Overcoming the challenges of interpreting complex and uncommon RH alleles from whole genomes

Abstract

Background and objectives Rh is one of the most diverse and complex blood group systems. Recently, next generation sequencing (NGS) has proven to be a viable option for RH genotyping. We have developed automated software (blood-Typer) for determining alleles encoding RBC antigens from NGS-based whole genome sequencing (WGS). The bloodTyper algorithm has not yet been optimized and evaluated for complex and uncommon RH alleles. Materials and methods Twenty-two samples with previous polymerase chain reaction (PCR) and Sanger sequencing-based RH genotyping underwent WGS. bloodTyper was used to detect RH alleles including those defined by structural variation (SV) using a combination of three independent strategies: sequence read depth of coverage, split reads and paired reads. Results bloodTyper was programmed to identify D negative and positive phenotypes as well as the presence of alleles encoding weak D, partial D and variant RHCE. Sequence read depth of coverage calculation accurately determined RHD zygosity and detected the presence of RHD/RHCE hybrids. RHCE*C was determined by sequence read depth of coverage and by split read methods. RHD hybrid alleles and RHCE*C were confirmed by using a paired read approach. Small SVs present in RHCE*CeRN and RHCE*ceHAR were detected by a combined read depth of coverage and paired read approach. Conclusions The combination of several different interpretive approaches allowed for automated software based-RH genotyping of WGS data including RHD zygosity and complex compound RHD and RHCE heterozygotes. The scalable nature of this automated analysis will enable RH genotyping in large genomic sequencing projects.

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

“…35 Long-read sequencing also allowed us to determine more precise translocation breakpoints for structural variants (Figure 5). Unlike short-read NGS technologies that depend on read depth of coverage or copy number variation within translocated regions to determine structural variants and predict breakpoints, 15,18,36,37 long-read sequencing does not rely on copy number variation. As a result, translocation breakpoints can be narrowed down to the individual base as opposed to a range within hundreds of bases as determined by sequence-specific PCR or predicted by various algorithms with short-read NGS assays.…”

“…As a result, translocation breakpoints can be narrowed down to the individual base as opposed to a range within hundreds of bases as determined by sequence-specific PCR or predicted by various algorithms with short-read NGS assays. 18,29,[37][38][39] One such example is the segment of RHD translocated into RHCE responsible for C expression. In 1997, Carritt et al reported a 4.26 kb translocation by using sanger sequencing, 29 similar to the 4.1 kb translocation we identified.…”

Genome Reporting for Healthy Populations—Pipeline for Genomic Screening from the GENCOV COVID‐19 Study

“…have rare blood type results (Halls et al, 2020, Lane et al, 2018. Viral lineage was also determined for each participant and included on the final genome report.…”

“…Two other analyses were implemented in our genome reports according to existing protocols developed by collaborators within the GENCOV study. These include polygenic risk score calculation for six diseases (atrial fibrillation, coronary artery disease, type 2 diabetes, prostate cancer, colorectal cancer, breast cancer; Hao et al., 2022 ) and ABO, Rh, and other blood group genotyping (Halls et al., 2020 , Lane et al., 2018 ).…”

“…The paired read method in which aligned regions are abnormally spaced is useful for observing breakpoints and evaluating read alignments, deletions, and duplications. The split read method detects when a single read matches alignments from disparate, nonsequential locations, indicating breakpoint transition (Halls et al., 2020 , Lane et al., 2018 ).…”