Computational protocol: Hidden genomic MHC disparity between HLA matched sibling pairs in hematopoietic stem cell transplantation

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Protocol publication

[…] Genomic sequencing of the full MHC region in samples of cohort 2 was performed at the McGill Genome Centre, McGill University, Montreal, Canada, using Roche SeqCap EZ Human MHC Design capture, which captures approximately 95% of the MHC region. Sequencing was performed with an Illumina HiSeq. 2000, yielding 100 bp paired-end reads and a median on-target coverage of 27.5 × per sample. The generated reads were aligned to the GRCH37/hg19 reference genome. Base quality score recalibration and SNP and INDEL discovery were performed using GATK v.3.6-0 VariantRecalibrator and ApplyRecalibration tools with the default settings. The used ts_filter_level setting was 99.0–. The data were filtered using the hard cutoffs of the total depth of coverage per sample >7 and genomic quality >19.For HLA typing, the fastq read data were quality checked using FastQC, and adapters were trimmed using Cutadapt. The Omixon Explore program version 1.2.0 (Omixon, Budapest, Hungary) was used for allele assignment of 30 genes at six-digit resolution level comprising both classical HLA genes, non-classical HLA genes and pseudogenes in the MHC region using the IMGT/HLA database’s HLA nomenclature release 3.25.0: HLA-F, HLA-V, HLA-G, HLA-H, HLA-K, HLA-A, HLA-J, HLA-L, HLA-E, HLA-C, HLA-B, HLA-DRA, HLA-DRB1-9, HLA-DQA1, HLA-DQB1, HLA-DOB, HLA-DMB, HLA-DMA, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DPB2. In addition, the alleles of the two MHC class I chain-related genes MICA and MICB as well as two ATP binding cassette transporter genes, TAP1 and TAP2, were assigned. […]

Pipeline specifications

Software tools GATK, FastQC, cutadapt
Databases IMGT HLA DB
Organisms Homo sapiens
Chemicals Nucleotides