Allelic decomposition and exact genotyping of highly polymorphic and structurally variant genes

Abstract

High-throughput sequencing provides the means to determine the allelic decomposition for any gene of interest—the number of copies and the exact sequence content of each copy of a gene. Although many clinically and functionally important genes are highly polymorphic and have undergone structural alterations, no high-throughput sequencing data analysis tool has yet been designed to effectively solve the full allelic decomposition problem. Here we introduce a combinatorial optimization framework that successfully resolves this challenging problem, including for genes with structural alterations. We provide an associated computational tool Aldy that performs allelic decomposition of highly polymorphic, multi-copy genes through using whole or targeted genome sequencing data. For a large diverse sequencing data set, Aldy identifies multiple rare and novel alleles for several important pharmacogenes, significantly improving upon the accuracy and utility of current genotyping assays. As more data sets become available, we expect Aldy to become an essential component of genotyping toolkits.

Added Value of Clinical Sequencing: WGS-Based Profiling of Pharmacogenes

“…Three command-line-based bioinformatic tools, Astrolabe (previously Constellation) [15], Aldy [16], and Stargazer [17] were used to call CYP2D6 variants, including SVs. Using downloaded genetic reference data, we compared the CYP2D6 variant calls of these three tools to the GeT-RM 2019 consensus genotypes [21].…”

“…For the SV detection in CYP2D6, we evaluated the software tools Astrolabe (previously Constellation) v0.8.6.1 [15], Aldy v2.2.3 [16], and Stargazer v1.0.7 [17]. To compare the accuracy of these three variant callers, we downloaded FASTQ and/or BAM files of 20 human PGx reference WGS samples from the European Nucleotide Archive (ENA, ebi.ac.uk) (HG00436, NA07029, NA18959, NA19109, NA21781, NA12873, NA18861, HG00589, NA19917, NA07019, NA12717, HG00276, NA18524, NA18540, NA07348, NA18519; NA18966, NA18992 and NA19226) [47] or from ftp-trace.ncbi.nih.gov/1000genomes/ftp/phase3/data/NA12892 (NA12892).…”

“…For resolving ambiguous calls, BAM files were manually evaluated using the Integrative Genomics Viewer v2.4.19 [22]. Moreover, we used Aldy [16] to assess the frequencies of CYP2D6 star alleles in our in-house WGS cohort.…”

Statistical Binning for Barcoded Reads Improves Downstream Analyses

“…In addition, the copy number derived from EMA’s alignments in this problematic region (spanning from exon 7 up to exon 9 in CYP2D6 and CYP2D7 ) was closer to the “expected” copy number by 20% compared to the copy number derived from Lariat’s alignments (we used Aldy [ Numanagić et al, 2018 ] to obtain this data). We further ran Aldy on our high-coverage NA12878 v2 dataset, where it correctly detected the *3/*68+*4 allelic combination on both EMA’s and Lariat’s alignments, and EMA’s overall copy number error over the whole region was around 4% better than Lariat’s.…”

Deciphering highly similar multigene family transcripts from Iso-Seq data with IsoCon

“…Classification 35 or construction 36 of allele-specific transcripts with Iso-Seq have been described, but these approaches require a reference and can only separate two alleles of a single gene. Genotyping approaches for multigene families have also been proposed 37 , 38 , but they require prior knowledge of the isoform sequences. A de novo approach for clustering highly similar isoforms is described in ref.…”