Journal: Genome Biology

Article Title: Transformation of alignment files improves performance of variant callers for long-read RNA sequencing data

doi: 10.1186/s13059-023-02923-y

Figure Lengend Snippet: Alignment file transformation for optimized calling of genetic variants from lrRNA-seq data and variant calling performance across the best pipelines on PacBio Iso-Seq reference datasets. A Alignment file (BAM) transformations to make spliced lrRNA-seq alignments suitable for variant calling. First, GATK’s SNCR function is used to split the reads at Ns in their cigar string, such that exons become distinct reads. Second, the flagCorrection function attributes the flag of the original read to all corresponding fragment reads. B The number of genetic variants kept in the ground-truth (Illumina DNA-seq) variant call format (VCF) files (for Jurkat and WTC-11 datasets) after filtering; y -axis refers to variant sites that are successively retained, as follows: All variants , all sites in the VCF files; Low-density regions , sites residing in regions such that there is a maximum of 3 variants in a 201-bp window; Exonic regions , sites where the Iso-Seq coverage is at least 1; High read coverage , sites where the short-read coverage is at least 20 and 72 for Jurkat and WTC-11, respectively; see the “ ” section for more details. C Schematic with proportion of intron-containing reads (N-cigar reads) at four variant sites (red boxes). D Precision-recall curves; point sizes indicate the filtering ranges for read coverage; dashed lines represent F1-scores. “Clair3-mix” denotes using Clair3 to call SNPs and SNCR + flagCorrection + Clair3 to call indels. SNCR-SplitNCigarReads; fC-flagCorrection; DV-DeepVariant. Additional file : Table S1 gives the number of covered true variants in each interval range. E , F UpSet plots show the intersection of variants called by the pipelines with the ground truth for Jurkat ( E ) and WTC-11 ( F ) datasets; sites shown here were filtered according to a minimum Iso-Seq read coverage of 20

Article Snippet: E , F UpSet plots show the intersection of variants called by the pipelines with the ground truth for Jurkat ( E ) and WTC-11 ( F ) datasets; sites shown here were filtered according to a minimum Iso-Seq read coverage of 20 To assess the performance of variant callers, we assembled a set of ground-truth variants for two datasets (Jurkat and WTC-11 cell lines) from Illumina DNA-seq data, retaining only variants from high confidence regions (see the “ ” section) and for which there is sufficient corresponding lrRNA-seq coverage (numbers after filtering are shown in Fig. B; we use the term “coverage” to refer to exonic coverage throughout this paper).

Techniques: Transformation Assay, Variant Assay, DNA Sequencing

Journal: Genome Biology

Article Title: Transformation of alignment files improves performance of variant callers for long-read RNA sequencing data

doi: 10.1186/s13059-023-02923-y

Figure Lengend Snippet: Variant calling performance according to splice junction proximity, homopolymers, or allele-specific expression. n indicates the number of true variants covered by Iso-Seq data for each calculated performance measure. SNCR-SplitNCigarReads; fC-flagCorrection; DV-DeepVariant. In Clair3-based pipelines, only the pileup model was used. Performance measures for SNP ( A ) and indel ( B ) calling of sites far from ( No ) and near to ( Yes ) splice junctions for datasets Jurkat and WTC-11. C Performance measures of indel calling of sites in non-homopolymers ( non-hp ) and within homopolymers of a specified length; results only from WTC-11 dataset. D FN and TP rates of heterozygous SNP calling from sites in allele-specific expressed (ASE) genes and non-ASE genes; results only from WTC-11 dataset; only sites with a minimum RNA short-read coverage of 40 and minimum Iso-Seq read coverage of 20 were considered

Article Snippet: E , F UpSet plots show the intersection of variants called by the pipelines with the ground truth for Jurkat ( E ) and WTC-11 ( F ) datasets; sites shown here were filtered according to a minimum Iso-Seq read coverage of 20 To assess the performance of variant callers, we assembled a set of ground-truth variants for two datasets (Jurkat and WTC-11 cell lines) from Illumina DNA-seq data, retaining only variants from high confidence regions (see the “ ” section) and for which there is sufficient corresponding lrRNA-seq coverage (numbers after filtering are shown in Fig. B; we use the term “coverage” to refer to exonic coverage throughout this paper).

Techniques: Variant Assay, Expressing