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When silent mutations speak up: Fine granularity in linkage disequilibrium
Abstracts / Human Immunology 76 (2015) 38–167
P078
UNAMBIGUOUS HLA GENOTYPING USING LONG-RANGE PCR AND NEXT-GENERATION SEQUENCING (NGS) ON BUCCAL-EXTRACTED DNA: RESULTS FROM 4000 DONOR RECRUITMENT SAMPLES. James H. Lan a,b, Yuxin Yin a, David Nguyen a, Yung-Tsi Bolon c, Brianna Springer c, Katsuyuki Saito d, David Berman d, Tim Hague e, Gyorgy Horvath e, Krisztina Rigo e, Elaine F. Reed a, Qiuheng Zhang a. a UCLA Immunogenetics Center, Los Angeles, CA, United States; b University of British Columbia, Vancouver, BC, Canada; c National Marrow Donor Program, Minneapolis, MN, United States; d One Lambda, Thermo Fisher Scientific, Canoga Park, CA, United States; e Omixon LTD, Budapest, Hungary. Aim: Here we report the feasibility and results of using long-range PCR and clonal sequencing by NGS to deliver unambiguous HLA typing on over 4000 buccal-based donor recruitment samples. Methods: Multiplex long-range PCR primers co-amplified HLA-A, -B, -C from promoter to 3’-UTR. DRB1 and DQB1 were amplified in separate reactions to cover exons 2–3. Library construction was performed using Illumina TruSeq Nano, followed by 2 250 bp paired-end sequencing on MiSeq. HLA typing was assigned using a combination of two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in HML 0.97 format for data consumption. Results: Initial NGS validation was accomplished on 72 DNA Exchange samples isolated from blood and 154 donor recruitment buccal-derived DNA. Overall, NGS-HLA typing was 99.6% concordant with reference results. Primer- and software-related allele mis-assignments accounted for the small number of discrepancies observed, which were later optimized to further strengthen typing accuracy. Next, we introduced robotics to streamline the complex library construction process, achieving an impressive typing throughput of 384 samples (1920 loci) per week. Using this workflow, over 4000 samples to date have been typed under high-resolution in a bone marrow donor recruitment pilot study. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, our assay was successful in typing 97.1% of sample loci. Overall, NGS typing accuracy was 99.02% based on blind QC of 256 loci. N = 59 rare alleles were identified during this period and were all confirmed to be accurate by an alternative method. Finally, long-range sequencing delivered unambiguous 6–8 digit typing resolution in HLA-A, -B, -C, and allele-level G group results in -DRB1 and -DQB1 in all samples reported. Conclusion: Long-range, unambiguous HLA genotyping is achievable on clinical buccal-extracted DNA. Importantly, full-length gene sequencing and the ability to curate raw sequence data in a standardized HML format could permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.
P079
WHEN SILENT MUTATIONS SPEAK UP: FINE GRANULARITY IN LINKAGE DISEQUILIBRIUM. Pedro Cano, Art Hoffman. Blood Systems Inc., Scottsdale, AZ, United States. Aim: Silent mutations and mutations in non-coding regions are traditionally considered clinically irrelevant and ignored in clinical testing. Are we missing any valuable information in the data we discard? Methods: Sequences obtained by standard SBT methods are reanalysed with BLAST and ad hoc sequence databases to study sequence segments typically overlooked. Results: Examples of some observations made: C⁄03:03:01 goes with B⁄15:01:01:01, B⁄55:01:01 and ⁄ B 07:02:01, but C⁄03:03:04 goes with B⁄44:03:01. C⁄03:04:01:01 goes preferentially with B⁄40:01:02 and less frequently with B⁄15:01:01:01, while C⁄03:04:02 goes commonly with B⁄15:10:01 and with B⁄53:01:01, which is also seen with the first allele. C⁄05:01:01:02 goes with B⁄44:02:01:01 and C⁄05:01:01:01 goes with B⁄18:01:01:01. C⁄06:02:01:01 goes with, B⁄13:02:01, B⁄57:01:01, B⁄37:01:01 and B⁄47:01:01:01; C⁄06:02:01:02 goes with B⁄50:01:01 and B⁄50:02; and C⁄06:02:01:03 goes with B⁄45:01. C⁄07:02:01:03 goes with B⁄07:02 and C⁄07:02:01:01 goes with a variety of B alleles including B27:05, B⁄39:01 and B⁄38:02. C⁄15:05:02 goes with both B⁄07:05 and B⁄07:06, two alleles that are themselves difficult to tell apart; and C⁄15:05:01 goes with B⁄73:01 [Full results to be presented at ASHI 2015.] Conclusions: Most of the time allele variants with silent mutations for a common allele are very rare or even poorly documented, but from time to time we see a common allele present in 2 silent-mutation forms. (Here we include intronic mutations with silent mutations.) Typically these variants are ignored and alleles are reported only with the first 2 fields or first 4 digits because only the protein product, and not the DNA sequence, is relevant in a clinical setting. When we look at linkage disequilibrium between HLA loci we find however that these silent-mutation variants of common alleles sometimes have strikingly different associations with alleles at other loci. Genotype analysis and linkage disequilibrium analysis should be based on information provided by the full DNA sequence, including silent mutations and intronic mutations, and not just on the coding sequence that defines a protein with a unique sequence of amino acids. Next generation sequencing will optimize this process.
91
P078
UNAMBIGUOUS HLA GENOTYPING USING LONG-RANGE PCR AND NEXT-GENERATION SEQUENCING (NGS) ON BUCCAL-EXTRACTED DNA: RESULTS FROM 4000 DONOR RECRUITMENT SAMPLES. James H. Lan a,b, Yuxin Yin a, David Nguyen a, Yung-Tsi Bolon c, Brianna Springer c, Katsuyuki Saito d, David Berman d, Tim Hague e, Gyorgy Horvath e, Krisztina Rigo e, Elaine F. Reed a, Qiuheng Zhang a. a UCLA Immunogenetics Center, Los Angeles, CA, United States; b University of British Columbia, Vancouver, BC, Canada; c National Marrow Donor Program, Minneapolis, MN, United States; d One Lambda, Thermo Fisher Scientific, Canoga Park, CA, United States; e Omixon LTD, Budapest, Hungary. Aim: Here we report the feasibility and results of using long-range PCR and clonal sequencing by NGS to deliver unambiguous HLA typing on over 4000 buccal-based donor recruitment samples. Methods: Multiplex long-range PCR primers co-amplified HLA-A, -B, -C from promoter to 3’-UTR. DRB1 and DQB1 were amplified in separate reactions to cover exons 2–3. Library construction was performed using Illumina TruSeq Nano, followed by 2 250 bp paired-end sequencing on MiSeq. HLA typing was assigned using a combination of two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in HML 0.97 format for data consumption. Results: Initial NGS validation was accomplished on 72 DNA Exchange samples isolated from blood and 154 donor recruitment buccal-derived DNA. Overall, NGS-HLA typing was 99.6% concordant with reference results. Primer- and software-related allele mis-assignments accounted for the small number of discrepancies observed, which were later optimized to further strengthen typing accuracy. Next, we introduced robotics to streamline the complex library construction process, achieving an impressive typing throughput of 384 samples (1920 loci) per week. Using this workflow, over 4000 samples to date have been typed under high-resolution in a bone marrow donor recruitment pilot study. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, our assay was successful in typing 97.1% of sample loci. Overall, NGS typing accuracy was 99.02% based on blind QC of 256 loci. N = 59 rare alleles were identified during this period and were all confirmed to be accurate by an alternative method. Finally, long-range sequencing delivered unambiguous 6–8 digit typing resolution in HLA-A, -B, -C, and allele-level G group results in -DRB1 and -DQB1 in all samples reported. Conclusion: Long-range, unambiguous HLA genotyping is achievable on clinical buccal-extracted DNA. Importantly, full-length gene sequencing and the ability to curate raw sequence data in a standardized HML format could permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.
P079
WHEN SILENT MUTATIONS SPEAK UP: FINE GRANULARITY IN LINKAGE DISEQUILIBRIUM. Pedro Cano, Art Hoffman. Blood Systems Inc., Scottsdale, AZ, United States. Aim: Silent mutations and mutations in non-coding regions are traditionally considered clinically irrelevant and ignored in clinical testing. Are we missing any valuable information in the data we discard? Methods: Sequences obtained by standard SBT methods are reanalysed with BLAST and ad hoc sequence databases to study sequence segments typically overlooked. Results: Examples of some observations made: C⁄03:03:01 goes with B⁄15:01:01:01, B⁄55:01:01 and ⁄ B 07:02:01, but C⁄03:03:04 goes with B⁄44:03:01. C⁄03:04:01:01 goes preferentially with B⁄40:01:02 and less frequently with B⁄15:01:01:01, while C⁄03:04:02 goes commonly with B⁄15:10:01 and with B⁄53:01:01, which is also seen with the first allele. C⁄05:01:01:02 goes with B⁄44:02:01:01 and C⁄05:01:01:01 goes with B⁄18:01:01:01. C⁄06:02:01:01 goes with, B⁄13:02:01, B⁄57:01:01, B⁄37:01:01 and B⁄47:01:01:01; C⁄06:02:01:02 goes with B⁄50:01:01 and B⁄50:02; and C⁄06:02:01:03 goes with B⁄45:01. C⁄07:02:01:03 goes with B⁄07:02 and C⁄07:02:01:01 goes with a variety of B alleles including B27:05, B⁄39:01 and B⁄38:02. C⁄15:05:02 goes with both B⁄07:05 and B⁄07:06, two alleles that are themselves difficult to tell apart; and C⁄15:05:01 goes with B⁄73:01 [Full results to be presented at ASHI 2015.] Conclusions: Most of the time allele variants with silent mutations for a common allele are very rare or even poorly documented, but from time to time we see a common allele present in 2 silent-mutation forms. (Here we include intronic mutations with silent mutations.) Typically these variants are ignored and alleles are reported only with the first 2 fields or first 4 digits because only the protein product, and not the DNA sequence, is relevant in a clinical setting. When we look at linkage disequilibrium between HLA loci we find however that these silent-mutation variants of common alleles sometimes have strikingly different associations with alleles at other loci. Genotype analysis and linkage disequilibrium analysis should be based on information provided by the full DNA sequence, including silent mutations and intronic mutations, and not just on the coding sequence that defines a protein with a unique sequence of amino acids. Next generation sequencing will optimize this process.
91