Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion

Zhe Zhang, Hyun Hyung An, Sunitha Vege, Taishan Hu, Shiping Zhang, Timothy Mosbruger, Pushkala Jayaraman, Dimitri Monos, Connie M. Westhoff, Stella T. Chou

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Next-generation sequencing (NGS) technologies have transformed medical genetics. However, short-read lengths pose a limitation on identification of structural variants, sequencing repetitive regions, phasing of distant nucleotide changes, and distinguishing highly homologous genomic regions. Long-read sequencing technologies may offer improvements in the characterization of genes that are currently difficult to assess. We used a combination of targeted DNA capture, long-read sequencing, and a customized bioinformatics pipeline to fully assemble the RH region, which harbors variation relevant to red cell donor-recipient mismatch, particularly among patients with sickle cell disease. RHD and RHCE are a pair of duplicated genes located within an ∼175 kb region on human chromosome 1 that have high sequence similarity and frequent structural variations. To achieve the assembly, we utilized palindrome repeats in PacBio SMRT reads to obtain consensus sequences of 2.1 to 2.9 kb average length with over 99% accuracy. We used these long consensus sequences to identify 771 assembly markers and to phase the RHD-RHCE region with high confidence. The dataset enabled direct linkage between coding and intronic variants, phasing of distant SNPs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints. A limiting factor in phasing is the frequency of heterozygous assembly markers and therefore was most successful in samples from African Black individuals with increased heterogeneity at the RH locus. Overall, this approach allows RH genotyping and de novo assembly in an unbiased and comprehensive manner that is necessary to expand application of NGS technology to high-resolution RH typing.

Original languageEnglish (US)
Pages (from-to)180-191
Number of pages12
JournalAmerican Journal of Human Genetics
Volume109
Issue number1
DOIs
StatePublished - Jan 6 2022
Externally publishedYes

Keywords

  • RH genes
  • de novo assembly
  • long-read sequencing
  • sickle cell disease
  • targeted capture
  • transfusion

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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