, all later HLA-I gene sequences other than the HLA-A sequences are downloaded as a comparison dataset. Such two data sets are compared to look for conservative and specific sequences for each gene site at the two termini and inside Exons 2, 3, 4, and the designated PCR primer sequence is compared to the total human genome sequence for homology. Since the HLA-A/B gene is highly similar to other genes belonging to HLA-I molecules in terms of sequence, when designating PCR primers, the 3' terminus of the primer should be specific as much as possible in order to ensure specificity of the amplification of the HLA-A/B gene with the primers. However, the length of the PCR products is less than 700bp, and the annealing temperature of forward and reverse primers is substantially the same.
[027] Multiple pairs of candidate HLA-A/B primers meeting the design requirements are used to amplify the DNA templates of HLA-A/B serotypes. Among them, two sets of HLA-A/B PCR Primers (six pairs for each set) with the best conservatism and specificity, for the amplification of Exons 2, 3 and 4, respectively, are screened.
[028] The two sets of PCR Primers (6 pairs for each set) are used as basic primers, on the basis of which, 95 sets of indexing primers which are used for the amplification of 95 and 950 DNA templates from common serotypes of HLA-A/B (the serotypes of these models include all common serotypes of HLA-A/B), respectively, are designated. All PCR products are sequenced with Illumina GA end-to-end in 100 pairs after mixing in an equal amount, and the sequencing results after assembly are compared to the original typing results to confirm the conservatism and specificity of the Primers. of PCR.
[029] The HLA-A, B primers are designated in the present invention, that is, two sets of HLA-A/B PCR primers (6 pairs for each set) for the amplification of Exons 2, 3 and 4, respectively, are shown in Table 1 and 2. Table 1: HLA-A, B PCR Primers

Table 2: HLA-A, B PCR Primers

[030] Degenerate primers refer to a mixture of all possible different sequences representing all different bases encoding a single amino acid. In order to increase specificity, degeneration must be reduced according to the trend of base usage in different organisms by referring to a codon table, where R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, W=A/T, H =A/C/T, B = C/G/T, V=A/C/G, D=A/G /T, N=A/C/G/T.
[031] The present invention designates 2 sets of PCR primers (three pairs for each set) for amplification of Exons 2, 3 and 4 of HLA-C using the PCR Primer designation method for amplification of Exons 2, 3 and 4 from the HLA-A/B gene.
[032] In the following examples, 95 and 950 blood samples with known HLA genotypes are subjected to PCR amplification for HLA-C using the two selected sets of PCR Primers (3 pairs for each set), respectively. The amplified products are sequenced by the Sanger method and by the second generation sequencing method. Sequencing results are applied to HLA-C typing, and are compared to the original typing results to confirm the conservatism and specificity of the PCR Primers.
[033] The present invention provides 2 sets of PCR Primers (three pairs for each set) for amplification of Exons 2, 3 and 4 of HLA-C gene, which are SEQ ID NOs: 25 and 26, 27 and 28, and 29 and 30 as shown in Table 3, and SEQ ID NOs: 31 and 32, 33 and 34, and 35 and 36 as shown in Table 4. Such 6 pairs of PCR Primers have good conservatism and specificity, and can cover the sequences lengths for Exons 2, 3, and 4 of HLA-C, where the length of all PCR products is less than 700bp, which meets the requirement for normal Illumina Solexa sequencing. Furthermore, the primers of the present invention are also applicable for Sanger sequencing. Table 3: HLA-C Gene Exons 2, 3, and 4 PCR Primers
Table 4: HLA-C Gene Exons 2, 3, and 4 PCR Primers

[034] According to the methods as described above, in order to apply the second generation sequencing technique to the HLA-DQB1 genotyping, the present invention provides the PCR Primers for the amplification of Exons 2 and/or 3 of HLA- DQB1, which are SEQ ID NOs: 37-40 as shown in Table 5. PCR Primers have good conservatism and specificity, and can cover the full length sequences of Exons 2, 3 of HLA-DQB1, where the length of all PCR products is less than 700bp, which meets the normal Illumina Solexa sequencing requirement. Furthermore, the primers of the present invention are also applicable to Sanger sequencing. Table 5 PCR Primers for Amplification of the corresponding HLA-DQB1 Exons

[035] Genotyping can be performed based on the amplification of Exons 2 and/or 3 of HLA-DQB1 using primer pairs for the amplification and genotyping methods as provided in the present invention. Relative to the prior art, the genotyping methods use an Illumina Solexa sequencing technique, which is characterized by the ability to obtain high resolution HLA typing results with high throughput and low cost. SPECIFIC WAY TO CARRY OUT THE INVENTION
[036] A method for sequencing nucleic acid
[037] In one aspect, the present invention provides a method for determining a nucleotide sequence of a nucleic acid of interest in a sample, comprising: 1) providing n samples, where n is an integer of >1, the samples are preferably from mammals, more preferably from humans, particularly are human blood samples; optionally the n samples to be analyzed are divided into m groups, m is an integer and n>m>1; 2) amplification: one pair or multiple pairs of indexing primers are used for each sample, when there are sample templates, PCR amplification is performed under conditions compatible for the amplification of the nucleic acid of interest, where each pair of indexing primers it consists of a forward indexing primer and a reverse indexing primer can be identical or different: the indexing primers in the indexing primer pairs used for different samples are different; 3) association: when n>1, association of PCR products from each of the samples together; 4) shear: subject the amplified products to incomplete shear, and purify coat; 5) sequencing: submit the coated DNA mixture to sequencing using the second generation sequencing technique, preferably a paired end technique (eg Illumina GA, Illumina Hiseq 2000), to obtain the sheared DNA sequences ; and 6) assembly: corresponding to the sequencing data obtained in the samples one by one based on the unique indexing primer for each sample, aligning each sequence read to the DNA reference sequence corresponding to the PCR products using an alignment program (such as Blast program, BWA), assembling a complete nucleic acid sequence of interest from the DNA sequences sheared by virtue of sequence overlap and binding relationship.
[038] In one aspect of the present invention, each pair of indexing primers and a pair of PCR primers from a pair of indexing primers, forward and reverse PCR Primers has a forward indexing primer and an indexing primer reverse at the 5' end (or optionally linked by a linker sequence), respectively.
[039] In an embodiment of the present invention, such PCR Primers are PCR Primers for amplifying an HLA gene, particularly PCR Primers for amplifying the HLA-A/B gene, preferably PCR Primers for amplifying Exons 2, 3 and 4 of HLA-A/B and Exon 2 of HLA-DRB1, preferably of PCR Primers for amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or preferably of PCR Primers for HLA-DRB1 Exon 2 Amplification as shown in Table 7.
[040] In an embodiment of the present invention, such PCR Primers are PCR Primers for HLA gene amplification, particularly PCR Primers for HLA-C gene amplification, preferably PCR Primers for Exons 2 amplification , 3 and/or 4 of HLA-C; preferably, such PCR Primers are shown in Table 3 or Table 4.
[041] In an embodiment of the present invention, such PCR Primers are PCR Primers for the amplification of HLA gene, preferably PCR Primers for the amplification of Exon 2 and/3 of HLA-DQB1 gene; preferably, such PCR Primers are shown in Table 5.
[042] In one aspect of the present invention, such indexing primers are designed for PCR Primers, preferably for PCR Primers for HLA-specific gene amplification, more preferably for PCR Primers for Exons 2 amplification , 3 and 4 of HLA-A/B and Exon 2 for HLA-DRB1, particularly for the PCR Primers as shown in Table 1, Table 2 or Table 7; such indexing primers particularly comprise at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or 95 pairs of 95 pairs of indexing primers as shown in Table 6 (or the set of indexing primers consisting of 10-95 pairs (eg 10-95 pairs, 20-95 pairs, 30-95 pairs, 40- 95 pairs, 50-95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs) of the 95 indexing primer pairs as shown in Table 6); and the set of indexing primers preferably comprise at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 to PI-40, or PI-41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, or PI-91 to PI-95 of 95 pairs of indexing initiators as shown in Table 6, or combinations of any two or more of them.
[043] In one embodiment of the present invention, such DNA shear includes chemical shear methods and physical shear methods, where chemical shear methods include an enzymatic digestion, and physical shear methods include ultrasonic shear methods or methods of mechanical shear.
[044] In an embodiment of the present invention, after such DNA shear, all DNA strands between the maximum read length of the sequencer and the maximum applicable DNA length of the sequencer are purified and coated, where such methods of purification and recovery include, but are not limited to, electrophoresis and sliced gel recovery, and magnetic bead recovery.
[045] In another embodiment of the present invention, a method for sequencing the nucleotide sequence of a nucleic acid of interest in a test sample, comprising steps 1) to 4) of claim 1, and the following steps : 7) construction of a library: build a free PCR sequencing library using the sheared PCR products library, where different library adapters can be added to distinguish the different free PCR sequencing libraries, all strands of DNA between the maximum read length of the sequencer and the maximum applicable DNA length of the sequencer, preferably 450 to 750 bp DNA fragments, are purified and recovered; 8) sequencing: submit the recovery of the DNA mixture to sequencing using the second-generation sequencing technique, preferably the end-of-pair technique (for example, Illumina GA, Illumina Hiseq 2000), obtaining the sequences of the sheared DNAs; 9) assembly: corresponding to the sequencing data obtained for the samples one by one based on the different library adapter sequences and the unique indexing primer for each sample, aligning each sequence read to the DNA reference sequence corresponding to the PCR products using an alignment program (such as Blast program, BWA), assemble a complete nucleic acid sequence of interest from sheared DNA sequences based on sequence overlap and binding relationship.
[046] In one aspect, the present invention further provides the use of the aforementioned method in HLA typing, characterized in that it comprises: sequencing a sample (particularly blood sample) from a patient portal method, and aligning the sequencing results with the sequence data of HLA Exons, preferably, Exons 2, 3, 4 of HLA-A/B, Exons 2, 3 and/or 4 of HLA-C, Exon 2 and/or 3 of HLA-DQB1 gene and /or HLA-DRB1 Exon 2 in an HLA database (such as IMGT HLA professional database); where if the result of the sequence alignment shows 100% equivalence, the HLA genotype of the corresponding sample is determined.
[047] A set of indexing initiators
[048] In another aspect, the present invention provides a set of indexing primers, comprising at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least minus 70, or at least 80, or at least 90, or 95 pairs of 95 indexing primer pairs as shown in Table 6 (or the indexing primer set consisting of 10-95 pairs (eg 10-95 pairs , 20-95 pairs, 30-95 pairs, 4095 pairs, 50-95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs) of the 95 indexing primer pairs as shown in Table 6); and the set of indexing primers preferably comprise at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 to PI-40, or PI-41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, or PI-91 to PI-95 of 95 pairs of indexing initiators as shown in Table 6, or combinations of any two or more of them.
[049] The present invention further provides the use of such a set of indexing primers in PCR sequencing methods, where in particular, each pair of indexing primers and a pair of PCR Primers for the amplification of a sequence of interest to to be tested forms a pair of indexing primers, where the PCR Primers forward and reverse are at the 5' end (or optionally linked by a linker sequence), respectively.
[050] In one aspect of the present invention, such PCR Primers are PCR Primers for the amplification of a specific HLA gene, preferably PCR Primers for the amplification of Exons 2, 3, 4 of HLA-A/B gene and Exon 2 of HLA-DRB1, preferably the PCR Primers for the amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or preferably the PCR Primers for the amplification of Exon 2 of HLA-DRB1 as shown in Table 7; or preferably PCR Primers for the amplification of Exons 2, 3 and/or 4 of HLA-C, preferably such PCR Primers are shown in Table 3 or Table 4; or preferably PCR Primers for the amplification of Exon 2 and/or 3 of HLA-DQB1, preferably such PCR Primers are shown in Table 5.
[051] In another aspect, the present invention provides a set of indexing primers comprising such sets of indexing primers and a pair of PCR Primers for the amplification of a sequence of interest to be tested, where a pair of indexing primers comprises a pair of indexing primers and a pair of PCR Primers, the forward and reverse PCR primer have a forward and reverse indexing primer at the 5' end (or optionally linked by a linker sequence), respectively.
[052] In another embodiment of the present invention, such PCR Primers are PCR Primers for the amplification of a specific HLA gene, preferably PCR Primers for the amplification of Exons 2, 3, 4 of HLA-A/B gene and Exon 2 of HLA-DRB1, preferably the PCR Primers for the amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or preferably the PCR Primers for the amplification of Exon 2 of HLA-DRB1 as shown in Table 7; preferably PCR Primers for the amplification of Exons 2, 3 and/or 4 of HLA-C, preferably such PCR Primers are shown in Table 3 or Table 4; or preferably PCR Primers for the amplification of Exon 2 and/or 3 of HLA-DQB1, preferably such PCR Primers are shown in Table 5.
[053] In another aspect, the present invention further provides the use of such indexing primers in PCR sequencing methods.
[054] An HLA Typing Method
[055] In one aspect, the present invention provides a method of HLA typing, comprising: 1) providing n samples, where n is an integer of >1, the samples are preferably from mammals, more preferably from humans, particularly are human blood samples; 2) divide the n samples to be analyzed into m groups, m is an integer and n>m>1; 3) amplification: one pair or multiple pairs of indexing primers are used for each sample, when there are sample templates, PCR amplification is performed under conditions compatible for the amplification of the nucleic acid of interest, where each pair of indexing primers consists of a forward indexing primer and a reverse indexing primer (both of which can be degenerate primers) comprising indexing primers, where the indexing primers comprised in the forward indexing primer and the reverse indexing primer can be identical or different: the index primers in the index primer pairs used for different samples are different; 4) association: combine the amplified PCR products from each of the samples together to obtain the PCR product libraries; 5) shear: subjecting the resulting PCR product libraries to incomplete shear; 6) construction of a library: build a free PCR sequencing library from the sheared PCR products library with a library adapter index technique, where different library adapters can be added to distinguish the different free PCR sequencing libraries , all DNA lanes between the maximum read length of the sequencer and the maximum applicable DNA length of the sequencer, particularly 450 to 750 bp DNA fragments, are recovered; 7) sequencing: submit the recovery of the DNA mixture to sequencing using the second-generation sequencing technique, preferably the end-of-pair technique (for example, Illumina GA, Illumina Hiseq 2000), obtaining the sequences of the sheared DNAs; 8) assembly: corresponding to the sequencing data obtained for the samples one by one based on the different library adapter sequences of the libraries and the unique indexing primer for each sample, aligning each sequence read to the DNA reference sequence corresponding to the products PCR using an alignment program (such as Blast program, BWA), assembling a complete nucleic acid sequence of interest from sheared DNA sequences based on sequence overlap and binding relationship; and 9) typing: alignment of sequencing results with sequence data from HLA Exons, preferably, Exons 2, 3, 4 of HLA-A/B, Exons 2, 3 and/or 4 of HLA-C, Exon 2 and/or HLA-DQB1 gene 3 and/or HLA-DRB1 Exon 2 in the HLA database (such as the professional IMGT HLA database), where if the sequence alignment result shows 100% equivalence, the HLA genotype of the corresponding sample is determined.
[056] In the HLA typing method of the present invention, a pair of indexing primers comprises a pair of indexing primers and a pair of PCR Primers, the forward and reverse PCR primer have a forward and a reverse indexing primer at the 5' end (or optionally linked by a linker sequence), respectively.
[057] In an embodiment of the present invention, such PCR Primers are PCR Primers for the amplification of a specific HLA gene, preferably of PCR Primers for the amplification of Exons 2, 3, 4 of HLA-A/B gene and Exon 2 from HLA-DRB1, preferably from PCR Primers for the amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or preferably from PCR Primers for the amplification of Exon 2 from HLA-DRB1 as shown in Table 7; preferably of PCR Primers for the amplification of Exons 2, 3 and/or 4 of HLA-C, preferably such PCR Primers are shown in Table 3 or Table 4; or preferably of PCR Primers for the amplification of Exon 2 and/or 3 of HLA-DQB1, preferably such PCR Primers are shown in Table 5.
[058] In an embodiment of the present invention, such indexing initiators are sets of indexing initiators as described above.
[059] In one embodiment of the HLA typing method of the present invention, such DNA shear includes chemical shear methods and physical shear methods, where chemical shear methods include an enzymatic digestion, and physical shear methods include ultrasonic shear methods or mechanical shear methods.
[060] In one embodiment of the HLA typing method of the present invention, such purification and recovery methods include, but are not limited to, electrophoretic and sliced gel recovery, and magnetic bead recovery.
[061] In one embodiment of the HLA typing method of the present invention, the construction of the free PCR sequencing libraries from the sheared PCR product libraries with library adapter indexing techniques comprises adding m library adapters to the libraries of PCR products m obtained in 2), where each of the PCR product libraries uses a different library adapter, thus building m adapter indexed sequencing libraries; m-adapter-indexed sequencing libraries are joined together in an equal mole to build a mixture of adapter-indexed sequencing libraries, where the method of ligating library-adapters refers to direct ligation using a DNA ligase without a PCR procedure .
[062] PCR primers for HLA genotyping
[063] In one aspect, the present invention provides PCR Primers for HLA genotyping, characterized in that such PCR Primers are PCR Primers for the amplification of Exons 2, 3, 4 of HLA-A/B and Exon 2 gene of HLA-DRB1, preferably PCR Primers for the amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or preferably PCR Primers for the amplification of Exon 2 of HLA-DRB1 as shown in Table 7; preferably of PCR Primers for the amplification of Exons 2, 3 and/or 4 of HLA-C, preferably such PCR Primers are shown in Table 3 or Table 4; or preferably of PCR Primers for the amplification of Exon 2 and/or 3 of HLA-DQB1, preferably such PCR Primers are shown in Table 5.
[064] The present invention further provides a sequencing method using such PCR Primers, comprising: providing a sample, particularly a blood sample, such blood sample is preferably from a mammal, particularly a human; amplification: amplify DNA from blood samples with PCR Primers to obtain PCR products, and purify PCR products; sequencing: subjecting the PCR products to sequencing, the sequencing method can be a Sanger sequencing method, or a second generation sequencing method (such as Hiseq 2000, Illumina GA and Roche 454).
[065] In another aspect, the present invention still provides the use of such PCR Primers in HLA genotyping, characterized by the use of such PCR Primers, performing the alignment and assembly analysis on the results obtained by the above sequencing method, and comparing sequencing results with the standard sequences in the database to obtain HLA genotyping results.
[066] In another aspect, the present invention further provides a kit for HLA genotyping, comprising such PCR Primers.
[067] PCR primers for HLA-A,B genotyping
[068] In one aspect, the present invention provides a set of PCR Primers for HLA-A,B genotyping, comprising: providing a sample, particularly a blood sample, such blood sample is preferably from a mammal, particularly from a human; amplification: amplify DNA from blood samples with PCR Primers to obtain PCR products, and purify PCR products; sequencing: subjecting the PCR products to sequencing, the sequencing method can be a Sanger sequencing method, or a second generation sequencing method (such as Hiseq 2000, Illumina GA and Roche 454).
[069] In another aspect, the present invention still provides the use of such PCR Primers in HLA genotyping, characterized by the use of such PCR Primers, performing the alignment and assembly analysis on the results obtained by the above sequencing method, and comparing sequencing results with the standard sequences in the database to obtain HLA genotyping results.
[070] In another aspect, the present invention further provides a kit for HLA genotyping, comprising such PCR Primers for HLA-A,B genotyping of the present invention.
[071] PCR primers for HLA-C genotyping
[072] The present invention still provides a new method for the amplification of Exons 2, 3 and 4 of HLA-C gene, characterized by performing a PCR amplification using the amplification primer pairs of the present invention, the sequences of the pairs of amplification primers are shown in Table 3 or Table 4.
[073] Since Exons 2, 3 and 4 of HLA-C can be amplified by a PCR reaction, the method of the present invention is particularly compatible for HLA-C genotyping. when compared to previous HLA-C genotyping methods, since the products obtained using the method and the primary amplifications of the present invention are controlled to within 700 bp, the HLA-SBT based on the Illumina Solexa sequencing technique can be used even during genotyping.
[074] The present invention further provides a method for sequencing Exons 2, 3 and 4 of HLA-C gene in samples, comprising the following steps: 1) providing a sample and extracting the DNA from the sample; 2) amplify the DNA with the HLA-C genotyping PCR primer pair of the present invention to obtain the PCR products, preferably purify the PCR products, such PCR primer pairs are preferably selected from a group that consists of a pair of primers of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, or SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; 3) submit the PCR products to sequencing, preferably by the second generation sequencing method such as Illumina Solexa or Roche454.
[075] The present invention further provides a method of HLA-C genotyping, comprising: 1) PCR amplification of Exons 2, 3 and/or 4 of HLA-C gene from the sample to be tested with the PCR primer pair for genotyping HLA-C of the present invention, such a pair of PCR primers is preferably selected from a group consisting of a pair of primers of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO :28, SEQ ID NO:29 and SEQ ID NO:30, or SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO: 36; 2) submit the amplified Exons to sequencing, comparing the sequencing results with the standard sequences in the database in order to determine the genotyping results, where sequencing is performed by a Sanger sequencing method, or a second sequencing method generation, such as Illumina GA and Roche 454.
[076] In another aspect, the present invention further provides a set for HLA-C genotyping, comprising a pair of PCR primers for HLA- genotyping of the present invention, preferably selected from a group consisting of a pair of primers of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, or SEQ ID NO: 31 and SEQ ID NO: 32 , SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36. In another embodiment, such kit further comprises additional agents, e.g., agents for the amplification of DNA, the purification of DNA , and/or DNA sequencing.
[077] Genotyping can be performed based on the amplification of Exons 2, 3 and 4 of HLA-C, using the amplification primer pair and the genotyping method as provided in the present invention. Therefore, when compared to the prior art, genotyping uses the Illumina Solexa sequencing technique, increases throughput, simplifies the procedure, and in turn saves time and cost.
[078] PCR primers for HLA-DQB1 genotyping
[079] The present invention still provides a new method for the amplification of Exon 2, and/or 3 of HLA-DQB1, characterized by performing a PCR amplification with the amplification primer pairs of the present invention, such primer pairs of amplification are shown in Table 5.
[080] Since Exons 2, and/or 3 of HLA-DQB1 can be amplified by a PCR reaction, the method of the present invention is particularly compatible for HLA-DQB1 genotyping. When compared to previous HLA-DQB1 genotyping methods, since the products obtained using the method and the primary amplifications of the present invention are controlled within 300-400 bp, the HLA-SBT based on the Illumina sequencing technique Solexa can be used even during genotyping.
[081] The present invention further provides a method for sequencing Exon 2 and/or 3 of HLA-DQB1 in samples, comprising the following steps: 1) providing a sample and extracting the DNA from the sample; 2) amplify the DNA with the PCR primer pair for HLA-DQB1 genotyping of the present invention, preferably the PCR primer pairs shown in Table 5, to obtain the PCR products, preferably purify the PCR products; 3) submit the PCR products to sequencing, preferably by the second generation sequencing method such as Illumina Solexa or Roche454.
[082] In another aspect of the present invention, the present invention provides an improved method for HLA-DQB1 genotyping, comprising: 1) amplifying HLA-DQB1 Exon 2 and/or 3 to be tested with PCR primer pairs for the HLA-DQB1 genotyping of the present invention, preferably the PCR primer pairs as shown in Table 5; 2) submit the amplified Exons to sequencing, comparing the sequencing results with the standard sequences in the database in order to determine the genotyping results, where the sequencing method can be a Sanger sequencing method, or the sequencing method of second generation, such as Illumina GA and Roche 454.
[083] In another aspect, the present invention further provides a kit for HLA-DQB1 genotyping, comprising a pair of PCR primers for HLA-DQB1 genotyping of the present invention, preferably, the PCR amplification primer pairs as shown in Table 5. In one embodiment, such a kit further comprises additional agents, for example, agents for DNA amplification, DNA purification, and/or DNA sequencing. DESCRIPTION OF DRAWINGS
[084] Figure 1: A drawing illustrates the assembly of the sequence after being labeled with the indexing primers, the DNA shear and the DNA sequencing. Index-N-F/R (1) forward and reverse indexing primer sequences are introduced at two ends of the PCR products of sample No. N. The PCR products after shear by the physical shear method, comprise products which carry indexing primer sequences at one end, products carrying no indexing primer sequences at both ends, and products not completely sheared. All strands of DNA between the maximum read length of the sequencer and the maximum applicable DNA length are purified and recovered by sliced gel, and used for sequencing (2). Sequencing data of PCR products belonging to sample No. N are plotted using Index-N-F/R. The known reference sequences of the PCR products are used to locate the relative positions of the read sequences, and the sequencing results of the complete PCR products are assembled based on the overlap and binding relationship between the read sequences (3, 4) .
[085] Figure 2: A drawing illustrating the results of the electrophoresis of the PCR products of the corresponding Exons of HLA-A/B/DRB1 in Sample #1 of Example 2. It can be seen from the electrophoretogram that the PCR products are a series of bands unique 300bp-500bp, where Line M is a molecular weight marker ((DL 2000, Takara Co.), Lines 1-7 are the PCR products of the Exons (A2, A3, A4, B2, B3, B4 , DRB1-2) of HLA-A/B/DRB1 from Sample No.1, and there is no band amplification in the Negative Control (N), the results for the other samples are similar.
[086] Figure 3: A drawing illustrating the results of a DNA electrophoresis after HLA-Mix shear in Example 4 (before and after the sliced gel), where the sliced gel area is the 450-750bp area. Line M is a molecular weight marker (NEB-50bp DNA ladder), and Line 1 shows the electrophoretic result of HLA-Mix before the sliced gel, and Line 2 is a drawing showing the HLA-Mix gel after sliced .
[087] Figure 4: A screenshot of a program for constructing the consensus schema of Sample No. 1 in Example 6, illustrating the assembly of the complete sequence of the PCR products based on indexing primers and overlapping relationship between the DNA fragments. Please refer to http://www.ebi.ac.uk/imgt/hla/align.html for the nomenclature of HLA genotypes. One can find the results of all sequence codes from A*02:03:01 A*11:01:01 in the output column result on the left, where the Exon 2 sequence is identical to the original result known in the Model 1.
[088] Figure 5: A drawing illustrating the PCR product after being labeled with the indexing primers and the adapter index. During the experiment, indexing primers are introduced at both ends of the PCR product of each PCR sample simultaneously; multiple PCR products carrying different indexing primers are associated together to build a multiple sequencing library. During the construction of the multiple sequencing libraries, when the multiple sequencing library has been built, the sequencing libraries can be labeled with the library adapters carrying different adapter indices. After you finish building the libraries, multiple sequencing libraries labeled with different adapter indices are bound together and sequenced by Illumina GA simultaneously (index primers can be identical across sequencing libraries labeled with different adapter indices). After taking the sequencing results, the DNA sequence information for each sample can be obtained by sorting the sequence information from the adapter indices and the indexing primers in the sequencing results.
[089] Figure 6: A drawing illustrating the electrophoretic result of the HLA-C Exons 2, 3, 4 PCR products from some samples in Example 8. It can be seen from the electrophoretogram that the PCR products are a series of single bands 400bp-500bp, where Line M is a standard DNA molecular weight marker (DL 2000, Takara Co.).
[090] Figure 7: A drawing illustrating the results of electrophoretic DNA sliced gel after HLA-Mix shear in Example 8, where the sliced gel area is the 450-750 bp area. Line M is a molecular weight marker (NEB-50bp DNA ladder), and Line 1 is a drawing showing the HLA-Mix gel before slicing, and Line 2 is a drawing showing the HLA-Mix gel after of sliced.
[091] Figure 8: A screenshot of the Exon 2 consensus sequence construction program from the HLA-C site of Sample #2 in Example 8. First, the sequences read from the C site of the sample are aligned with the sequence of reference by the BWA software, thus constructing the consensus sequences of Exons 2, 3, 4 from sample C site; further, the haplotype sequence of each C-site Exon is determined based on the binding relationship between SNPs; and finally the sample type is determined by the intersection of the Exon haplotype sequences. As shown in the figure, two SNP heterozygotes are comprised in areas 695-764 of the C gene sequence of sample No. 2, and it can be determined by reading 1 or reading 2 that the SNP binding ratio is AC, GA (" ..." in the figure represents the bases identical to those of the reference sequence). Sequences correspond to the shaded parts of sequences of types C*010201 and C*07020101, respectively. Judgment of linkage relationships from other areas is similar.
[092] Figure 9: A drawing illustrating the electrophoresis results of PCR products from Exons 2, 3 and 4 from HLA-C site of 26 samples in Example 9. As shown in the figure, all PCR products are of one length less than 500bp; the electrophoretic band is unique; there is no obvious unspecific band; and the efficiency in amplifying the same pair of primers is the same across multiple samples.
[093] Figure 10: A drawing illustrating the analytical results of the sequencing data from Model 1 PCR amplification products using Type-u software in Example 9. The output column output on the left shows the result, C*08 :01:01 C*15:05:01, which are identical to the original type known in model 1.
[094] Figure 11: A drawing illustrating the electrophoretic result of the HLA-DQB1 Exon 2+3 PCR products in 94 samples from Example 10. It can be seen from the electrophoretogram that the PCR products are a series of single 250-bands. 500bp, where Line M is reference for standard DNA molecular weights (DL 2000, Takara Co.), Lines PI-1 to PI-94 are PCR amplification products of HLA-DQB1 Exon 2+3 in 94 samples , and there is no amplification band in the negative control (N).
[095] Figure 12 shows the results of an electrophoretic DNA slicing gel after the HLA-Q-Mix shear in Example 10, where the sliced gel area is an area of 350-550bp. Line M is a standard DNA molecular weight marker (NEB-50bp DNA Escada), and Line 1 is a drawing showing the HLA-Q-Mix gel before slicing, and Line 2 is a drawing showing the gel of HLA-Q-Mix after slicing.
[096] Figure 13 shows a screen shot of the program for building the consensus sequence of Sample #7 in Example 10, illustrating the main data analysis procedure. First, the sequences read from the DQB1 site of the sample are aligned with the reference sequence, by a BWA software, thus constructing the consensus sequences of Exons 2, 3 of the DQB1 of the sample; and the DQB1 Exons 2, 3 haplotype sequences are determined based on the binding relationship between SNPs. As shown in the figure, six heterozygous SNPs are comprised in area 2322-2412 of the DQB1 gene sequence of Sample No. 7, and it can be determined from reading 1 that the binding relationship of SNP1-SNP5 is T-G-T-C-C; it can be determined by reading 2 that the binding relationship of another SNP1-SNP5 is C-C-A-G-T; it can be determined from reading 3 that the SNP3-SNP6 binding relationship is A-G-T-G; it can be determined that from reading 4 the binding relationship of another SNP3-SNP6 is T-C-C-A; and it can be determined from the above binding relationships of such SNPs that reading 1 is linked to reading 4, reading 2 is linked to reader 3, the complete SNP combination in this area is TGTCCA and CCAGTG, and the sequences correspond to shaded parts of sequences of type DQB1*0303 and DQB1*0602. The judgment of the relationship for the other areas is similar.
[097] Figure 14 shows the electrophoretogram of the products in Example 11, results of amplification of each of Exons 2 and 3 from the HLA-DQB1 site and amplification of Exons 2 and 3 with two pairs of PCR Primers, respectively. The electrophoretogram shows three sets of PCR products from seven DNA templates, where all DNA products are less than 500bp in length; the electrophoretic ranges are unique; and there is no obvious unspecific band. There is no amplification in the negative control (N), and Line M is the reference for standard DNA molecular weights (DL2000, Takara Co.).
[098] Figure 15 illustrates the analytical results of the sequencing data of PCR products resulting from the amplification of Exons 2 and 3 of HLA-DQB1 from Model 7, using u-type software in Example 11. The result of the column output à left shows the result, DQB1*03:03 DQB1*06:02, which is identical to the known original result of Model 7.
[099] Figure 16 shows the electrophoretic results of the PCR products of the corresponding HLA-A/B/C/DQB1 Exons in Sample #1 in Example 12. It can be seen from the electrophoretogram that the PCR products are a series of 300bp-500bp single bands, where Line M is a molecular weight marker (DL 2000, Takara Co.); Lanes 1-10 are PCR products amplified from the Exons (A2, A3, A4, B2, B3, B4, C2, C3, C4, DQB1) of HLA-A/B/C/DQB1 of Sample #1; no amplification range is present in the negative control (N). Results from other samples are similar.
[0100] Figure 17 illustrates the result of recovering an agarose gel after pooling HLA-1-Mix, HLA-2-Mix, HLA-3-Mix, HLA-4-Mix, HLA-5-Mix, HLA- 6- Mix, HLA-7-Mix, HLA-8-Mix, HLA-9-Mix and HLA-10-Mix in equal moles in Example 12. Line M is a molecular weight marker, and Line 1 is a result electrophoretogram, and Line 2 is a Line electrophoretogram after slicing the gel containing the DNA fragments of average length from 450 to 750 bp.
[0101] Figure 18 shows a screen shot of the program for constructing Exon 2 consensus sequence from the HLA-C site of Sample #1 in Example 12. First, the sequences read from the C site of the sample are aligned with the reference sequence by a BWA software, thus constructing the consensus sequences of Exons 2, 3, 4 from sample C sites; further, the halotype sequences of the C-site Exons are determined based on the binding relationship between the SNPs; and finally the type of the sample is determined by the intersection of the halotype sequences of the Exons. As shown in the figure, the two heterozygous SNPs are comprised in area 695-764 of the C gene sequence of Sample No. 1, and it can be determined from reading 1 and reading 2 that the binding relationship of SNPs is AC, GA (“...” in the figure represents the bases identical to those of the reference sequence). The sequences correspond to the shared parts of sequences of type C*010201 and C*07020101, respectively. Judgment of the binding relationship from other areas is similar. EXAMPLES
[0102] The embodiments of the present invention are described in detail in the following examples. However, one skilled in the art would understand that the following examples are used to illustrate the present invention rather than limiting the restriction on the scope of the present invention.
[0103] In examples 1-6 of the present invention, Exons 2, 3, 4 of HLA-A/B and Exon 2 of HLA-DRB1 in 95 samples were genotyped using the combination of indexing primers + shear strategy of Incomplete DNA + Illumia GA paired end sequencer 100 sequencing technique (PCR products have a length in the range of 290bp to 500bp), demonstrating that the method of the invention can perform typing of gene fragments of a length exceeding the maximum length read from the sequencer while sufficiently utilizing the characteristics of the second generation sequencer, such as high throughput and low cost.
[0104] Principles: For the sample to be analyzed, indexing primers were introduced to both ends of the PCR products of HLA-A/B Exons 2, 3, 4 and HLA-DRB1 Exon 2 by a PCR reaction of to specifically label the sample information of the PCR products. The three-site PCR amplification products (HLA-A/B/DRB1) in each sample group were pooled together to obtain a library of PCR products; after incomplete ultrasonic shear of the PCR product library, a PCR-free sequencing library was constructed. The sequencing library was subjected to a 2% low melting point agarose gel electrophoresis, and all DNA strands of a length in the range of 450bp to 750bp were purified and recovered by a sliced gel (during construction of the sequencing library of PCR-free, since library adapters were added to the two ends of the DNA fragments, the length of a strand of DNA as shown in the electrophoretogram was about 250 bp longer than the actual length of the DNA fragments. DNA; therefore, fragments of a length in the range of 450bp to 700bp as recovered here actually correspond to DNA fragments of an original length in the range of 200bp to 500bp). The recovered DNA was sequenced by Illumina GA PE-100. The sequence information of all tested samples can be traced by the indexing primer sequences, and the sequences of the entire PCR product can be assembled based on the known reference sequences and the overlap and overlapping relationship between the sequences. DNA fragments, the complete sequence of the original PCR products can be assembled with the standard database of corresponding HLA-A/B/DRB1 Exons, thus performing HLA-A/B/DRB1 genotyping. Example 1
[0105] Sample extraction
[0106] DNAs were extracted from 95 blood samples with known HLA-SBT typing results (China Bone Marrow Donor Program referred to herein as (CMDP)) using a KingFisher Automated Extraction Instrument (US Thermo Co.). The main steps were as second: as directed in the book, a certain amount of self-contained agents was added to six very deep plates and one very shallow plate equipped by the KingFisher Automatic Extraction Instrument and all plates, to which agents were added, were placed in the corresponding positions as required. The program “Bio-easy_200ul Blood DNA_KF.msz” was selected, and it was implemented to extract the nucleic acids by pressing “start”. Approximately 100 μl of eluted products (ie the extracted DNA) were collected from the Elution plate after the program ended. Example 2
[0107] PCR amplification
[0108] Different PCR indexing primers were made by synthesizing PCR Primers having different indexing primers at the 5' end, and such different PCR indexing primers can be applied to different samples, where PCR Primers were PCR Primers PCR for Exons 2, 3, 4 of HLA-A/B and Exon 2 of HLA-DRB1. Therefore, indexing primers were introduced to the two ends of PCR products by a PCR reaction, thus specifically labeling PCR products from different samples.
[0109] 95 PCR indexing primer sets were used to amplify 95 DNA samples, respectively, where each PCR indexing primer set consisted of a pair of bidirectional indexing primers (Table 6) and PCR Primers for the amplification of HLA-A/B Exons 2, 3, 4 (Table 1) and HLA-DRB1 Exon 2 (Table 7), each PCR Forward Primer has the forward indexing primer in the pair of indexing primers linked in the 5' end, and the reverse PCR primer has a reverse indexing primer in the pair of indexing primers attached at the 5' end. During primer synthesis, indexing primers were directly added to the 5' end of the PCR Primers.
[0110] The 95 DNAs obtained from the sample extraction steps of Example 1 were designated as No. 1-95. The PCR reaction took place in 96 good plates, 7 plates in total, designated as HLA-P-A2, HLA-P-A3, HLA-P-A4, HLA-P-B2, HLA-P-B3, HLA-P -B4 and HLA-P-DRB1-2 (A2/A3/A4, B2/B3/B4, DRB1-2 represent the amplified sites), where a negative control without the addition of any template was configured on each plate, and the primers used in the negative control were the same as those used in Model 1. During the experimentation, the amount of sample information corresponding to each pair of indexing primers was recorded. Table 6: Relevant Information on Indexing Initiators



Table 7: PCR Primers for Amplification of the corresponding Exons of DRB1 and without the Indexing Primers

[0111] D2-F1, D2-F2, D2-F3, D2-F4, D2-F5, D2-F6, D2-F7 were forward primers for the HLA-DRB1 Exon 2 amplification, D2-R was a primer reverse HLA-DRB1 Exon 2 amplification. The PCR procedure for HLA-A/B/DRB1 was as followed: 96OC 2min 95OC 30s ^60oC 30s ^72OC 20s (32cycles) 15°C ~
[0112] PCR reaction system for HLA-A/B was as follows, where all agents were purchased from Promega (Beijing) Bio-Tech Co.


[0113] The PCR reaction system for HLA-DRB1 was as follows:

[0114] Where PInf-A/B/D2-F1/2/3/3/4/5/6/7 represents the F primer of HLA-A/B/DRB1 having a forward indexing primer sequence No. n (Table 6) at the 5' end, PInr-A/B/D2-R2/3/4 represents the R primer HLA-A/B/DRB1 having the reverse indexing primer sequence No. n at the 5' end (here n <95), and the rest can be deduced similarly. However, each sample corresponds to a specific set of PCR Primers (PInf-A/B/D2-F1/2/3/4/5/6/7, PInr-A/B/D2-R2/3/4) .
[0115] The PCR reactions were performed in a PTC-200 PCR machine from Bio-Rad Co.. After the PCR reaction, 2ul of PCR products were subjected to 1% agarose gel electrophoresis. Figure 2 is shown in the electrophoretic result of the PCR products of the corresponding Exons of HLA-A/B/DRB1 from Sample No.1, and the DNA molecular weight markers were DL 2000 (Takara Co.). There are a series of single bands of an average length of 300 bp to 500 bp on the electrophorogram, indicating successful PCR amplification of the Exons (A2, A3, A4, B2, B3, B4, DRB1-2) of HLA-A/ B/DRB1 of Sample No.1. There is no amplification in the negative control range (N). The results for the other samples were similar. Example 3
[0116] Association and purification of PCR products
[0117] 20 μl of the remaining PCR products were taken from each of the 96 good HLA-P-A2 plates (except for the negative control), and was mixed homogeneously under agitation in a 3 ml EP tube (designated as HLA -A2-Mix). The same operation was applied to the other 6 good 96 plates, designated as HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix and HLA-D2-Mix. 200ul was taken from each HLA-A2-Mix, HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix and HLA-D2-Mix, and was mixed in a 3 ml EP tube, designated as HLA-Mix. 500ul of HLA-Mix DNA mix was subjected to column purification with a Qiagen DNA Purification kit (QIAGEN Co.) (For specific purification steps, please refer to the manufacturer's instructions). It was determined by Nanodrop 8000 (Thermo Fisher Scientific Co.) that the 200ul of DNA obtained by purification has an HLA-Mix DNA concentration of 48ng/ul. Example 4
[0118] Shearing of PCR products, and construction of Illumina GA PCR-free sequencing libraries 1. DNA Shear
[0119] A total amount of 5 ug of DNA, taken from the purified HLA-Mix, was contained in a Covaris microtube with an AFA fiber and a snap cap and was sheared in the S2DNA Covaris Shear (Covarisv Co.). The shear conditions were as follows: Scan frequency
2. Purification after shear
[0120] All HLA-Mix shear products were recovered and purified by a QIaquick PCR Purification Kit, and were dissolved in 37.5 ul EB (QIAGEN Elution Buffer), respectively. 3. Terminal repair reaction
[0121] The purified HLA-Mix after shear was subjected to a DNA repair reaction, and the reaction system was as follows (all agents were purchased from Enzymatics Co.):

[0122] Reaction conditions: 20X incubation for 30 min in a Thermomixer (Thermomixer, Eppendorf Co.).
[0123] Reaction products were recovered and purified by a QIAquick PCR Purification Kit, and were dissolved in 34 μl EB (QIAGEN Elution Buffer). 4. Addition of A at the 3rd end
[0124] It was added at the 3' end of the DNA recovered in the last step, and the reaction system was as followed (all agents were purchased from Enzymatics Co.):

[0125] Reaction conditions: incubation at 37X for 30 min in a Thermomixer (Thermomixer, Eppendorf Co.).
[0126] Reaction products were recovered and purified by a MiniElute PCR Purification Kit (QIAGEN Co.), and were dissolved in 13 μl EB (QIAGEN Elution Buffer). 5. Illumina GA free PCR library adapter binding
[0127] The term "free PCR library adapter" refers to a segment of the designated bases whose main role lies in the auxiliary attachment of a DNA molecule to the sequencing chip and lies in providing the binding sites for the sequencing universal primers, where the free PCR library adapter can be directly attached to two ends of the DNA fragments in the sequencing library; since no PCR was involved in the introduction of the library adapter, the library adapter was called a free PCR library adapter.
[0128] Products having A added were attached to Illumina GA free PCR library adapters, and the reaction system was as follows (all agents were purchased from Illumina Co.):


[0129] Reaction conditions: incubation at 20X for 15 min in a Thermomixer (Thermomixer, Eppendorf Co.).
[0130] The reaction products were purified by Ampure Beads (Beckman Coulter Genomics), and were dissolved in 50ul of deionized water, and the DNA concentration determined by a quantitative Fluorescent PCR (QPCR) was as follows:
6. Sliced gel recovery
[0131] 30μL of HLA-Mix was subjected to 2% low melting point agarose gel electrophoresis. The electrophoretic conditions were 100V, 100min. The DNA marker was the NEB Co. 50bp DNA marker. The gel containing the average DNA fragments of 450 to 750bp were sliced (Figure 3). The products on the sliced gel were recovered and purified by a QIAquick PCR Purification Kit (QIAGEN Co.), the volume after purification was 32ul, and the DNA concentration measured by quantitative Fluorescent PCR (QPCR) was 10.16nM . Example 5
[0132] Illumina GA Sequencing
[0133] According to the QPCR results, 10pmol DNA was taken and subjected to sequencing by the Illumina GA PE-100 program. For the specific operating procedure, please refer to the operating instructions of illumina GA (Illumina GA IIx). Example 6
[0134] Analysis of results
[0135] The sequencing results of Illumina GA were a series of DNA sequences, and looking for the forward and reverse primary sequences and the primary sequences in the sequencing results, the database comprising the sequencing results of the PCR products of several HLA-A/B/DRB1 exons for each sample corresponding to the respective indexing primer were constructed. The sequencing results of each Exon were aligned to the reference sequence (reference sequences were from http://www.ebi.ac.uk/imgt/hla/) of the corresponding Exon by BWA (Burrows-Wheeler Aligner), and therefore, consensus sequences from each database were constructed, and the DNA sequences in the database were selected and corrected. The corrected DNA sequences were assembled into the corresponding sequences of HLA-A/B/DRB1 Exons based on the sequence of overlap and ligation relationship (end ligation in pairs). The resulting DNA sequence was aligned with the database sequence of the corresponding HLA-A/B/DRB1 Exons in a professional IMGT HLA database. If the sequence alignment result shows 100% binding, the HLA-A/B/DRB1 genotype of the corresponding sample has been determined. Please refer to the screenshot of the program for building the local HLA-A Exon 2 consensus sequence in Sample No.1 as illustrated in Figure 4.
[0136] For all 95 samples, the typing results obtained by the above methods were completely consistent with the known typing results, where the results for Samples No. 1-32 were as follows:



Note: among the HLA-DRB1 types, DRB1*1201 does not exclude the possibility of DRB1*1206/1210/1217, and DRB1*1454 does not exclude the possibility of DRB1*1401, because such alleles have been fully identified in the sequence of Exon 2 of HLA-DRB1. Example 7
[0137] HLA-A,B and DRB1 genotyping using the second generation sequencing technique (Illumina GA)
[0138] Sample Extraction
[0139] DNAs were extracted from 950 blood samples with known HLA-SBT typing results (China Bone Marrow Donor Program referred to herein as (CMDP)) using a KingFisher Automated Extraction Instrument (US Thermo Co.) . The method was as described in Example 1.
[0140] PCR Amplification
[0141] The 950 DNAs obtained from the sample extraction step were designated as No. 1-950, and were divided into 10 groups (95 DNAs for each), which were designated as HLA-1, HLA-2, HLA-3 , HLA-4, HLA-5, HLA-6, HLA-7, HLA-8, HLA-9, HLA-10. For each sample group, 95 DNA samples were amplified by 95 sets of PCR Primers (Table 1) carrying bi-directional indexing primers (Table 6) for amplification of HLA-A/B Exons 2, 3, 4 and Primers. PCR (Table 7) loading the bidirectional indexing primers (Table 6) for the HLA-DRB1 Exon 2 amplification. The PCR reaction took place in 96-good plates, using 70 plates in total, designated as HLA-XP-A2, HLA-XP-A3, HLA-XP-A4, HLA-XP-B2, HLA-XP-B3, HLA -XP-B4 and HLA-XP-DRB1-2 ("X" represents the information of the group number 1/2/3/4/5/6/7/8/9/10, "A2/3/4" , “B2/3/4”, “DRB1-2” represents the amplification sites), where a negative control without the addition of any template was configured on each plate, and the primers used for the negative control were PI-labeled primers -1 (Table 6). During the experiment, the information of each sample on the group number and on the indexing initiators was recorded. The method was described in Example 2.
[0142] Association and purification of PCR products
[0143] For Group X samples ("X" is 1/2/3/4/5/6/7/8/9/10), 20 μl of the rest of the PCR products were taken from each good sample of 96 HLA-XP-A2 good plates (except for the negative control), and was mixed homogeneously under shaking in a 3 ml EP tube (designated as HLA-X-A2-Mix). The same operation was applied to the other 96 good plates from the Group X samples, designated as HLA-X-A3-Mix, HLA-X-A4-Mix, HLA-X-B2-Mix, HLA-X-B3-Mix , HLA-X-B4-Mix and HLA-X-D2-Mix. 200ul was taken from each HLA-X-A2-Mix, HLA-X-A3-Mix, HLA-X-A4-Mix, HLA-X-B2-Mix, HLA-X-B3-Mix, HLA-X-B4 -Mix and HLA-X-D2-Mix, and was mixed in a 3ml EP tube, designated as HLA-X-Mix. 500ul of HLA-X-Mix DNA Mix was subjected to column purification with a Qiagen DNA DNA Purification Kit (QIAGEN Co.) (For specific purification steps, please refer to manufacturer's instructions ) to obtain 200ul of DNA, and its DNA concentration was determined by Nanodrop 8000 (Thermo Fisher Scientific Co.). The same operation was also applied to the other groups. The DNA concentrations determined finally went as follows.

[0144] The method was described in Example 3.
[0145] Construction of the Illumina GA Sequencing Libraries was performed by the method of Example 4. The corresponding relationships between sample groups and library adapters were as followed.

[0146] The reaction products were purified by Ampure Beads (Beckman Coulter Genomics), and were dissolved in 50ul of deionized water, and the molar concentrations of DNA determined by a Fluorescent Quantitative PCR (QPCR) were as follows:


[0147] Sliced gel recovery
[0148] HLA-1-Mix, HLA-2-Mix, HLA-3-Mix, HLA-4-Mix, HLA-5-Mix, HLA-6-Mix, HLA-7-Mix, HLA-8-Mix , HLA-9-Mix and HLA-10-Mix were mixed in an equal mole (final concentration was 72.13 nM/ul), designated as HLA-Mix-10. 30μL HLA-Mix-10 was subjected to a 2% low melting point agarose gel electrophoresis. The electrophoretic conditions were 100V, 100min. the DNA marker was the 50bp DNA of NEB Co.. The gel containing the DNA fragments in the average of 450 to 750bp was sliced. The products on the sliced gel were recovered and purified by a QIAquick PCR Purification Kit (QIAGEN Co.), the volume after purification was 32ul, and the DNA concentration measured by the Fluorescent Quantitative PCR (QPCR) was 9.96nM .
[0149] The sequencing and analysis of the results were performed as described in Examples 5 and 6. For all 950 samples, the typing results obtained by the above method were completely consistent with the known typing results. Example 8
[0150] HLA-C genotyping using the second generation sequencing technique (Illumina GA) 1. DNA sample extraction The steps were described in Example 1. 2. PCR amplification
[0151] The steps were described in Example 2, except that the PCR Primers used were PCR Primers for Exons 2, 3 and 4 of HLA-C, as shown in Table 3.
[0152] 95 indexing PCR primer sets were used to amplify 95 DNA samples, respectively, where each indexing primer set consisted of PCR primer for the amplification of HLA-C Exons 2, 3, 4 (Table 3) and a pair of bi-directional indexing primers (as described below), each Forward PCR Primer has a forward indexing primer of a pair of indexing primers linked at the 5' end, and a Reverse PCR Primer has the forward primer. reverse indexing of a pair of reverse indexing primers connected at the 5' end. During primer synthesis, indexing primers were added directly to the 5' end of the PCR Primers.
[0153] The 95 DNAs obtained from the sample extraction step were designated as No. 1-95. The PCR reaction took place in 96-good plates, 3 plates in total, designated as HLA-P-C2, HLA-P-C3, HLA-P-A4 (C2/3/4 represent the amplification sites), where a control negative without adding any template was configured on each plate, and the primers used in the negative control were the same as the PI-96 primer. During the experiment, the amount of sample information corresponding to each pair of indexing primers was retrieved.
[0154] The indexing primers used were the PI-1 to PI-95 indexing primers as listed in Table 6, and the following PI-96 indexing primer negative controls (Table 8) Table 8: Relevant information of the PI-96 indexing primers indexing used for negative control

[0155] The DNAs, extracted using the KingFisher Automated Extraction Instrument in step 1, were used as a template, and the PCR amplification was performed in single tubes using the primers for Exons of HLA-C, where the primers have indices at 5 ' far end. The PCR procedure was as follows: C2: 96OC 2 min 95OC 30s ^62OC 30s 72OC 20s (35 cycles) 15℃ ∞ C3: 96C 2 min 95OC 30s 56OC 30s 72OC 20s (35 cycles) 15℃ ∞ C4: 96C 2 min 95OC 30s ^60OC 30s ^72OC 20s (35 cycles) 15℃ ∞
[0156] HLA-C PCR reaction system was as follows:

[0157] Where, PInf-C-F2/3/4 represents the F initiator of HLA-C having a forward prime sequence No. n (Table 2) at the 5' end, PInr-C-R2/3/4 represents the R HLA-C primer having a forward primer sequence No. n at the 5' end (here n<96), and the rest can be similarly deduced. In addition, each sample corresponds to a set of PCR Primers.
[0158] The PCR reaction was performed in the PTC-200 PCR apparatus from Bio-Rad Co.. After the PCR reaction, 2ul of the PCR products were subjected to a 1.5% agarose gel electrophoresis. Figure 6 showed the electrophoretic result of the PCR products of the corresponding HLA-C Exons from the first 20 samples, and the DNA molecule marker was DL 2000 (Takara Co.). There were a series of single bands of an average length of 400 bp to 500 bp on the electrophorogram, indicating success in PCR amplification of Exons (C2, C3, C4) of HLA-C from the samples. The results for the other samples were similar.
[0159] Association and purification of PCR products
[0160] 20 μl of the rest of the PCR products were taken from each good of the 96-good HLA-P-C2 plates (except for the negative control), and was homogeneously combined under agitation in a 3 ml EP tube (designated as HLA-C2-Mix). The same operation was applied to the other 2 of the 96 good plates, designated as HLA-C3-Mix and HLA-C4-Mix. 200ul was taken from each HLA-C2-Mix, HLA-C3-Mix and HLA-C4-Mix, and was mixed in a 1.5 ml EP tube, designated as HLA-Mix. 500ul of HLA-Mix DNA mix was subjected to column purification with a Qiagen DNA Purification Kit (QIAGEN Co.) (For specific purification steps, please refer to manufacturer's instructions). It was determined by Nanodrop 8000 (Thermo Fisher Scientific Co.) that the 200ul of DNA obtained by purification has an HLA-Mix DNA concentration of 50ng/ul. 4. Construction of Illumina GA Free PCR Sequencing Libraries 4.1 Shear of PCR products
[0161] A total amount of 5 ug of DNA, taken from the purified HLA-Mix, was contained in a Covaris microtube with an AFA fiber and a snap cap and was subjected to S2 Covaris shear (Covarisv Co.). The shear conditions were as follows: F sweep frequency

4.2 Purification after shear
[0162] All HLA-Mix shear products were recovered and purified by a QIaquick PCR Purification Kit, and were dissolved in 37.5 ul EB (QIAGEN Elution Buffer), respectively. 4.3 Terminal Repair Reaction
[0163] The purified products were subjected to a DNA repair reaction, and the reaction system was as follows (all agents were purchased from Enzymatics Co.):

[0164] Reaction conditions: 20X incubation for 30 min in a Thermomixer (Thermomixer, Eppendorf Co.).
[0165] Reaction products were recovered and purified by a QIAquick PCR Purification Kit, and were dissolved in 32 μl EB (QIAGEN Elution Buffer). 4.4 Addition of A at the 3rd end
[0166] It was added at the 3' end of the DNA recovered in the last step, and the reaction system was as follows (all agents were purchased from Enzymatics Co.):

[0167] Reaction conditions: incubation at 37X for 30 min in a Thermomixer (Thermomixer, Eppendorf Co.).
[0168] Reaction products were recovered and purified by a MiniElute PCR Purification Kit (QIAGEN Co.), and were dissolved in 38 μl EB (QIAGEN Elution Buffer). 4.5 Illumina GA Free PCR Library Adapter Linking
[0169] Products having A added were attached to Illumina GA free PCR library adapters, and the reaction system was as follows (all agents were purchased from Illumina Co.):

[0170] Reaction conditions: incubation at 16X overnight in a Thermomixer (Thermomixer, Eppendorf Co.).
[0171] The reaction products were purified by Ampure Beads (Beckman Coulter Genomics), and were dissolved in 50ul of deionized water, and the DNA concentration determined by a quantitative Fluorescent PCR (QPCR) was as follows:
4.6 Sliced gel recovery
[0172] 30μL of HLA-Mix was subjected to 2% low melting point agarose gel electrophoresis. The electrophoretic conditions were 100V, 100min. The DNA marker was the NEB Co. 50bp Ladder DNA marker. The gel containing the average DNA fragments of 400 to 750bp were sliced (Figure 7). The products on the sliced gel were recovered and purified by a QIAquick PCR Purification Kit (QIAGEN Co.), the volume after purification was 32ul, and the DNA concentration measured by the Fluorescent Quantitative PCR (QPCR) was 17.16nM . 5. Illumina GA sequencing
[0173] According to the QPCR results, 10pmol DNA was taken and subjected to sequencing by the Illumina GA PE-100 program. For the specific operating procedure, please refer to the operating instructions of illumina GA (Illumina GA IIx). 6. Analysis of results
[0174] The sequencing results of Illumina GA were a series of DNA sequences, and looking for the forward and reverse primary sequences and the primary sequences in the sequencing results, the database comprising the sequencing results of the PCR products of several HLA-C exons for each sample corresponding to the respective indexing primer were constructed. The sequencing results of each Exon were aligned to the reference sequence (reference sequences were from http://www.ebi.ac.uk/imgt/hla/) of the corresponding Exon by BWA (Burrows-Wheeler Aligner), and the consensus sequences from each database were constructed, and the read DNA sequences were selected and corrected based on the quality value of the sequencing base and difference between the read sequence and the consensus sequences. Corrected DNA sequences were assembled into the corresponding sequences of HLA-C Exons based on overlapping sequence and linkage relationship (end ligation in pairs). The screenshot of Figure 8 illustrates the procedure for constructing the HLA-C Exon 2 consensus sequence in place in Sample No.2.
[0175] The resulting DNA sequence was aligned with the corresponding HLA-C in IMGT HLA professional Exon database sequence. If the result of the alignment sequence showed 100% match, the HLA-C genotype of the corresponding sample was determined. For all 95 samples, the typing results obtained by the above methods were completely consistent with the known typing results, where the results for Samples No. 1-32 were as follows: (as shown in Table 9, all results obtained were identical to the original results). Table 9: Comparison of the typing results obtained by the above method with the original known typing results of the samples

Note: Among the HLA-C types, C*0303 does not exclude the possibility of C*0320N, C*0401 does not exclude the possibility of C*0409N/0430, C*0702 does not exclude the possibility of C*0750 , C*0801 does not exclude the possibility of *0822, C*1505 does not exclude the possibility of C*1529, because such alleles have been fully identified in the Exon 2 sequence of HLA-C. Example 9: HLA-C genotyping using a Sanger sequencing method 1. DNA sample extraction
[0176] As described in Example 1, DNAs were extracted using a KingFisher Automated Extraction Instrument from 26 to 95 samples with the known HLA genotypes. 2. PCR amplification
[0177] The above DNAs, extracted using the KingFisher Automated Extraction Instrument, were used as a template, and PCR amplification was performed in single tubes using three pairs of PCR Primers C-F2/C-R2, C-F3/ C-R3, C-F4/C-R4 (Table 3), respectively. The PCR procedure for each primer pair was as follows: C2: 96OC 2 min 95OC 30s ^62OC 30s ^72OC 20s (35 cycles) 15℃ ∞ C3: 96C 2min 95OC 30s ^56OC 30s ^72OC 20s (35 cycles) 15 ℃ ∞ C4: 96C 2min 95OC 30s ^60oC 30s ^72OC 20s (35 cycles) 15℃ ∞
[0178] The HLA-C PCR reaction system was as follows:


[0179] The PCR products were subjected to agarose gel electrophoresis (Figure 9) before purification. 3. Purification of PCR products
[0180] PCR products were purified using Millipore purification plates. The main steps were as follows. The sources to be used were marked with a marker pen on the 96 purifying plate sources for the PCR products, and 50 μl of ultrapure water was added to each of the sources to be used. The rest of the fonts were sealed by a sealing film. The plate was kept for 15 min or was connected to a filtration and extraction system (-10 pa) for 5 min. When the purification plate was taken from the filtration and extraction system, the liquid in the discharge port at the bottom of the purification plate was sucked into an absorbent paper.
[0181] The PCR products to be purified were centrifuged at 4000 rpm for 1 min; the silica gel cover or pad for the PCR products to be purified was removed, and 100 µl of ultrapure water was added to each PCR reaction system. Then, the purification plate, to which the PCR products to be purified were added, was connected to the filtration and extraction system, and the degree of vacuum was adjusted to -10 pa as shown in the barometer. Filtration and extraction was continued until the liquid was left in the micropore regenerable cellulose film at the bottom of the purification plate, and no reflection of gloss from the intact liquid surface was found when viewing under light.
[0182] In the sources containing PCR products to be purified, 50 μl of ultrapure water or TE were added to the micropore regenerable cellulose film; the purification plate was vibrated at room temperature on a trace vibrator for 5 min; and all liquids contained in the corresponding sources were transferred to corresponding sources of new 96 PCR plate sources. 4. Carrying out the sequencing reaction and purification of sequencing reaction products
[0183] The purified PCR products were used as a template for the sequencing reaction. Conditions for Sequencing Reaction 96OC 2 min 96OC 10s ^ 55C 5s ^ 60C 2min (25 cycles) 15℃ ∞
[0184] The system for the sequencing reaction was

[0185] The sequencing reaction products were purified by the following steps: the sequencing reaction plate was balanced, and centrifuged 3000 g for 1 min. In the 96-source plate, for every 5 µL of reaction system, 2 µL 0.125 mol/L EDTA-Na2 solution, 33 µL 85% ethanol was added, and the plate was covered with a silica gel pad and sufficiently vibrated for 3 min. The plate was then centrifuged at 4C, 3000 g for 30 min. The sequencing plate was taken after centrifugation, the silica gel pad was removed, and the sequencing plate was blotted down on absorbent paper, and was then subjected to an inverted centrifugation until the centrifugation force reached 185g. For each source plate 96, 50 µl 70% ethanol was added. The plate was covered with a silica gel pad, vibrated for 1.5 min, and centrifuged at 4 °C, 3000 g for 15 min. The sequencing reaction plate was then placed in a dark place and ventilated for 30 min in order to be air dried until the ethanol odor was no longer felt. For each source plate source 96, 10 µL HI-DI formamide was added (alternatively, for each source plate source 384, 8 µL was added), and then the plate was covered with a sealing film, and centrifuged at 1000 rpm after vibrated for 5s. 5. Sequencing and result analysis
[0186] The purified products of the sequencing reaction were subjected to a capillary electrophoresis sequencing in ABI 3730XL. Peaks from the sequencing were analyzed by Type-u software (Invitrogen) to obtain HLA typing results (Figure 10). All results obtained by the above method were identical to the original known results, as shown in Table 10. Table 10: Comparison of the typing results obtained by the above method with the known original typing results

Example 10: HLA-DQB1 Genotyping Using Second Generation Sequencing Technique (Illumina Solexa)
[0187] 94 blood samples with known HLA-SBT typing results were subjected to HLA-DQB1 genotyping according to the methods as described in Example 8, except for the following items.
[0188] 94 Indexing Primer PCR Sets were used to amplify 94 DNA samples, respectively, where each Indexing Primer PCR Set consisted of PCR Primers for HLA-DQB1 Exon 2 or 3 amplification (Table 5) and a pair of bi-directional indexing primers (as described above), each PCR Primer has a forward indexing primer of a pair of indexing primers linked at the 5' end, and the PCR primer has an indexing primer reverse of a pair of indexing primers attached 5' end. During primer synthesis, indexing primers were added directly to the 5' end of PCR Primers, where primers were synthesized by Shanghai Invitrogen Co.
[0189] The 94 DNAs obtained in the sample extraction step were designated as No. 1-94. The PCR reaction was carried out in a 96 source plate, Exons 2, 3 of DQB1 in which the sample was amplified in the same source. Two negative controls without adding any template have been set up on each plate, and the indexing primers used on the negative controls are PI-95 and PI-96. During the experiment, the quantity information of the samples corresponding to each pair of indexing initiators was recorded.
[0190] The indexing primers used were the indexing primers PI-1 to PI-94 as listed in Table 6, and the following indexing primers PI-95 and PI-96 (Table 11) for negative controls. Table 11: Relevant information on indexing primers used for negative controls
PCR procedure for HLA-DQB1 was as follows: 96OC 2min 95OC 30s^60oC 30s^72oC 20s (32 cycles) 15℃ ∞ PCR reaction system for HLA-DQB1 was as follows:

where, PInf-Q-F2/3 represents the F primer of HLA-DQB1 having forward indexing primer sequence No. n (Table 1) at the 5'end; PInf-QR 2/3 represents the R initiator of HLA-DQB1 having a reverse primer sequence No. n at the 5' end (here n<96); and the rest can be deduced similarly. In addition, each sample corresponds to a specific set of PCR primers.
[0191] The PCR reactions were performed in the PTC-200 PCR apparatus from Bio-Rad Co.. After the PCR reaction, 2ul of PCR product was subjected to 1.5% agarose gel electrophoresis. Figure 11 showed the electrophoretic result of HLA-DQB1 Exons 2+3 PCR products from 94 samples, and the DNA molecular marker was DL 2000 (Takara Co.).
[0192] Association and purification of PCR products
[0193] 20 μl of the rest of the PCR products were taken from each source of the 96 HLA-P-DQB1 source plate (except for the negative control), and was mixed homogeneously in a 3 ml EP tube (designated as HLA-Q -Mix). 500ul of HLA-Q-Mix DNA Mix was subjected to column purification with a Qiagen DNA Purification Kit (QIAGEN Co.) (For specific purification steps, please refer to the manufacturer's instructions). It was determined by Nanodrop 8000 (Thermo Fisher Scientific Co.) that the 200ul of DNA obtained by purification has a DNA HLA-Q-Mix DNA concentration of 48ng/ul.
[0194] Conditions for shear was as follows: F requency of sweep


[0195] Reaction products were subjected to an end-repair reaction, then recovered and purified by a QIAquick PCR Purification Set, and were dissolved in 34ul EB (QIAGEN Elution Buffer).
[0196] The reaction products were further subjected to the addition of A at the 3' end, and then were recovered and purified by a MiniElute PCR purification kit (QIAGEN Co.), and were dissolved in 13 μl EB solution (QIAGEN Elution Buffer).
[0197] After binding the library adapters, the reaction products were purified by Ampure Beads (Beckman Coulter Genomics), and were dissolved in 50 μl of deionized water, and the DNA concentration determined by a quantitative Fluorescent PCR (QPCR). ) was as follows:

[0198] The gel containing the DNA fragments on average from 350 to 550bp was sliced (Figure 12). After purification and recovery of the gel products, the DNA concentration, as determined by the Fluorescent Quantitative Fluorescence PCR (QPCR), was 18.83 nM.
[0199] Analysis of results
[0200] The sequencing results of Illumina GA were a series of DNA sequences, and looking for the forward and reverse primary sequences and the primary sequences in the sequencing results, the database comprising the sequencing results of the PCR products of several HLA-DQB1 exons for each sample corresponding to the respective indexing primer were constructed. The sequencing results of each Exon were aligned to the reference sequence (reference sequences were from http://www.ebi.ac.uk/imgt/hla/) of the corresponding Exon by BWA (Burrows-Wheeler Aligner), and the consensus sequences were constructed, the read DNA sequences were selected and corrected based on the quality value of the sequencing base and the difference between the read sequence and the consensus sequences. Corrected DNA sequences were assembled into the corresponding sequences of Exons 2, 3 of HLA-DQB1 based on overlapping sequence and linkage relationship (end ligation in pairs). The screenshot of Figure 13 illustrates the procedure for constructing the HLA-DQB1 Exon 2 consensus sequence in place in Sample No.7.
[0201] The resulting DNA sequence for Exons 2, 3 of HLA-DQB1 was aligned with the corresponding HLA-DQB1 Exon Professional Database sequence in IMGT HLA. If the result of the alignment sequence showed 100% match, the HLA-DQB1 genotype of the corresponding sample was determined.
[0202] For all 94 samples, the typing results obtained by the above methods were completely consistent with the known typing results, where the results of Samples No. 1-32 were shown in Table 12. Table 12: The typing results of Samples No. 1-32
Example 11: HLA-DQB1 genotyping using a sequencing method using Sanger 1. DNA sample extraction
[0203] As described in example 1, DNAs were extracted using a KingFisher Automated Extraction Instrument from 20 to 94 samples with the known HLA genotypes. 2. PCR amplification
[0204] The above DNAs, extracted by the KingFisher Automated Extraction Instrument, were used as templates, and the PCR amplifications were performed in single tubes using two pairs of PCR Primers (Q-F2 and Q-R2, Q-F3 and Q-R3) as listed in Table 5, respectively. The PCR procedure for each primer pair was as follows: 96OC 2min 95OC 30s^56oC 30s^72oC 20s (35 cycles) 15℃ ∞ PCR reaction system for HLA-Q was as follows:

[0205] The PCR products were subjected to an agarose gel electrophoresis prior to purification. 3. Purification of PCR products
[0206] The methods and steps were the same as those described in Example 9. 4. Carrying out the sequencing reaction and purifying the sequencing reaction products
[0207] The method and steps were the same as those described in Example 9 5. Results analysis and sequencing
[0208] The purified products of the sequencing reaction were subjected to a capillary electrophoresis sequencing in ABI 3730XL. Sequencing peaks were analyzed by Type-u software (Invitrogen) to obtain HLA typing results (Figure 15). All results obtained by the above method were identical to the original known results, as shown in Table 13. Table 13: Comparison of the typing results obtained by the above method with the typing results. known originals

Example 12: Genotyping of Exons 2, 3, 4 of HLA-A/B/C and Exons 2, 3 of HLA-DQB1 in 950 samples
[0209] In the present examples, Exons 2, 3, 4 of HLA-A/B/C and Exons 2, 3 of HLA-DQB1 in 950 samples were genotyped using a combination of indexing primers, incomplete DNA shear strategy , library indices, free PCR library preparation, and Illumia GA paired end 100 sequencing technique (PCR products having an average length of 300bp to 500bp), demonstrating that the method of the present invention can perform genotyping of fragments of a gene of a length exceeding the maximum length read from the sequencer, and also demonstrates that the present invention can perform HLA genotyping with low cost, high throughput, high precision, high security and high resolution.
[0210] Principle: the samples to be analyzed were divided into 10 groups; for sample from each group, indexing primers were introduced to both ends of PCR products from HLA-A/B/C Exons 2, 3, 4 and HLA-DQB1 Exons 2, 3 by PCR reaction in order to specifically label the sample information for Dops PCR products. The four-site PCR amplification products (HLA-A/B/C/DQB1) in each sample group were mixed together to obtain a library of PCR products; after ultrasonic shear were constructed (where for the PCR product libraries from each group, a different adapter was used, thus building 10 indexed sequencing libraries). The 10 indexed sequencing libraries were joined together in an equal mole to build a scrambled index sequencing library. The mix index sequencing library was subjected to a 2% low melting point, and all DNA strands of an average length of 450bp a were sequenced by the Illumina GA PE-100 method. The sequence information of all tested samples can be traced by the indexing primer sequences and the library index sequences, and the sequence of the entire PCR product can be assembled based on the known reference sequences and the overlapping relationship and linking between the DNA fragment sequences, the complete sequence of the original PCR product can be aligned with the standard database of the corresponding HLA-A/B/C/DQB1 Exons, thus performing the HLA-A/B/C genotyping /DQB1. 1. Sample extraction
[0211] DNAs were extracted from 950 blood samples with known HLA-SBT typing results (China Bone Marrow Donor Program referred to herein as (CMDP)) using an Automated Extraction Instrument (US Thermo Co.). the process was the same as that described in Example 1. 2. PCR amplification
[0212] The 950 DNAs obtained from the sample extraction step were designated as No. 1-950, and were divided into groups of 10 (95 DNAs for each group), which were designated as HLA-1, HLA-2, HLA -3, HLA-4, HLA-5, HLA-6, HLA-7, HLA-8, HLA-9, HLA-10. For each sample group, 95 DNA samples were amplified by 95 sets of PCR Primers carrying bidirectional indexing primers (Table 6) for the amplification of HLA-A/B Exons 2, 3, 4 (Table 2), to the amplification of Exons 2, 3, 4 of HLA-C (Table 4), and for the amplification of Exons 2, 3 of HLA-DQB1 (Table 5), respectively. The PCR reaction took place in source 96 plates, using 100 plates in total, designated as HLA-XP-A2, HLA-XP-A3, HLA-XP-A4, HLA-XP-B2, HLA-XP-B3, HLA -XP-B4, HLA-XP-C2, HLA-XP-C3, HLA-XP-C4 and HLA-XP-DQB1 ("X" represents group number information 1/2/3/4/5/6 /7/8/9/10, "A2/3/4", "B2/3/4", "C2/3/4", "DQB1" represents the locations of), where a negative control without the addition of any template was configured on each plate, and the primers used for the negative control were PI-1-labelled primers (Table 6). During the experiment, the information of each sample on the group number and the indexing initiators were recorded. For example, the relevant information in indexing primers PI-1 and PI-2 was as follows, and the rest can be similarly deduced.

[0213] The PCR procedure and the PCR reaction system for HLA-A/B/C were the same as those described in Example 2. The PCR Primers for the amplification of the corresponding Exons of HLA-A/B were shown in Table 2, and the PCR Primers for the amplification of the corresponding HLA-C Exons were shown in Table 4.
[0214] The PCR procedure for HLA-DQB1 was as follows. 96OC 2min 95OC 30s^55oC 30s^72oC 20s (32 cycles) 15℃ ∞
[0215] The multiple PCR reaction system for HLA-DQB1 (amplification of Exons 2, 3 simultaneously) is the same as that described in Example 10, and the PCR Primers for the amplification of the corresponding Exons of HLA-DQB1 were as shown in Table 5.
[0216] Where, PInf-A/B/C-F2/3/4 and PInf-Q-F2/F3 represent the F primers of HLA-A/B/C/DQB1 having a forward indexing primer sequence No. n (Table 6) at the 5' end, PInr-A/B/C-R2/3/4 and PInr-Q-R2/R3 represent the R primers of HLA-A/B/C/DQB1 having an index sequence reverse No. n at the 5' end (here n<95), and the rest can be similarly deduced. In addition, each sample corresponds to a specific set of PCR Primers (PInf-A/B/C-F2/3/4, PInr-A/B/C-R2/3/4, PInf-Q-F2/F3 , PInr-Q-R2/R3).
[0217] The PCR reaction was performed in the PTC-200 PCR apparatus from Bio-Rad Co.. After the PCR reaction, 3ul of PCR products were subjected to a 2% agarose gel electrophoresis. Figure 16 showed the electrophoretic result of the PCR products of the corresponding Exons of HLA-A/B/C/DQB1 from Sample No. 1, and the DNA molecular marker was DL 2000 (Takara Co.). There were a series of single bands of an average length of 300 bp to 500 bp on the electrophorogram, indicating success in PCR amplification of Exons (A2, A3, A4, B2, B3, B4, C2, C3, C4, DRB1) of HLA-A/B/C/DQB1 from Sample No.1. There was no amplification range in the negative control (N). the results of the other samples were similar. 3. Association and purification of PCR products
[0218] For Group X samples ("X" is 1/2/3/4/5/6/7/8/9/10), 20 μl of the rest of the PCR products were taken from each plate source source 96 HLA-XP-A2 (except for the negative control), and was mixed homogeneously under agitation in a 3 ml EP tube (designated as HLA-X-A2-Mix). The same operation was applied to another 9 source plates 96 of the Group X samples, designated as HLA-X-A3-Mix, HLA-X-A4-Mix, HLA-X-B2-Mix, HLA-X-B3-Mix , HLA-X-B4-Mix, HLA-X-C2-Mix, HLA-X-C3-Mix, HLA-X-C4-Mix, and HLA-X-DQB1-Mix. 200ul was taken from each HLA-X-A2-Mix, HLA-X-A3-Mix, HLA-X-A4-Mix, HLA-X-B2-Mix, HLA-X-B3-Mix, HLA-X-B4 -Mix, HLA-X-C2-Mix, HLA-X-C3-Mix, HLA-X-C4-Mix, and HLA-X-DQB1-Mix, and was mixed in a 3ml EP tube, designated as HLA- X-Mix. 500ul of HLA-X-Mix DNA mix was subjected to column purification with a Qiagen DNA Purification Kit (QIAGEN Co.) (For specific purification steps, please refer to manufacturer's instructions) to obtain 200ul of purified DNA, each DNA concentration was determined by Nanodrop 8000 (Thermo Fisher Scientific Co.). The same operation was also applied to another 9 groups of samples. The finally determined DNA concentrations of the 10 sample groups were as follows.
4. Construction of Illumina GA sequencing libraries
[0219] As described in Example 4, a total amount of 5ug of DNA, taken from the purified HLA-X-Mix, was subjected to a DNA shear, purification after shear, end repair reaction, addition of A in 3' far end; and Illumina GA free PCR library adapter ligation.
[0220] The corresponding relationship between sample groups and library adapters was as follows.
The reaction products obtained were purified by Ampure Beads (Beckman Coulter Genomics), and were dissolved in 50ul of deionized water, and the DNA concentrations determined by quantitative Fluorescent PCR (QPCR) were as follows:
6. Sliced gel recovery
[0221] HLA-1-Mix, HLA-2-Mix, HLA-3-Mix, HLA-4-Mix, HLA-5-Mix, HLA-6-Mix, HLA-7-Mix, HLA-8-Mix , HLA-9-Mix and HLA-10-Mix were mixed in an equal mole (final concentration was 70.86nM/ul), designated as HLA-Mix-10, 30μL HLA-Mix-10 was subjected to 2% electrophoresis low melting point of agarose gel. The electrophoretic condition was 100V, 100min. The DNA marker was the NEB Co.'s 50bp DNA marker. The gel containing the DNA fragments averaging 450 to 750bp was sliced (Figure 17). The products on the sliced gel were recovered and purified by a QIAquick PCR Purification Kit (QIAGEN Co.), the volume after purification was 32ul, the DNA concentration measured by the Fluorescent PCR quantitative (QPCR) was 10.25nM . 5. Results analysis and sequencing Illumina GA
[0222] Result analyzes and sequencing were performed according to the methods as described in Examples 5 and 6.
[0223] The database, comprising the results of the sequencing of the PCR products of several Exons of HLA-A/B/C/DQB1 for each sample corresponding to a respective indexing primer was constructed. The resulting DNA sequence was aligned with the database sequence of the corresponding Exons from the professional database HLA-A/B/C/DQB1 in IMGT HLA. If the result of the sequence alignment showed 100% match, the HLA-A/B/C/DQB1 genotype of the corresponding sample was determined. Please refer to the screenshot of the program for the construction of the HLA-C site Exon 2 consensus sequence in Sample No.1, as illustrated in Figure 18. For all 950 samples, the typing results obtained by above method were fully consistent with the original known typing results, where the results for Samples No. 1-32 were as follows:


Note: in the case where the sequences of Exons 2, 3, 4 of HLA-A/B/C were completely identical, a common type was selected.
[0224] 950 samples with known HLA-SBT typing results that were genotyped at HLA-A/B/C/DQB1 sites by the technical strategy of the present invention were completely consistent with the original known results.
[0225] Although the embodiments of the present invention have already been described in detail, one skilled in the art would understand that based on all the teachings as disclosed, various modifications and substitutions can be made without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims and any equivalents thereto. References [1] . http://www.ebi.ac.uk/imgt/hla/stats.html [2] . Tiercy J M. Molecular basis of HLA polymorphism: implications for clinical transplantation. [J]. Transpl Immunol, 2002, 9: 173-180. [3] . C.Antoine, S.Müller, A.Cant, et al. long-lived survival and hemopoietic stem cell transplantation for immunodeficiencies: report of the European experience. 1968-99. [J]. The Lancet, 2003, 9357:553-560. [4]. H.A. Erlich, G. Opelz, J. Hansen, et al. HLA DNA Typing and Transplantation.[J].Immunity, 2001, 14:347-356.http://www.sciencedirect.com/science ob=ArticleURL& udi=B6WSP- 42YW740- 2& user=10& coverDate=04%2F30%2F2001& rdoc=1& fmt=high& orig=search& sort=d& docanchor=&view=c& searchStrId=1194405262& rerunOrigin=00005021 accol. =1& urlVersion=0& userid=10&md5=bbe8e010b8 bc54730458f34cddd8ec1d - aff2 [5] . Lillo R, Balas A, Vicario JL, et al. Two new classes of HLA alleles, DPB1*02014, by typing based on sequencing. [J]. tissue antigens, 2002, 59: 47-48. [6] . A. Dormoy, N. Froelich. Leisenbach, et al. Mono-Allelic Amplification of Exons 2-4 Using Specific Primer Allele Groups for Sequence-Based Typing (SBT) of HLA-A, -B, and -C genes: Preparation and validation of ready-to-use pre-SBT minikits. [J]. Tissue antigens, 2003, 62: 201-216. [7] . Elaine R. Mardis. The impact of new generation sequencing technologies on genetics. [J].Trends in Genetics.2008, 24:133-141. [8]. Christian Hoffmann1, Nana Minkah1, Jeremy Leipzig. Pyrosequencing DNA barcode to identify a rare drug resistant HIV mutation. [J]. Nucleic acid research, 2007, 1-8. [9] . Shannon J. Odelberg, Robert B. Weiss, Akira Hata. Template switching during DNA synthesis by Thermus aquaticus I DNA polymerase. [J]. Search for nucleic acids. 1995, 23: 2049-2057. [10] . Sayer D, Whidborne R, Brestovac B. HLA-DRB1 DNA sequencing-based typing: a compatible approach to high-throughput typing including registered non-bone marrow donors. [J]. Tissue antigens. 2001, 57(1):46-54. [11] . Iwanka Kozarewa, Zemin Ning, Michael A Quail. Preparation of free amplifying Illumina sequencing library facilitates improved mapping and assembly of biased (G+C) genomes. [J]. Natural methods. 2009, 6:291-295. [12] Marsh, S.G.E., Parham, P. & Barber, L.D. The HLA 3-91 Factbook (Academic Press, London, 2000). [13] Campbell, K.J. et al. Characterization of 47 MHC class I sequences in Filipino cynomolgus monkeys. Immunogenetics 61, 177-187 (2009). [14] Goulder, P.J.R. & Watkins, D.I. Impact of class MHC I diversity on immune control of immunodeficiency virus replication. Nat. Rev. Immunol. 8, 619-630 (2008). [15] O'Leary, C.E. et al. Identification of novel MHC I class sequences in monkeys with pig radon by amplified pyrosequencing of full-length cDNA sequencing and cloning. Immunogenetics 61, 689-701 (2009). [16] Robinson J, Malik A, Parham P, Bodmer JG, Marsh SGE.IMGT/HLA database - a sequence database for most human histocompatibility complexes. Tissue antigens. 55, 80-7 (2000). [17] Hoffmann C, Minkah N, Leipzig J, Wang G, Arens MQ, Thebes P, Bushman FD. Barcode and DNA pyrosequencing to identify rare HIV drug resistant mutations. Search for nucleic acids. 2007; 35(13):e91. [18] . WU, D.L. et al. Comparative analysis of serological typing and HLA-II typing by micro-PCR-SSP. Di Yi Jun Yi Da Xue Xue Bao, 2002, 22:247-249. [19] . Al-Hussein K A, Rama N R, Butt A I, et al. HLA class II sequence based on typing in normal Saudi subjects. Tissue antigens, 2002, 60: 259261. [20]. D.C. Sayer, D.M. Goodridge. Pilot study: assessment of interlaboratory typing variability based on DNA sequencing data quality. Tissue Antigens, 2007, 69 Suppl: 66-68. [21] . Horton V, Stratton I, Bottazzo G.F. et al. Genetic heterogeneity of autoimmune diabetes: age at presentation in adults is influenced by HLA DRB1 and DQB1 genotypes. Diabetologia, 1999, 42:608-616. [22] . ONE HUNDRED. Voorter, M.C. Kik1, E.M. van den Berg-Loonen et al. High resolution HLA typing for the DQB1 gene by sequencing-based typing. Tissue antigen, 2008, 51:80-87. [23] . G. Bentley, R. Higuchi, B. Hoglund et al. High resolution, high throughput HLA genotyping by next-generation sequencing. Tissue Antigens, 2009, 74: 393-403.

权利要求:
Claims (8)
[0001]
1. Method for determining the nucleotide sequence of a nucleic acid of interest in a sample, the method CHARACTERIZED by the fact that it comprises: 1) providing n samples, where n is an integer, and n>1; 2) divide the n samples into m groups, where m is an integer, and n>m>1; 3) perform PCR amplification on samples under conditions suitable for amplification of the nucleic acid of interest when sample templates are available, where one pair or multiple pairs of indexing primers are used for each sample, where each pair of indexing primers is available. indexing consists of a forward indexing initiator and a reverse indexing initiator and the indexing initiators used for different samples are different; 4) associate PCR amplification products from each of the samples together; 5) subjecting the amplified products to incomplete shear, purify and recover, wherein said incomplete shear yields intact unsheared PCR products and partially sheared PCR products; 6) build a free PCR sequencing library based on intact unsheared PCR products and partially sheared PCR products, in which different library adapters are added to distinguish different free PCR sequencing libraries; 7) purify and recover DNA lanes between the maximum read length and an applicable maximum DNA length of a sequencer; 8) subjecting the recovered DNA mixture to sequencing to obtain the sheared DNA sequences; and 9) match the obtained sequencing data with the corresponding samples based on a single indexing primer for each sample, align the sequences obtained from the sheared DNA against the reference DNA sequences corresponding to the PCR products, and assemble a complete sequence of the nucleic acid of interest from sequences obtained from the sheared DNA based on sequence overlap and linkage ratio, wherein a length of the complete sequence of the nucleic acid of interest exceeds a maximum reading length of a sequencer used for said sequencing .
[0002]
2. Method according to claim 1, CHARACTERIZED by the fact that: each pair of indexing primers and a pair of PCR primers form a pair of indexing primers, and the forward and reverse PCR primers have a primer of forward indexing and a 5' end reverse indexing primer, respectively.
[0003]
3. Method according to claim 1, CHARACTERIZED by the fact that: a) said PCR primers are PCR primers for amplification of HLA gene, HLA-A/B gene, Exons 2, 3 and 4 of HLA- A/B, or Exon 2 of HLA-DRB1; b) said PCR primers are PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11 and 12; c) said PCR primers are PCR primers for amplifying Exons 2, 3 and 4 of HLA-A/B as shown in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; d) said PCR primers are PCR primers for HLA-DRB1 Exon 2 amplification as shown in SEQ ID NO: 231, 232, 233, 234, 235, 236, 237 and 238; e) said PCR primers are PCR primers for amplification of HLA gene, HLA-C gene, or Exons 2, 3 and/or 4 of HLA-C; f) said PCR primers are PCR primers for amplifying Exons 2, 3 and/or 4 of HLA-C as shown in SEQ ID NO: 25, 26, 27, 28, 29 and 30; g) said PCR primers are PCR primers for amplifying Exons 2, 3 and/or 4 of HLA-C as shown in SEQ ID NO: 31, 32, 33, 34, 35 and 36; h) said PCR primers are PCR primers for amplification of HLA gene, HLA-DQB1 gene, or Exon 2 and/or 3 of HLA-DQB1 gene; i) said PCR primers are PCR primers for amplification of HLA gene, HLA-DQB1 gene or Exon 2 and/or HLA-DQB1 gene 3 as shown in SEQ ID NO: 37, 38, 39 and 40.
[0004]
4. Method according to claim 1, CHARACTERIZED by the fact that said indexing primers are designed for: a) PCR primers for amplification of a specific HLA gene; b) PCR primers for the amplification of Exons 2, 3 and 4 of HLA-A/B and Exon 2 of HLA-DRB1; c) PCR primers as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; d) PCR primers as shown in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24; or e) PCR primers as shown in in SEQ ID NO: 231, 232, 233, 234, 235, 236, 237 and 238.
[0005]
5. Method according to claim 1, CHARACTERIZED by the fact that said indexing primers comprise at least 10 pairs of the following 95 indexing primer pairs:
[0006]
6. Method according to claim 1, CHARACTERIZED by the fact that said DNA shear is performed by chemical shear and physical shear, in which chemical shear includes enzymatic digestion, and physical shear includes ultrasonic shear or mechanical shear.
[0007]
7. Method according to claim 1, CHARACTERIZED by the fact that said methods of purification and recovery include recovery by electrophoresis and gel cutting, and recovery by magnetic beads.
[0008]
8. Method for determining the HLA genotype of a sample, the method CHARACTERIZED in that it comprises: sequencing a sample from a patient by the method as defined in any one of claims 1 to 7; align sequencing results with sequence data from HLA Exons 2, 3, 4 of HLA-A/B, Exons 2, 3 and/or 4 of HLA-C, Exon 2 and/or 3 of HLA gene -DQB1 and/or Exon 2 of HLA-DRB1 in an HLA database; and determining the HLA genotype of the sample if the sequence alignment result shows 100% match.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112012032586B1|2021-08-17|METHODS FOR DETERMINING THE NUCLEOTIDE SEQUENCE OF A NUCLEIC ACID OF INTEREST AND FOR DETERMINING THE HLA GENOTYPE IN A SAMPLE

---

US20190002979A1|2019-01-03|Haplotyping of hla loci with ultra-deep shotgun sequencing

---

WO2012000152A1|2012-01-05|Pcr-sequencing method based on technology of dna molecular index and strategy of dna-breaking incompletely

---

Liu et al.2014|Extended blood group molecular typing and next-generation sequencing

---

WO2012000153A1|2012-01-05|High resolution typing method of hla gene based on illumina ga sequencing technology

---

WO2012068955A1|2012-05-31|Mhc region nucleic acid library, construction method therfor, and use thereof

---

WO2015200701A2|2015-12-30|Software haplotying of hla loci

---

WO2012000150A1|2012-01-05|Pcr primers for determining hla-a,b genotypes and methods for using the same

---

Mondal et al.2011|Targeted sequencing of the human X chromosome exome

---

Dunn2015|Novel approaches and technologies in molecular HLA typing

---

JP2003500067A|2003-01-07|Method for analyzing a patient&#39;s genetic predisposition to at least one disease and amplification adapted to the method

---

TW201300528A|2013-01-01|Method for hla-dqb1 genotyping and related primers thereof

---

WO2017135396A1|2017-08-10|Probe set for hla genotyping by capture method without using pcr, and typing method in which same is used

---

Kulski et al.2014|In phase HLA genotyping by next generation sequencing-a comparison between two massively parallel sequencing bench-top systems, the Roche GS Junior and ion torrent PGM

---

WO2018147438A1|2018-08-16|Pcr primer set for hla gene, and sequencing method using same

---

JP2009261358A|2009-11-12|Method for typing allele polymorphism of human gene including hla-drb1 gene, and kit used in the method

---

TWI542696B|2016-07-21|HLA - C genotyping and its related primers

---

JP5143450B2|2013-02-13|New alleles in HLA-B locus

---

Yin et al.2021|Challenges in the application of NGS in the clinical laboratory

---

CN111187810B|2020-09-29|Method for detecting multiple tumor-associated genes for non-diagnostic therapeutic purposes

---

JP2010162020A|2010-07-29|New hla-drb1 gene, and use thereof

---

Yang et al.2019|Human leukocyte antigen-A* 33: 03-B* 58: 01-DRB1* 15: 140, a deduced probable human leukocyte antigen haplotype in association with a human leukocyte antigen low-incidence allele DRB1* 15: 140 in Taiwanese individuals: A case analysis

---

Yang et al.2017|Deduced probable human leukocyte antigen haplotypes associated with HLA-A* 11: 256Q and HLA-A* 02: 621 identified by case analyses of Taiwanese individuals

---

JP2011050377A|2011-03-17|New hla-drb1 gene and use thereof

同族专利:

---

公开号 | 公开日

---

RU2587606C2|2016-06-20|

---

RU2013103795A|2014-08-20|

---

WO2012000445A1|2012-01-05|

---

AU2011274090B2|2015-04-09|

---

US9957564B2|2018-05-01|

---

KR20130038353A|2013-04-17|

---

EP2599877A4|2014-01-01|

---

AU2011274090A1|2013-02-07|

---

CA2803940C|2019-07-02|

---

EP2599877A1|2013-06-05|

---

SG186876A1|2013-02-28|

---

JP2013529472A|2013-07-22|

---

CA2803940A1|2012-01-05|

---

EP2599877B1|2017-09-27|

---

US20130237432A1|2013-09-12|

---

BR112012032586A2|2019-07-30|

---

DK2599877T3|2017-11-20|

---

JP5968879B2|2016-08-10|

---

MY173793A|2020-02-24|

---

KR101709826B1|2017-02-24|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

WO1993009245A1|1991-10-31|1993-05-13|University Of Pittsburgh|Reverse dot blot hybridization using tandem head-to-tail monomers containing probes synthesized by staggered complementary primers|

---

US5580730A|1994-08-19|1996-12-03|Olympus America, Inc.|Enzyme digestion method for the detection of amplified DNA|

---

AU6321998A|1997-02-11|1998-08-26|William H. Hildebrand|Class i sequence based typing of hla-a, -b, and -c alleles by direct dna sequencing|

---

EP0953650A1|1998-04-20|1999-11-03|Innogenetics N.V.|Method for typing of HLA alleles|

---

CN101128601B|2003-01-29|2011-06-08|454生命科学公司|Methods of amplifying and sequencing nucleic acids|

---

US7300755B1|2003-05-12|2007-11-27|Fred Hutchinson Cancer Research Center|Methods for haplotyping genomic DNA|

---

WO2005042764A2|2003-10-28|2005-05-12|Dynal Biotech Llc|Primers, methods and kits for amplifying or detecting human leukocyte antigen alleles|

---

CA2612145A1|2005-05-10|2006-11-16|Eric A. Johnson|Methods of mapping polymorphisms and polymorphism microarrays|

---

EP1915618A4|2005-06-02|2009-09-30|Fluidigm Corp|Analysis using microfluidic partitioning devices|

---

EP1929039B2|2005-09-29|2013-11-20|Keygene N.V.|High throughput screening of mutagenized populations|

---

ES2422288T3|2005-11-14|2013-09-10|Keygene Nv|Method for ultrafast screening of transposon labeling populations and massive identification of the parallel sequence of insertion sites|

---

DK2789696T3|2005-12-22|2016-02-29|Keygene Nv|A method for high-throughput AFLP-based polymorphism|

---

WO2007140540A2|2006-06-09|2007-12-13|Conexio 4 Pty Ltd|Identification of a nucleic acid molecule|

---

AT509123T|2007-10-16|2011-05-15|Hoffmann La Roche|HIGH-RESOLUTION HIGH-SPEED HLA GENOTYPIZATION USING CLONED SEQUENCING|

---

US20100086914A1|2008-10-03|2010-04-08|Roche Molecular Systems, Inc.|High resolution, high throughput hla genotyping by clonal sequencing|

---

RU2393231C1|2008-12-29|2010-06-27|Федеральное государственное учреждение здравоохранения "Российский научно-исследовательский противочумный институт "Микроб" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека |Method for determination of genetic relationship of comma bacilli strains by sequencing genes flanking cluster of o-antigen biosynthesis genes|

---

CN101921841B|2010-06-30|2014-03-12|深圳华大基因科技有限公司|HLA gene high-resolution genotyping method based on Illumina GA sequencing technology|

---

CN101921840B|2010-06-30|2014-06-25|深圳华大基因科技有限公司|DNA molecular label technology and DNA incomplete interrupt policy-based PCR sequencing method|

---

CN101921842B|2010-06-30|2013-08-07|深圳华大基因科技有限公司|HLA -A,B genotyping PCR primer and application method thereof|WO2014171898A2|2013-04-17|2014-10-23|Agency For Science, Technology And Research|Method for generating extended sequence reads|

---

JP6308724B2|2013-05-09|2018-04-11|ジェノダイブファーマ株式会社|Multiplex DNA typing method and kit for HLA gene|

---

US10508311B2|2013-08-26|2019-12-17|The Translational Genomics Research Institute|Single molecule-overlapping read analysis for minor variant mutation detection in pathogen samples|

---

GB201409282D0|2014-05-23|2014-07-09|Univ Sydney Tech|Sequencing process|

---

AU2014406026B2|2014-09-12|2018-08-23|Mgi Tech Co., Ltd.|Isolated oligonucleotide and use thereof in nucleic acid sequencing|

---

CN105400864B|2014-09-12|2020-04-14|深圳华大基因股份有限公司|Method for constructing sequencing libraries based on blood samples and use thereof in determining fetal genetic abnormalities|

---

US10233490B2|2014-11-21|2019-03-19|Metabiotech Corporation|Methods for assembling and reading nucleic acid sequences from mixed populations|

---

US9618474B2|2014-12-18|2017-04-11|Edico Genome, Inc.|Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids|

---

CA2971589C|2014-12-18|2021-09-28|Edico Genome Corporation|Chemically-sensitive field effect transistor|

---

US9859394B2|2014-12-18|2018-01-02|Agilome, Inc.|Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids|

---

US10020300B2|2014-12-18|2018-07-10|Agilome, Inc.|Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids|

---

US9857328B2|2014-12-18|2018-01-02|Agilome, Inc.|Chemically-sensitive field effect transistors, systems and methods for manufacturing and using the same|

---

US10006910B2|2014-12-18|2018-06-26|Agilome, Inc.|Chemically-sensitive field effect transistors, systems, and methods for manufacturing and using the same|

---

CN107002153B|2015-02-04|2020-10-27|深圳华大生命科学研究院|Method for constructing long fragment sequencing library|

---

KR101651817B1|2015-10-28|2016-08-29|대한민국|Primer set for Preparation of NGS library and Method and Kit for making NGS library using the same|

---

WO2017147294A1|2016-02-23|2017-08-31|Novozymes A/S|Improved next-generation sequencing|

---

WO2017201081A1|2016-05-16|2017-11-23|Agilome, Inc.|Graphene fet devices, systems, and methods of using the same for sequencing nucleic acids|

---

EP3626835A1|2018-09-18|2020-03-25|Sistemas Genómicos, S.L.|Method for genotypically identifying both alleles of at least one locus of a subject's hla gene|

---

RU2703394C1|2018-10-24|2019-10-16|Федеральное государственное бюджетное образовательное учреждение высшего образования "Кубанский государственный аграрный университет имени И.Т. Трубилина"|Method for detecting and genotyping rna of porcine reproductive-respiratory syndrome virus|

---

RU2703401C1|2018-10-24|2019-10-16|Федеральное государственное бюджетное образовательное учреждение высшего образования "Кубанский государственный аграрный университет имени И.Т. Трубилина"|Test system for detecting and genotyping rna of swine reproductive-respiratory syndrome virus|

---

FR3100820A1|2019-09-12|2021-03-19|Bionobis|Genotyping process suitable for processing a large number of samples, especially in cases of high polymorphism|

---

FR3106596A1|2020-01-27|2021-07-30|Bionobis|SIMPLE AND FAST HLA GENOTYPING PROCESS|

---

FR3110178A1|2020-05-18|2021-11-19|Bionobis|Genotyping process suitable for the simultaneous treatment of a large number of patients|

法律状态:
2019-11-05| B25C| Requirement related to requested transfer of rights|Owner name: BGI SHENZHEN CO., LIMITED (CN) Free format text: A FIM DE ATENDER A TRANSFERENCIA, REQUERIDA ATRAVES DA PETICAO NO 870160018312 DE 06/05/2016, E NECESSARIO APRESENTAR A TRADUCAO JURAMENTADA DO DOCUMENTO, ALEM DA GUIA DE CUMPRIMENTO DE EXIGENCIA. |

---

2019-11-12| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2020-02-11| B25A| Requested transfer of rights approved|Owner name: BGI GENOMICS CO., LTD (CN) |

---

2020-03-31| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2021-04-06| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

---

2021-07-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-08-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/06/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

---

申请号 | 申请日 | 专利标题

---

CN201010213719.5|2010-06-30|

---

CN201010213717.6|2010-06-30|

---

CN201010213721.2|2010-06-30|

---

CN201010213719.5A|CN101921841B|2010-06-30|2010-06-30|HLAgene high-resolution genotyping method based on Illumina GA sequencing technology|

---

CN201010213717.6A|CN101921840B|2010-06-30|2010-06-30|DNA molecular label technology and DNA incomplete interrupt policy-based PCR sequencing method|

---

CN 201010213721|CN101921842B|2010-06-30|2010-06-30|HLA -A,B genotyping PCRprimer and application method thereof|

---

CNPCT/CN2010/002149|2010-12-24|

---

CNPCT/CN2010/002150|2010-12-24|

---

PCT/CN2010/002149|WO2012083505A1|2010-12-24|2010-12-24|Method for hla-c genotyping and related primers thereof|

---

PCT/CN2010/002150|WO2012083506A1|2010-12-24|2010-12-24|Method for hla-dqb1 genotyping and related primers thereof|

---

PCT/CN2011/076688|WO2012000445A1|2010-06-30|2011-06-30|New pcr sequencing method and use thereof in hla genotyping|

---