We previously reported on the use of the Ion PGM next generation sequencing (NGS) platform to genotype HLA class I and class II genes by a super-high resolution, single-molecule, sequence-based typing (SS-SBT) method (Shiina et al. 2012). However, HLA alleles could not be assigned at the field 4 level at some HLA loci such as DQA1, DPA1 and DPB1 because the SNP and indel densities were too low to identify and separate both of the phases. In this regard, we have now added the single molecule, real-time (SMRT) DNA sequencer PacBio RS II method to our analysis in order to…
HLA-DPB1 mismatching between patients and unrelated donors is known to increase the risk of acute graft-versus-host-disease (GvHD) after hematopoietic stem cell transplantation. If only HLA-DPB1 mismatched donors are available, the genotype defined by the Single Nucleotide Polymorphism (SNP) rs9277534 can be used to select mismatched donors that are well-tolerated. However, since rs9277534 resides within the 3’ untranslated region (UTR), it usually is not analyzed during DPB1 routine typing.
The killer-cell immunoglobulin-like receptor (KIR) gene family are involved in immune modulation during viral infection, autoimmune disease and in allogeneic stem cell transplantation. Most KIR gene diversity studies and their impact on the transplant outcome is performed by gene absence/presence assays. However, it is well known that KIR gene allelic variations have biological significance. Allele level typing of KIR genes has been very challenging until recently due to the homologous nature of those genes and very long intronic sequences. SMRT (Single Molecule Real-Time) Sequencing generates average long reads of 10 to 15 kb and allows us to obtain in-phase long…
Q&A session following the 2014 ASHI PacBio Workshop with speakers Martin Maiers, Steven Marsh and Nezih Cereb
KIR haplotypes can be determined by physical and computational and statistical methods. Martin Maiers from National Bone Marrow Donor Program (NMDP) presents a summary of their work to determine KIR genomic content for use in clinical transplantation, outcomes of HLA sequencing of KIR region across a variety of methods and shares their data from recent experiments using PacBio single-molecule sequencing of fosmid libraries.
Fully phased allele-level sequencing of highly polymorphic HLA genes is greatly facilitated by SMRT Sequencing technology. In the present work, we have evaluated multiple DNA barcoding strategies for multiplexing several loci from multiple individuals, using three different tagging methods. Specifically MHC class I genes HLA-A, -B, and –C were indexed via DNA Barcodes by either tailed primers or barcoded SMRTbell adapters. Eight different 16-bp barcode sequences were used in symmetric & asymmetric pairing. Eight DNA barcoded adapters in symmetric pairing were independently ligated to a pool of HLA-A, -B and –C for eight different individuals, one at a time and…