Dual redundant sequencing strategy: Full‐length gene characterisation of 1056 novel and confirmatory HLA alleles

Abstract

The high‐throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5′‐ to 3′‐UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory – European Nucleotide Archive (EMBL‐ENA) and the IPD‐IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real‐time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full‐length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi‐automated web service TypeLoader, to aid in the submission of sequences to the EMBL‐ENA and the IPD‐IMGT/HLA Database. In the IPD‐IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full‐length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database.

Single molecule real‐time DNA sequencing of HLA genes at ultra‐high resolution from 126 International HLA and Immunogenetics Workshop cell lines

“…This coupled with the limitations of Sanger, 454 pyrosequencing and Illumina sequencing by synthesis to correctly call all bases in long repeat regions has made them particularly notorious. Recent combination uses of SMRT and Illumina sequencing perform better at sequencing through these regions than either platform alone . We did observe some discrepancies to allele reference sequences in homopolymer regions.…”

Identification of the novel HLA‐B*57:91 allele, by next‐generation sequencing in a Spanish individual

“…In addition, most phase ambiguities can be solved with this technology, making possible to determine the allele where the new nucleotide change belongs. In this regard, NGS technology has allowed the characterization of a large number of new alleles in the last years . In this article we report the identification of a novel allele, the HLA‐B*57:91 , which has been found during routine HLA typing with NGS technology of cord blood donors for hematopoietic stem cell transplantation.…”

Immunogenetics in stem cell donor registry work: The DKMS example (Part 2)

“…Since then, we have only submitted whole‐gene sequences and also plan to do so in the future. To avoid submitting erroneous sequences, we developed a dual redundant reference sequencing strategy (DR2S) that combines Illumina shotgun with Pacific Biosciences single molecule real‐time (SMRT) sequencing data (Albrecht et al, ; Klasberg et al, ). Both sequencing methods have specific weaknesses: possible loss of phasing when two heterozygous positions are separated by more than 500 base pairs for Illumina shotgun sequencing, high error rates especially in homopolymeric and repetitive sequence regions for SMRT sequencing.…”