Download Henry Erlich

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
• 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
Schematic of PCR Crossover Formation
Increased probability in long amplicon PCR
3’
PCR cycle “n”
5’
PCR cycle “n+1”
3’
5’
Template 1
5’
Incompletely Extended
PCR Product
5’
5’
Template 2
3’
Crossover Product
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 (pseudogenes, 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 3 seq 1
2-1, 3-1
2-1, 3-2
Exon 2 seq 2
Exon 3 seq 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