Full length phased sequencing of HLA class I samples reveals deeper insight in sequence variety

摘要: Aim The vast majority of donor typing relies on sequencing exons 2 and 3 HLA class I genes (HLA-A, -B, -C). With such an approach certain allele combinations do not result in the anticipated “high resolution” (G-code) typing, due to lack exon-phasing information. To resolve ambiguous results for a haplotype frequency project, we established whole gene I, facilitating also estimation degree sequence variability outside commonly sequenced exons. Methods Primers were developed flanking UTR regions resulting similar amplicon lengths 4.2–4.4 kb. Using 4-primer approach, secondary primers containing barcodes combined with specific obtain barcoded full-gene amplicons single amplification step. Amplicons pooled, purified, ligated SMRT bells (i.e. annealing points primers) following standard protocols from Pacific Biosciences. Taking advantage chemistry, pools 48–72 full length phased runs Biosciences RSII instrument. Demultiplexing was achieved using portal. Sequence analysis performed NGSengine software (GenDx). Results We successfully full-length 1003 samples, harboring typings either HLA-A (n = 46), HLA-B (n = 304) or HLA-C (n = 653). Despite high per-read raw error rates typical ( ∼ 15%) consensus proved highly reliable. All sequences accordance their MiSeq-derived sequences. Unambiguous allelic resolution all samples. observed novel intronic, exonic as well variations many alleles covered by our data set. This included 600 individuals HLA-C∗07:01/C∗07:02 genotype revealing extent variation 3. Conclusion Here present genes. maturity this demonstrated more than 1000 achieving fully Extensive one common combination hints at yet discover diversity system analyzed