Cost-efficient multiplex PCR for routine genotyping
Cost-efficient multiplex PCR for routine
genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing
Yuki Ozaki 1?,Shingo Suzuki 1?,Koichi Kashiwase 2,Atsuko Shigenari 1,Yuko Okudaira 1,Sayaka Ito 1,Anri Masuya 1,Fumihiro Azuma 2,Toshio Yabe 2,Satoko Morishima 3,Shigeki Mitsunaga 1,Masahiro Satake 2,Masao Ota 4,Yasuo Morishima 5,Jerzy K Kulski 1,6,Katsuyuki Saito 7,Hidetoshi Inoko 1and Takashi Shiina 1*
*Correspondence:tshiina@is.icc.u-tokai.ac.jp ?
Equal contributors 1
Department of Molecular Life Science,Division of Basic Medical Science and Molecular Medicine,Tokai University School of Medicine,Isehara,Kanagawa 259-1143,Japan
Full list of author information is available at the end of the article
?2015Ozaki et al.;licensee BioMed Central.This is an Open Access article distributed under the terms of the Creative
Ozaki et al.BMC Genomics (2015) 16:318 DOI 10.1186/s12864-015-1514-4
Background
The Human Leukocyte Antigen(HLA)or the Major Histocompatibility Complex(MHC)is a highly poly-morphic region of the human genome(on the short arm of chromosome6)that is critically involved in the rejection and graft-versus-host disease(GVHD)of hematopoietic stem cell transplants[1,2],the pathogenesis of numerous autoimmune diseases[3-6],infectious diseases[7]and drug adverse reactions[8,9].Many variations of the con-ventional HLA genotyping methods such as incorporating restriction fragment polymorphisms(RFLP)[10],single strand conformation polymorphism(SSCP)[11],sequence specific oligonucleotides(SSOs)[12],sequence specific primers(SSPs)[13]and sequence based typing(SBT),like the Sanger method[14],have been used for the efficient and rapid HLA matching in transplantation therapy [15-18],research into autoimmunity and HLA related dis-eases[19,20],population diversity studies[21-23]and in forensic and paternity testing[24].The HLA genotyping methods mainly applied today are PCR-SSOP,such as the Luminex commercial methodology[25,26],and SBT by the Sanger method employing capillary sequencing based on chain-termination reactions[14,27].However,both methods often detect more than one pair of unresolved HLA alleles because of chromosomal phase(cis/trans) ambiguity[28-30].To solve the phase ambiguity problem, we previously reported the development and application of the super high resolution-single molecule-sequence-based typing(SS-SBT)method using long-range PCR of the sample DNA from the promoter-enhancer region to the3′untranslated region(3′UTR)for11classical HLA loci,HLA-A,HLA-B,HLA-C,HLA-DRB1,HLA-DRB3/ 4/5,HLA-DQA1,HLA-DQB1,HLA-DPA1,and HLA-DPB1in combination with next generation sequencing (NGS)platforms such as Ion PGM(Life Technologies) and GS Junior(Roche)[31-33].Other long-range PCR and NGS-based HLA genotyping methods using454GS-FLX(Roche)and MiSeq(Illumina)platforms[30,34-36] also resolved the phase ambiguities.Thus,the NGS methods combined with the long-range PCR technology are expected to produce genotyping results to the field4 level(formerly known as8-digit typing)allelic resolution to efficiently detect new and null alleles without phase ambiguity.
The NGS methods are usually divided into three basic steps,long-range PCR of the DNA samples,NGS,and allele assignment step(Figure1A)[25].Before performing the NGS step there are at least five sub-steps for PCR, such as preparation of DNA template and PCR mixes,the PCR runs,electrophoresis,purification,and quantitative determination of the PCR products(Figure1B).Multiple micro-tubes are required for the singleplex PCR.For ex-ample,at least six micro-tubes are required to amplify of NGS library preparation processes can be used after performing the long-range singleplex PCR procedure. One process is to prepare a number of single locus tagging NGS libraries and then pool all of them into a single NGS library(singleplex PCR/singleplex NGS library model:Figure1B(1)).The other process is to pool all of the PCR products and prepare a single NGS library for each of the tagged multiple loci as a multiplex NGS library (singleplex PCR/multiplex NGS library model:Figure1B (2)).However,the long-range singleplex PCR amplifica-tion and NGS library preparation as outlined in Figures1B (1)and B(2)are extremely labor intensive and time con-suming.Furthermore,it is easy to make human errors at the pooling stage that negatively influence the sequence read numbers.Therefore,simplification,acceleration and cost-saving in the NGS protocols are required if they are to become routine DNA typing methods and replace the conventional HLA genotyping methods such as SBT and PCR-SSOP(e.g.Luminex methodology).
In this paper,we describe the development and evalu-ation of four types of multiplex PCR methods that geno-type multiple HLA loci to the field3level(6-digit typing) using combinations of locus specific PCR primers for up to nine classical HLA loci(HLA-A,HLA-B,HLA-C, HLA-DRB1/3/4/5,HLA-DQB1,and HLA-DPB1).We evaluated the uniformity and accuracy of NGS-based HLA genotyping among the nine HLA loci and between HLA alleles obtained by one of the multiplex PCR methods(the nine loci[9LOCI]multiplex method)in a single NGS run with the Ion PGM sequencer using46 genomic DNA reference samples from Japanese subjects who represented a distribution of more than99.5%HLA alleles in each of the HLA locus in the Japanese popula-tion.In addition,we investigated template DNA amounts as low as1ng to evaluate the smallest amounts of gen-omic DNA samples that we could use successfully in our multiplex PCR methods for NGS-based HLA genotyping. Results
Characteristics of four types of multiplex PCR methods Four types of multiplex PCR methods were developed after optimization of primer composition and PCR con-ditions such as annealing and extension temperatures using the HLA-A,HLA-B,HLA-C,HLA-DRB1/3/4/5, HLA-DQB1,and HLA-DPB1specific primer pair sets (Figure2).These four types were CI:A/B/C,CII:DRB1/ 3/4/5/DQB1/DPB1,7LOCI:A/B/C/DRB1/3/4/5,and 9LOCI:A/B/C/DRB1/3/4/5/DQB1/DPB1.Two,three, three,and four bands that reflect the targeted PCR prod-ucts were observed in CI,CII,7LOCI,and9LOCI multi-plex PCR methods,respectively.Although most of the bands overlapped because of their similar PCR lengths as for HLA-B and HLA-C in CI(Figure2A),it is note-
in CII and9LOCI(Figure2B,D)were clearly observed as unique bands.In addition,the PCR product from the HLA-DRB1gene varies in size depending on the DR sub-type such as5.2kb in the DR4sub-type and4.0-4.1kb in the other DR sub-types.
When we applied the nine loci multiplex PCR(9LOCI) method using46genomic DNA samples(JPN01to JPN46), the PCR products with similar band patterns were ob-served in all samples,although weak bands were also ob-served in some samples such as JPN23,JPN24,JPN25, and JPN29(Additional file1:Figure S1).
Sequence read information obtained from46genomic DNA samples
Sequence read information was obtained for all the46 genomic DNA samples after sequencing of the9LOCI products using the Ion PGM system in a single sequen-cing run after gathering the46barcode-labeled DNA li-braries into one tube.Draft read numbers in total were 5,284,570sequence reads with a range of reads from 83,680in JPN21to156,157sequence reads in JPN45that were high quality sequence reads with more than10 quality values(QV)and an average QV of28.0±0.2in the high quality sequence reads.The draft read bases in total were1,447Mb with a range between21.7Mb in JPN021and43.4Mb in JPN041(31.4±6.0Mb on aver-age),with an overall average read length of273.3±9.9 bases and an overall mode read length of358.6±16.7 bases(Additional file2:Table S1).Therefore,the sequence reads had high quality and sufficient sequence volume for further HLA genotyping analysis.
Genotyping to the field3level on the nine HLA loci Nucleotide similarity searches of the sequenced HLA al-leles to the field3level using the BLAT program identi-fied276alleles at the six HLA loci(Additional file3: Table S2),except for DRB3/4/5,that were consistent with known HLA alleles assigned by the conventional Luminex method(Additional file4:Table S3).Of the heterozygous 242alleles,224were defined as two separate HLA alleles without any ambiguities.However,one locus observed in JPN15(DPB1*05:01:01/DPB1*135:01and DPB1*25:01)
patterns of the multiplex PCR products.Electrophoresis images of PCR products from four unrelated genomic DNA using four types of multiplex PCR methods,CI(A),CII(B),7LOCI(C)and9LOCI(D),respectively.Numbers1to DNA samples WW035,WW090,WW102,and WW104,respectively.The lanes labeled M are bands of the1kb the bands and the HLA loci amplified by PCR are indicated on the right side of the figure.
SNP in exon4for DPB1*25:01that was needed to as-sign the correct allele at the locus.Also,five types of ambiguous HLA alleles,such as DRB1*04:07:01/*04:92, DRB1*04:10:01/*04:10:03,DRB1*09:01:02/*09:21,DRB1* 12:01:01/*12:10,and DPB1*13:01/*107:01at a total of17 loci,were observed because the informative SNPs that differentiate between ambiguous alleles were located out-side of the PCR regions such as within exon1or exon4 of HLA-DRB1or HLA-DPB1(Additional file3:Table S2). From the results of genotyping to the field3level,five HLA-DRB3,three HLA-DRB4,and three HLA-DRB5al-leles were assigned in the45DNA samples(T able1and Additional file3:Table S2).There were thirty DRB1-DRB3/ 4/5haplotypes in total with15assigned as the DRB1-DRB3haplotype,12as DRB1-DRB4,and three as DRB1-DRB5. These haplotypes were identified by estimating HLA-DRB1 and HLA-DRB3/4/5alleles without any descrepancy to pre-viously reported DRB structures[32,37].
Moreover,mapping analysis including other exons and introns using the SEABASS program suggested that no recombinations were evident within the gene loci examined for the46genomic samples(data not shown).Through this process one synonymous substitution was newly de-tected in exon4of HLA-C*07:04of JPN16.
Evaluation of the9LOCI multiplex PCR method
To evaluate the9LOCI method,we compared the depth of redundancy derived from the sequence read numbers
Table1DRB1-DRB3/4/5haplotypes to the field3level
DR haplotype DR type DRB1type DRB1-DRB3/4/5haplotype Observed number
DRB1allele DRB3/4/5allele
DR52DR3DR3DRB1*03:01:01DRB3*02:02:011
DR5DR11DRB1*11:01:01DRB3*02:02:013
DRB1*11:19:01DRB3*02:02:011
DR12DRB1*12:01:01/*12:10DRB3*01:01:024
DRB1*12:01:01/*12:10DRB3*01:121
DRB1*12:02:01DRB3*03:01:033
DR6DR13DRB1*13:01:01DRB3*01:01:021
DRB1*13:02:01DRB3*03:01:012
DRB1*13:07:01DRB3*02:02:011
DR14DRB1*14:02:01DRB3*02:02:011
DRB1*14:03:01DRB3*01:01:022
DRB1*14:05:01DRB3*02:02:011
DRB1*14:06:01DRB3*02:02:011
DRB1*14:07:01DRB3*02:02:011
DRB1*14:54:01DRB3*02:02:011
DR53DR4DR4DRB1*04:01:01DRB4*01:021
DRB1*04:03:01DRB4*01:03:011
DRB1*04:04:01DRB4*01:03:011
DRB1*04:05:01DRB4*01:03:018
DRB1*04:05:01DRB4*01:03:022
DRB1*04:06:01DRB4*01:03:016
DRB1*04:06:01DRB4*01:03:021
DRB1*04:07:01/*04:92DRB4*01:03:021
DRB1*04:10:01/*04:10:03DRB4*01:03:011
DR7DR7DRB1*07:01:01DRB4*01:03:012
DR9DR9DRB1*09:01:02/*09:21DRB4*01:03:0210
DRB1*09:01:02/*09:21DRB4*01:03:012
DR51DR2DR15DRB1*15:01:01DRB5*01:01:014
DR15DRB1*15:02:01DRB5*01:025
DR16DRB1*16:02:01DRB5*02:021
between HLA alleles and among HLA loci.An observed average depth and range for six HLA loci was as follows:78.5±42.0from 31.6to 225.1for HLA-A,116.5±51.1from 33.7to 341.8for HLA-B,130.0±59.3from 62to 331.3for HLA-C,209.1±115.9from 44.1to 712.4for HLA-DRB1,194.7±104.9from 74.9to 614.4for HLA-DQB1,and 59.2±34.6from 25.3to 161.4for HLA-DPB1(Table 2and Additional file 5:Table S4).The aver-age depth ratio was mostly even for both alleles,ranging from 0.9±0.3in HLA-DRB1to 1.0±0.1in HLA-A,but allelic imbalances of 0.2-0.5were observed in eight DNA samples of HLA-B and 16DNA samples of HLA-DRB1(Additional file 5:Table S4).Most of the loci contained specific HLA allele groups such as B*39and DRB1*04.On the other hand,an observed average depth among the HLA locus was from 116.3±42.6in HLA-DPB1to 418.3±143.0in HLA-DRB1.When we normalized the values using the average sequence read numbers (114,882reads),the depth was from 118.4±37.8in HLA-DPB1to 416.7±114.3in HLA-DRB1(Table 2).A locus balance plot showed locus imbalance among the loci ranging from high at HLA-DRB1and HLA-DQB1to low at HLA-A and HLA-DPB1(Figure 3).However,the genotypes obtained at all the loci (276alleles)in this study,were consistent with known HLA alleles to the field 3level with more than 25depth units per allele in DPB1*05:01:01of JPN39,suggesting that the locus balance completely made up for the allelic imbalances observed for some spe-cific alleles.Taken together,the 9LOCI PCR and NGS is a precise HLA genotyping method with relevant locus bal-ance and without excessive allelic imbalance (<0.2)affect-ing the results deleteriously.
Investigation of template DNA amounts for the 9LOCI method
In order to achieve precise HLA genotyping for the 9LOCI method even with using extremely small amounts of genomic DNA samples,we tried the 9LOCI multiplex PCR using four different amounts of template DNA,1ng,5ng,and 10ng,along with standard amount of 25ng.Amplified PCR products were observed for all of the tem-plate DNA amounts ranging from 1to 25ng (Figure 4).
The DNA amounts after purification of the PCR products ranged from 119.7ng (1ng template)to 608.6ng (25ng template)in TU5,and from 112.5ng (1ng template)to 559.7ng (25ng template)in TU6.The purified PCR prod-ucts of 100ng were used for construction of Ion PGM libraries.The molarities after construction of Ion PGM li-braries ranged from 8,444pM (5ng template in TU5)to 26,772pM (25ng template in TU5),and the sequence read numbers ranged from 329,752(1ng template in TU5)to 651,450(25ng template in TU6).The genotype results obtained for the eight samples used in this test of template DNA amounts were consistent to the previously assigned HLA alleles [31].
Discussion
In this study,we used a reference set of 46Japanese sub-jects that represented a distribution of more than 99.5%of the Japanese HLA alleles at the nine HLA loci geno-typed by the multiplex PCR-NGS method using the Ion
Table 2Depth information of each allele and each locus
Locus Average depth (average ±SD)Average depth ratio (average ±SD)*
Average depth per locus (average ±SD)Observed Normalized*Observed Normalized*HLA-A 78.5±42.078.2±37.9 1.0±0.1156.9±48.5156.3±36.2HLA-B 116.5±51.1117.8±46.80.9±0.2233.0±75.2235.6±68.2HLA-C 130.0±59.3127.4±55.80.9±0.1257.1±90.9255.4±71.3HLA-DRB1209.1±115.9208.4±108.20.9±0.3418.3±143.0416.7±114.3HLA-DQB1194.7±104.9194.4±95.30.9±0.1389.3±115.9388.9±100.4HLA-DPB1
59.2±34.6
59.2±31.3
0.9±0.1
116.3±42.6
118.4±37.8
Figure 3Locus balance depth plot based on normalized average depth per allele.White and black dots indicate normalized depth per locus in each DNA sample and normalized depth on average,respectively.The Y-axis of the plot shows the units of sequence depth and the X-axis shows the HLA gene loci.The detailed information is provided in Additional file 5:Table S4.
PGM system.All of the genotypes and linkages of DRB1 and DRB3/4/5alleles were consistent with known alleles (Additional file3:Table S2)and previous publications [32,37],suggesting that a combination of our multiplex PCR methods and the Ion PGM system is an efficient and accurate HLA genotyping method for the detection of HLA alleles to the field3level of genotyping without phase ambiguity.In addition,PCR products were ob-tained from all of the HLA loci by the multiplex PCR-NGS methods in tests using400non-Japanese(mainly European),subjects,confirming that the methods will be useful for the Japanese as well as for other world-wide populations(data not shown).
The high density in the average depth of sequences by NGS suggests that an increase in the DNA sample num-bers for sequencing beyond46per run as described here is likely to contribute to even lower costs.For example, 85,879sequence reads for sample JPN33that was imbal-anced at DRB1*04:06:01was assigned with a sequence depth of58.9.When we assume that an average of85,879 sequence reads was obtained from a total of5,284,570 reads that have a similar quality to those described in Additional file2:Table S1,then at least61DNA samples could have been genotyped in a single run using Ion PGM.The multiplex PCR methods for HLA genotyping could also be used on other NGS platforms such as MiSeq (illumina),GS Junior and454GS-FLX(Roche),as well as on the3rd generation sequencing platform PacBio RS (Pacific Bioscience)that is based on single molecule real-time(SMRT)technology(unpublished data). Although,a few samples like JPN15(DPB1*05:01:01/ DPB1*135:01and DPB1*25:01)were not fully resolved by the multiplex PCR-NGS method,this problem could be solved in future by determining the full gene nucleo-tide sequence for the DPB1gene with the*25:01allele. Hence,it is necessary to comprehensively collect the HLA allele sequences for all of the PCR regions of all the HLA genes to avoid misidentifying the true locus because of potential problems of allele sharing be-tween different loci or PCR amplification of sequences ambiguous HLA alleles,DRB1*04:07:01/*04:92,DRB1*04:10:01/ *04:10:03,DRB1*09:01:02/*09:21,DRB1*12:01:01/*12:10, and DPB1*13:01/*107:01,were observed at17loci.In these cases,the ambiguities were not solved because the informative SNPs for these genes are outside of the PCR regions such as in some of the introns or the5′and3′non-coding regions or because the informative SNPs that differentiate between ambiguous alleles are located outside of the PCR regions such as within exon1or exon4of HLA-DRB1or HLA-DPB1(Figure5).It is noteworthy, however,that there was no problem with phase ambiguity for more than99%of the HLA alleles detected by the 9LOCI method that included the signature sequences of the highly polymorphic exon2that play an important role for antigen presentation.Therefore,the multiplex PCR-NGS HLA genotyping method that we have de-scribed here is highly effective,accurate and informative and provides an important alternative to the conventional HLA genotyping methods such as SBT and PCR-SSOP that are currently in use in the clinical laboratory.When we applied our previously published long-range PCR primer sets for the ambiguous loci[31],DPB1*05:01:01 and DPB1*25:01,DRB1*04:07:01,DRB1*04:10:03,DRB1* 09:01:02,DRB1*12:01:01,and DPB1*13:01were assigned without ambiguity.These alleles were consistent with known HLA alleles previously assigned by the con-ventional methods(Additional file4:Table S3).
To evaluate the sequencing parameters for the9LOCI PCR-NGS method,we compared the sequencing depth derived from the sequence read numbers between HLA alleles and among HLA loci.The lowest observed aver-age depth(59.2±34.6)was for HLA-DPB1among the six loci with more than a read depth of25(Table2), where a depth of at least30is necessary to identify gen-etic variants with the highest sensitivity and resolution [38].In this respect,it will be necessary to improve the sequence reads of HLA-DPB1by further optimization of primer composition.
In contrast to the one simple multiplex PCR step that is required for the9HLA loci,the singleplex PCR models described in Figure1B(1)and B(2)require many more repetitive steps to amplify the9HLA loci and at least five complicated PCR steps for each locus such as preparation of PCR reagents and DNA tem-plates,long range singleplex PCR,electrophoresis,puri-fication,and quantitative determination of the PCR products before preparing the single locus tagging NGS libraries and pooling of all libraries(singleplex PCR/sin-gleplex NGS library model,Figure1B(1)),and/or before pooling of all PCR products and preparing the multiple locus tagging NGS libraries(singleplex PCR/multiplex NGS library model,Figure1(2)).As a more efficient, economical and rapid alternative to the time-consuming
Figure4Electrophoresis images of PCR products from two unrelated genomic DNA samples TU05and TU06using the9LOCI method.Numbers1to4above the lanes indicate the template DNA amounts25ng,10ng,5ng,and1ng,respectively.The lanes labeled M are bands of the1kb DNA size marker ladder.
multiplex PCR methods(CI,CII,7LOCI,and9LOCI) for NGS-based HLA genotyping of polymorphic exons. All of the four types of multiplex PCR methods are useful for the HLA genotyping(this study and data not shown),but the9LOCI method is likely to be the more valuable method for future routine genotyping for the following reasons and technical advantages.(1)The 9LOCI genotyping method is capable not only for typing the specific HLA loci such as HLA-A,HLA-B,and HLA-DRB1,but also other classical HLA loci such as HLA-C,HLA-DRB3/4/5,HLA-DQB1,and HLA-DPB1 at the same time(Additional file3:Table S2).(2)The running cost and operation time for the PCR step in the 9LOCI method were reduced to one sixth of the single-plex PCR models.The use of only one micro-tube per sample for the PCR and NGS steps is time-saving and economically helpful for cost-savings on micro-tubes, DNA polymerase and other reagents,and it also elimi-nates the pooling operations(Figure1B(3)).If the quan-tity of all template DNA samples is adjusted accurately among the samples,then electrophoresis of the samples also could be omitted because the PCR products are quantified by the PicoGreen assay after their purifica-tion.Thus,omission of some processes such as the pool-ing step that can influence the sequence read numbers could help to a further reduce potential human experi-mental errors.(3)The PCR step of multiplex9LOCI PCR-NGS method for46DNA samples was performed in one day.In comparison,the singleplex methods using 46DNA samples for each of the nine HLA loci would have taken at least three days.(4)Investigation of the template DNA amount suggested that1ng of template DNA is sufficient for genotyping all nine HLA loci by the9LOCI method(Figure4).The1ng amount for the multiplex method is much less than that required for and therefore markedly reduced the progressive loss of valuable DNA samples that are required for genotyping of nine HLA loci.This small DNA amount also could be helpful for DNA typing from swab samples derived from oral mucosa cells,FACS derived lymphoma cells and other valuable clinical samples.(5)Therefore,the multi-plex PCR method for the nine HLA loci greatly simpli-fies the procedures required in preparing the DNA samples for NGS by reducing the time of preparation and the amount and costs of reagents,including the use of much smaller amounts of template DNA samples.In addition,the use of different NGS methods might further improve the simplicity and cost of the multiplex PCR-NGS method in the future.For example,a new protocol using Ion Isothermal Amplification Chemistry that enables se-quence reads of up to and beyond500bp,and Ion Hi-Q?Sequencing Chemistry that reduces consensus insertion and deletion(indel)errors including homopolymer errors will be available in the near future(personal communica-tion with Life Technologies),and might lead to further simplification and cost reduction with higher data quality. Conclusions
Our aim was to simplify and streamline the NGS-based HLA genotyping method as an alternative to the con-ventional HLA genotyping methods.Although46gen-omic DNA samples were used in the present study as an example of using multiple samples in a single genotyping run,we have recently applied the same methods for genotyping more than500DNA samples from Japanese, Indian and French populations in a number of different genotyping runs to unequivocally define the HLA-A, HLA-B,HLA-C,HLA-DRB1/3/4/5,HLA-DQB1,and HLA-DPB1loci to single HLA alleles to the field3level without ambiguity.Therefore,the multiplex PCR methods
the targeted PCR regions in nine HLA loci.Black,gray and white boxes indicate promoter regions,highly exons,respectively.
costs and reagents at the PCR step in the NGS-based HLA genotyping method.The methods also conserve on the amounts of DNA samples needed to genotype a multiple number of HLA loci.Overall,the multiplex PCR methods are a powerful tool that provides precise genotyping data without phase ambiguity and with a po-tential to replace the current routine genotyping methods to find polymorphisms.These methods may help to fur-ther activate many fields of medical research involved in the studies of transplantation,disease association,drug ad-verse reaction,peptide vaccination treatment for cancer and provide us with a better understanding about the di-versity and evolution of the human MHC.
Methods
Genomic DNA samples
A total of3,115donors for bone marrow transplantation through the Japan Marrow Donor Program(JMDP)be-tween2006and2010were retrospectively genotyped for HLA-A,HLA-B,HLA-C,HLA-DRB1,HLA-DQB1,and HLA-DPB1alleles to the field2level(4-digit typing)as described elsewhere[39].Of these genotyped donor sam-ples,46genomic DNA samples(JPN01to JPN46)were selected as a reference set based on the distribution of the HLA allele frequency data in the Japanese population (HLA laboratory:http://www.hla.or.jp/haplo/haplonavi. php?type=aril&lang=en)(Additional file6:Table S5). The reference samples represented more than99.5%of HLA alleles at each HLA locus with99.6%at HLA-A, 99.6%at HLA-B,99.7%at HLA-C,99.8%at HLA-DRB1, 100%at HLA-DQB1,and99.9%at HLA-DPB1.In this regard,the reference set included18HLA-A alleles, 37HLA-
B alleles,18HLA-
C alleles,31HLA-DRB1al-leles,14HLA-DQB1alleles,and18HLA-DPB1alleles (Additional file7:Table S6).In addition,approximately 200genomic DNA samples collected from populations in Africa and Europe were used for an initial study of the optimization of the multiplex PCR methods demonstrat-ing that the method works for various worldwide popula-tions as well as for Japanese.The Japanese HLA genotyping results using the Luminex method are shown in Additional file4:Table https://www.sodocs.net/doc/0813268989.html,rmed consents were obtained from do-nors in accordance with the Declaration of Helsinki,and the study protocol was approved from the institutional re-view board of JMDP and Tokai University.
PCR primer designation and multiplex PCR amplification To develop multiplex PCR systems we used previously designed HLA-A,HLA-B,HLA-C,and HLA-DPB1 locus-specific primer sets that cover the whole gene re-gions from the promoter-enhancer region to3′UTR with the product size of5.5kb in HLA-A,4.6kb in HLA-B,and4.8kb in HLA-C,and from intron1to3′Also,we newly designed an HLA-DRB1/3/4/5DRB-specific primer set and an HLA-DQB1locus-specific pri-mer set(available upon request)that cover polymorphic exons(exons2and3)from intron1to exon4with the product size of 4.0-5.2kb in HLA-DRB1, 4.1kb in HLA-DRB3,4.5kb in HLA-DRB4,4.1kb in HLA-DRB5, and3.9-4.3kb in HLA-DQB1based on the genomic se-quences released from GenBank/EMBL/DDBJ DNA data-bases(accession numbers NG_002392,NG_002433,and NG_002432)and conserved regions of1000genome se-quences(https://www.sodocs.net/doc/0813268989.html,/)(Figure5).Multi-plex PCR methods were constructed using the primer sets by carefully optimizing primer composition and PCR con-ditions and by comparing to sequence read data from NGS (data not shown).
For PCR amplification of the multiplex PCR methods, the20μL PCR amplification-reaction-volume contained 1–25ng of genomic DNA,1unit of PrimeSTAR GXL DNA polymerase(TaKaRa Bio,Shiga,Japan),4.0μL of5×PrimeSTAR GXL Buffer(5mM Mg2+),1.6μL of2.5mM of each dNTP and3.2-5.1μL(10pmol/μL)of each primer mixture.The cycling parameters were as follows:pri-mary denaturation94°C/2min.,followed by30cycles for 98°C/10sec.and70°C/4min.The PCR reactions were performed using the thermal cycler GeneAmp PCR sys-tem9700(Applied Biosystems/Life Technologies/Thermo Fisher Scientific,Foster City,CA).The DNA size was mea-sured by using a1kb DNA ladder marker(New England BioLabs,Ipswich,MA).The PCR products were puri-fied by the Agencourt AMPure XP(Beckman Coulter, Fullerton,CA)and quantified by the PicoGreen assay (Invitrogen/Life Technologies/Thermo Fisher Scientific, Carlsbad,CA)with a Fluoroskan Ascent micro-plate fluorometer(Thermo Fisher Scientific,Waltham,MA). NGS using Ion Torrent PGM system
Barcoded-library DNA samples were prepared with an Ion Xpress Plus Fragment Library Kit and Ion Xpress bar-code Adaptors1–96Kit according to the manufacturer’s protocol for400base-read sequencing(Life Technologies/ Thermo Fisher Scientific,Palo Alto,CA).One hundred nanograms of the multiplex PCR products were used for the preparation of each DNA library.DNA samples were fragmented with a M220Focused-ultrasonicator(Covaris, Woburn,MA).Each DNA library was amplified by eight cycles of PCR.The DNA size and quantitation for each library was measured by an Agilent2100Expert Bioanalyzer using the Agilent High Sensitivity DNA Kit(Agilent Technologies,Santa Clara,CA).Each barcoded-library was mixed at equimolar concentrations then diluted ac-cording to the manufacturer’s recommendation.Emulsion PCR(emPCR)was performed using the resulting pooled library with the Ion PGM Template OT2400Kit on an
with the following cycling parameters:primary denatur-ation95°C/10min.,followed by20cycles for95°C/30sec., 66°C/4min.,20cycles for95°C/30sec.,66°C/6min.and 10cycles for95°C/30sec.,and66°C/20min.After the emulsion was automatically broken with the OneTouch2 instrument,the beads carrying the single-stranded DNA templates were enriched according to the manufacturer’s recommendation.Sequencing was performed using the Ion PGM Sequencing400Kit and Ion316and318Chip Kit v2with a flow number of850for400base-read[40]. Data processing and allele assignment
The raw data processing and base-calling,trimming and output of quality-filter sequence reads that were binned on the basis of the Ion Xpress Barcodes into46separate sequence fastq files,were all performed by the Torrent Suite4.2.1software(Life Technologies)with full pro-cessing for shotgun analysis.These files were further quality trimmed to remove poor sequence at the end of the reads with QVs of less than10.The trimmed and barcode-binned sequence reads were used for HLA allele assignment to the field3and4levels by Sequence Alignment Based Assigning Software(SeaBass)(an in-house development of Tokai University,in preparation). HLA allele candidates and/or reference sequences used for mapping of the sequence reads were selected by nucleotide similarity searches with HLA allele sequences in the IMGT-HLA database using the BLAT program (https://www.sodocs.net/doc/0813268989.html,/),and thereafter,mapping of the sequence reads and the selected reference sequences were performed automatically with the GS Reference Mapper Ver.3.0software(Life Technologies).The map-ping parameter was set to a perfectly matched condition between the read sequences and the reference sequences to avoid mismapping among the HLA loci and contamin-ation of in vitro generated PCR crossover products[41].If a reference sequence covering the PCR region was not available,we constructed a new virtual sequence by de novo assembly using the Sequencher Ver.5.0.1DNA se-quence assembly software(Gene Code,Ann Arbor,MI), and used it as a reference sequence.
Calculation of uniformity among HLA loci and between HLA alleles
After assignment of the HLA alleles we calculated uni-formity among the HLA loci and between alleles using the sequence reads that separated to each allele.The read depth is the number of individual sequence reads that align to a particular nucleotide position[25].An aver-age depth of exons2and3in class I loci,HLA-A,HLA-B, and HLA-C,and exon2in class II loci,HLA-DRB1, HLA-DQB1,and HLA-DPB1,was calculated as an aver-age redundancy per nucleotide site(the sum of depth on Average depth ratio was calculated as an average depth of one allele/large average depth of the other allele.Depth per locus was calculated by the sum of average depth of both alleles.
Additional files
Abbreviations
BLAT:BLAST-like alignment tool;FACS:Fluorescence activated cell sorting; GVHD:Graft-versus-host disease;HLA:Human leukocyte antigen;
Indel:Insertion and deletion;MHC:Major histocompatibility complex; NGS:Next generation sequencing;PCR:Polymerase chain reaction;
QV:Quality value;RFLP:Restriction fragment polymorphisms;SBT:Sequence based typing;SD:Standard deviation;SeaBass:Sequence alignment based assigning software;SMRT:Single molecule real-time;SSCP:Single strand conformation polymorphism;SSOP:Sequence specific oligonucleotide primers;SSOs:Sequence specific oligonucleotides;SSPs:Sequence specific primers;SS-SBT:Super high resolution-single molecule-sequence-based typing.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
S.Mitsunaga,KS,HI and TS participated in the design of this study;Y.Ozaki, SS,AS,Y.Okudaira,SI and AM carried out most of the experiments and analyzed the data;KK,FA and TY were involved in HLA genotyping by conventional methods;S.Morishima,MS,MO and YM supported the study; Y.Ozaki,SS,JKK,HI and TS analyzed the data and wrote the manuscript.All authors read and approved the final manuscript.
Acknowledgements
This work was supported by grants from the Japanese Ministries of Health,
Author details
1Department of Molecular Life Science,Division of Basic Medical Science and Molecular Medicine,Tokai University School of Medicine,Isehara,Kanagawa 259-1143,Japan.2HLA Laboratory,Japanese Red Cross Kanto-Koshinetsu Block Blood Center,Koto-ku,Tokyo135-8639,Japan.3Department of Hematology,Fujita Health University School of Medicine,Toyoake,Aichi
470-1192,Japan.4Department of Legal Medicine,Shinshu University School of Medicine,Matsumoto,Nagano390-8621,Japan.5Division of Epidemiology and Prevention,Aichi Cancer Center Research Institute,Nagoya,Aichi
464-8681,Japan.6Centre for Forensic Science,The University of Western Australia,Nedlands,WA6008,Australia.7Research Department,One Lambda Inc,Part of Thermo Fisher Scientific,Kittridge Street,Canoga Park,CA 91303-2801,USA.
Received:26December2014Accepted:8April
2015
References
1.Zinkernagel RM,Doherty PC.The discovery of MHC restriction.Immunol
Today.1997;18(1):14–7.
2.Sasazuki T,Juji T,Morishima Y,Kinukawa N,Kashiwabara H,Inoko H,et al.
Effect of matching of class I HLA alleles on clinical outcome after
transplantation of hematopoietic stem cells from an unrelated donor.Japan Marrow Donor Program.N Engl J Med.1998;339(17):1177–85.
3.Cotsapas C,Voight BF,Rossin E,Lage K,Neale BM,Wallace C,et al.
Pervasive sharing of genetic effects in autoimmune disease.PloS Genet.
2011;7(8):e1002254.
4.International MHC,Autoimmunity Genetics N,Rioux JD,Goyette P,Vyse TJ,
Hammarstrom L,et al.Mapping of multiple susceptibility variants within the MHC region for7immune-mediated diseases.Proc Natl Acad Sci U S A.
2009;106(44):18680–5.
5.International Multiple Sclerosis Genetics C,Wellcome Trust Case Control C,
Sawcer S,Hellenthal G,Pirinen M,Spencer CC,et al.Genetic risk and a
primary role for cell-mediated immune mechanisms in multiple sclerosis.
Nature.2011;476(7359):214–9.
6.Raychaudhuri S,Sandor C,Stahl EA,Freudenberg J,Lee HS,Jia X,et al.Five
amino acids in three HLA proteins explain most of the association
between MHC and seropositive rheumatoid arthritis.Nat Genet.
2012;44(3):291–6.
7.International HIVCS,Pereyra F,Jia X,McLaren PJ,Telenti A,de Bakker PI,
et al.The major genetic determinants of HIV-1control affect HLA class I
peptide presentation.Science.2010;330(6010):1551–7.
8.McCormack M,Alfirevic A,Bourgeois S,Farrell JJ,Kasperaviciute D,
Carrington M,et al.HLA-A*3101and carbamazepine-induced hypersensitivity reactions in Europeans.N Engl J Med.2011;364(12):1134–43.
9.Illing PT,Vivian JP,Dudek NL,Kostenko L,Chen Z,Bharadwaj M,et al.
Immune self-reactivity triggered by drug-modified HLA-peptide repertoire.
Nature.2012;486(7404):554–8.
10.Ota M,Fukushima H,Kulski JK,Inoko H.Single nucleotide polymorphism
detection by polymerase chain reaction-restriction fragment length
polymorphism.Nat Protoc.2007;2(11):2857–64.
11.Arguello JR,Madrigal JA.HLA typing by Reference Strand Mediated
Conformation Analysis(RSCA).Rev Immunogenet.1999;1(2):209–19.
12.Saiki RK,Walsh PS,Levenson CH,Erlich HA.Genetic analysis of amplified
DNA with immobilized sequence-specific oligonucleotide probes.Proc Natl Acad Sci U S A.1989;86(16):6230–4.
13.Olerup O,Zetterquist H.HLA-DR typing by PCR amplification with
sequence-specific primers(PCR-SSP)in2hours:an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation.Tissue Antigens.1992;39(5):225–35.
14.Santamaria P,Lindstrom AL,Boyce-Jacino MT,Myster SH,Barbosa JJ,Faras AJ,
et al.HLA class I sequence-based typing.Hum Immunol.1993;37(1):39–50. 15.Sheldon S,Poulton K.HLA typing and its influence on organ
transplantation.Methods Mol Biol.2006;333:157–74.
16.Mahdi BM.A glow of HLA typing in organ transplantation.Clin Transl Med.
2013;2(1):6.
17.Erlich HA,Opelz G,Hansen J.HLA DNA typing and transplantation.
Immunity.2001;14(4):347–56.19.Shiina T,Inoko H,Kulski JK.An update of the HLA genomic region,
locus information and disease associations:2004.Tissue Antigens.
2004;64(6):631–49.
20.Shiina T,Hosomichi K,Inoko H,Kulski JK.The HLA genomic loci map:
expression,interaction,diversity and disease.J Hum Genet.
2009;54(1):15–39.
21.Fernandez Vina MA,Hollenbach JA,Lyke KE,Sztein MB,Maiers M,Klitz W,
et al.Tracking human migrations by the analysis of the distribution of HLA alleles,lineages and haplotypes in closed and open populations.Philos
Trans R Soc Lond B Biol Sci.2012;367(1590):820–9.
22.Gourraud PA,Khankhanian P,Cereb N,Yang SY,Feolo M,Maiers M,et al.
HLA diversity in the1000genomes dataset.PLoS One.2014;9(7):e97282. 23.Nakaoka H,Mitsunaga S,Hosomichi K,Shyh-Yuh L,Sawamoto T,Fujiwara T,
et al.Detection of ancestry informative HLA alleles confirms the admixed
origins of Japanese population.PLoS One.2013;8(4):e60793.
24.Grubic Z,Stingl K,Martinez N,Palfi B,Brkljacic-Kerhin V,Kastelan A.STR and
HLA analysis in paternity testing.Int Congr Ser.2004;1261:535–7.
25.Itoh Y,Mizuki N,Shimada T,Azuma F,Itakura M,Kashiwase K,et al.
High-throughput DNA typing of HLA-A,?B,?C,and-DRB1loci by a
PCR-SSOP-Luminex method in the Japanese population.Immunogenetics.
2005;57(10):717–29.
26.Itoh Y,Inoko H,Kulski JK,Sasaki S,Meguro A,Takiyama N,et al.Four-digit
allele genotyping of the HLA-A and HLA-B genes in Japanese patients with Behcet’s disease by a PCR-SSOP-Luminex method.Tissue Antigens.
2006;67(5):390–4.
27.Hutchison3rd CA.DNA sequencing:bench to bedside and beyond.Nucleic
Acids Res.2007;35(18):6227–37.
28.Adams SD,Barracchini KC,Chen D,Robbins F,Wang L,Larsen P,et al.
Ambiguous allele combinations in HLA Class I and Class II sequence-based typing:when precise nucleotide sequencing leads to imprecise allele
identification.J Transl Med.2004;2(1):30.
29.EH R.Collection and analysis of SBT results data.In:JA H,editor.13th IHWS
Technology Joint Report,Immunobiology of the Human MHC.Seattle:
IHWG Press;2006.p.413–6.
30.Lind C,Ferriola D,Mackiewicz K,Heron S,Rogers M,Slavich L,et al.
Next-generation sequencing:the solution for high-resolution,unambiguous
human leukocyte antigen typing.Hum Immunol.2010;71(10):1033–42.
31.Shiina T,Suzuki S,Ozaki Y,Taira H,Kikkawa E,Shigenari A,et al.Super high
resolution for single molecule-sequence-based typing of classical HLA loci at the8-digit level using next generation sequencers.Tissue Antigens.
2012;80(4):305–16.
32.Ozaki Y,Suzuki S,Shigenari A,Okudaira Y,Kikkawa E,Oka A,et al.
HLA-DRB1,?DRB3,?DRB4and-DRB5genotyping at a super-high
resolution level by long range PCR and high-throughput sequencing.
Tissue Antigens.2014;83(1):10–6.
33.Kulski JK,Suzuki S,Ozaki Y,Mitsunaga S,Inoko H,Shiina T.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.In:Xi Y,editor.HLA and Associated Important Diseases.
Croatia:Intech;2014.p.141–81.
34.Wang C,Krishnakumar S,Wilhelmy J,Babrzadeh F,Stepanyan L,Su LF,et al.
High-throughput,high-fidelity HLA genotyping with deep sequencing.Proc Natl Acad Sci U S A.2012;109(22):8676–81.
35.Hosomichi K,Jinam TA,Mitsunaga S,Nakaoka H,Inoue I.Phase-defined
complete sequencing of the HLA genes by next-generation sequencing.
BMC Genomics.2013;14:355.
36.Ehrenberg PK,Geretz A,Baldwin KM,Apps R,Polonis VR,Robb ML,et al.
High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging.BMC Genomics.2014;15:864.
37.Andersson G,Larhammar D,Widmark E,Servenius B,Peterson PA,Rask L.
Class II genes of the human major histocompatibility https://www.sodocs.net/doc/0813268989.html,anization and evolutionary relationship of the DR beta genes.J Biol Chem.
1987;262(18):8748–58.
38.Pleasance ED,Cheetham RK,Stephens PJ,McBride DJ,Humphray SJ,
Greenman CD,et al.A comprehensive catalogue of somatic mutations from
a human cancer genome.Nature.2010;463(7278):191–6.
39.Morishima Y,Kashiwase K,Matsuo K,Azuma F,Morishima S,Onizuka M,
et al.Biological significance of HLA locus matching in unrelated donor
bone marrow transplantation.Blood.2015;125(7):1189–97.
Neisseria meningitidis for rapid determination of multiple layers of typing information.J Clin Microbiol.2012;50(6):1889–94.
41.Holcomb CL,Rastrou M,Williams TC,Goodridge D,Lazaro AM,Tilanus M,
et al.Next-generation sequencing can reveal in vitro-generated PCR crossover products:some artifactual sequences correspond to HLA
alleles in the IMGT/HLA database.Tissue Antigens.2014;83(1):32–40.