HLA Analysis and Next Generation Sequencing Henry Erlich, Ph.D. Cherie Holcomb, Ph.D. Roche...
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Transcript of HLA Analysis and Next Generation Sequencing Henry Erlich, Ph.D. Cherie Holcomb, Ph.D. Roche...
![Page 1: HLA Analysis and Next Generation Sequencing Henry Erlich, Ph.D. Cherie Holcomb, Ph.D. Roche Molecular Systems picture placeholder NGS and EFI, May 14,](https://reader037.fdocuments.in/reader037/viewer/2022110211/56649ece5503460f94bdb8cc/html5/thumbnails/1.jpg)
HLA Analysis and Next Generation Sequencing
Henry Erlich, Ph.D.Cherie Holcomb, Ph.D.Roche Molecular Systems
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NGS and EFI, May 14, 2013
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Library preparation strategies for NGS for HLA typing
• Amplify gene (multiple Kb)/cleave and ligate universal 454 adaptors/emPCR/sequence
• Sequence capture (NimbleGen)/cleave and ligate universal 454 adaptors/emPCR/sequence
• Amplify exons (and introns) with MID primers/emPCR/sequence (“fusion” or Fluidigm system primers)
• Sequencing HLA genes vs. HLA typing by sequencing
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Incompletely ExtendedPCR Product
5’ 3’
Schematic of PCR Crossover FormationIncreased probability in long amplicon PCR
Crossover Product
5’
3’ 5’Template 1 PCR cycle
“n”
5’
3’ 5’ Template 2
PCR cycle “n+1”
3
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The Benefits of Clonal Sequencing for HLA Genotyping
• Sets phase for linked polymorphisms within amplicon (reduces ambiguity)
• Allows amplification and sequencing of multiple members of multi-gene family (ie DRB1, DRB3, DRB4, DRB5) with generic (DRB) primers
• Allows for the separation of co-amplified sequences (pseudo-genes, related genes as “noise”) from target sequence (“signal”)
• Allows for the “digital” analysis (counting sequence reads) of mixtures and, with “deep” sequencing, for the detection of rare variants
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Phasing across amplicons
Exon 2 seq 1
Exon 2 seq 2
Exon 3 seq 1
Exon 3 seq 2
2-1, 3-1
2-1, 3-2
2-2, 3-2
2-1, 3-2
Note: Assumes all HLA class I alleles in IMGT/HLA database have exon 2 and exon 3 sequences. If one of the 4 combinations is absent from the sequence database, an unambiguous inference of phase can be made. Physical direct phasing can be accomplished with longer amplicons or “bridging “amplicons.
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NGS Applications for HLA Sequencing
• High Resolution: many amplicons/many exons per gene
• High Throughput: many samples per run
• Deep Sequencing: many reads per sample for detection of rare variants; analysis of mixtures
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Neurosis is the inability to tolerate ambiguity
-Sigmund Freud