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NGS and EFI, May 14, 2013. HLA Analysis and Next Generation Sequencing. Henry Erlich, Ph.D. Cherie Holcomb, Ph.D. Roche Molecular Systems. picture placeholder. Library preparation strategies for NGS for HLA typing. - PowerPoint PPT Presentation
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HLA Analysis and Next Generation Sequencing
Henry Erlich, Ph.D.Cherie Holcomb, Ph.D.Roche Molecular Systems
picture placeholder
NGS and EFI, May 14, 2013
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
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
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
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.
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
Neurosis is the inability to tolerate ambiguity
-Sigmund Freud