GRIDSS2: comprehensive characterisation of somatic structural variation using single breakend variants and structural variant phasing

Abstract

<jats:p>Here we present GRIDSS2, a general purpose structural variant caller optimised for tumour/normal somatic calling. Using cell line, patient sample validation and cohort-level comparisons, we show GRIDSS2 outperforms recent state-of-the-art tools. We demonstrate GRIDSS2 retains high sensitivity and precision even for small events by identifying a small (32-100bp) duplication signature strongly associated with colorectal cancer using 3,782 metastatic cancers that have been deeply sequenced by the Hartwig Medical Foundation. Essential to the high precision achieved by GRIDSS2 is the novel reporting of single breakend variants: structural variants in which only one side can be unambiguously determined. We show that the inclusion of single breakends reduces the false negative rate from 10.4% to 3.4%. Demonstrating the power single breakend calling has in genomic regions traditionally considered inaccessible to short read callers, we find that 47% of somatic centromeric breaks are repaired to non-centromeric sequence, with chromosome 1 exhibiting a unique centromeric rearrangement signature. Finally, we show that somatic structural variants are highly clustered with GRIDSS2 able to phase 16% of somatic structural variants in the Hartwig cohort from short read sequencing alone.</jats:p>

A multi-platform reference for somatic structural variation detection

“…Our results indicate that these missing events typically involve centromeric regions that are not directly accessible by any current sequencing technology. Annotation data provided by the GRIDSS2 SV caller (Cameron et al, 2020) .…”

“…Reads were mapped to GRCh37 with BWA mem (version 0.7.5, (Li, 2013) ), followed by indel realignment with GATK (v3.4-46, (DePristo et al, 2011) ). SVs were called jointly for COLO829 and COLO829BL with GRIDSS (v2.0.1, (Cameron et al, 2020) ). Somatics SVs were filtered with the GRIDSS somatic SV filtering script (https://github.com/PapenfussLab/gridss/blob/master/scripts/gridss_somatic_filter.R).…”

An international virtual hackathon to build tools for the analysis of structural variants within species ranging from coronaviruses to vertebrates

“…Along with the manually-curated reference set, the panel of normal (PON) used for further filtering was generated from a compiled set of high-quality germline calls using 3,782 normal samples freshly-sequenced at a median depth of 38x by the Hartwig Medical Foundation. 49,50 Sniphles: The main idea is to phase the identified SVs. We use two approaches; the first is to extract the tagged reads from the bam file and use these reads to phase the SVs if not conflicted.…”

VIRUSBreakend: Viral Integration Recognition Using Single Breakends

“…SNVs called with bcftools and the viral reference modified to incorporate these SNVs. Viral read pairs are realigned to the updated reference and structural variants called using GRIDSS2 17 and filtered to single breakends. Single breakends are breakpoints in which one side cannot be unambiguously aligned to the (viral) reference.…”

“…As input, it uses a SAM/BAM/CRAM file of reads aligned to the host reference genome. Viral reads are classified using Kraken216 , aligned to the most abundant host-infecting virus with bwa26 , realigned to a modified viral reference that incorporates SNVs called by bcftools, single breakends identified with GRIDSS217,27 , aligned to the host to identify putative integration sites, and annotated with RepeatMasker to identify false positive and multi-mapping integration sites.All read sequences 20bp or longer that are not aligned to the host reference genome are classified using a Kraken2 database containing the human, viral and UniVec_Core sequences.For soft clipped reads, only the unaligned bases are classified. For split read alignments, only the bases not aligned to either location are classified.…”