Deciphering complex genome rearrangements in C. elegans using short-read whole genome sequencing

Abstract

Genomic rearrangements cause congenital disorders, cancer, and complex diseases in human. Yet, they are still understudied in rare diseases because their detection is challenging, despite the advent of whole genome sequencing (WGS) technologies. Short-read (srWGS) and long-read WGS approaches are regularly compared, and the latter is commonly recommended in studies focusing on genomic rearrangements. However, srWGS is currently the most economical, accurate, and widely supported technology. In Caenorhabditis elegans (C. elegans), such variants, induced by various mutagenesis processes, have been used for decades to balance large genomic regions by preventing chromosomal crossover events and allowing the maintenance of lethal mutations. Interestingly, those chromosomal rearrangements have rarely been characterized on a molecular level. To evaluate the ability of srWGS to detect various types of complex genomic rearrangements, we sequenced three balancer strains using short-read Illumina technology. As we experimentally validated the breakpoints uncovered by srWGS, we showed that, by combining several types of analyses, srWGS enables the detection of a reciprocal translocation (eT1), a free duplication (sDp3), a large deletion (sC4), and chromoanagenesis events. Thus, applying srWGS to decipher real complex genomic rearrangements in model organisms may help designing efficient bioinformatics pipelines with systematic detection of complex rearrangements in human genomes.

Case Report: Biallelic Loss of Function ATM due to Pathogenic Synonymous and Novel Deep Intronic Variant c.1803-270T > G Identified by Genome Sequencing in a Child With Ataxia–Telangiectasia

“…To be certain that the two variants in the ATM , the maternal synonymous and the paternal deep intronic, remained the best potential genetic explanation for this proband, we expanded our GS analyses beyond the ATM . Our trio-based genome sequencing analyses, where we consider single nucleotide variants (SNVs), structural variants (SVs) ( Maroilley et al, 2021 ), repeat expansions ( Dolzhenko et al, 2020 ), mitochondrial DNA ( Calabrese et al, 2014 ) according to various modes of inheritance, revealed no additional findings of significance beyond the ATM variants.…”

“…First, we scrutinized the GS data of the ATM locus. We looked for rare or novel complex structural variants ( Maroilley et al, 2021 ), mobile element insertions ( Tarailo-Graovac et al, 2017a ), 5′ and 3′ UTR ( Whiffin et al, 2020 ; Wright et al, 2021 ; Zhang et al, 2021 ), deep exonic ( Casadei et al, 2019 ), and deep intronic variants either inherited from father or absent from both parents. These analyses led to the identification of a paternal deep intronic variant NG_009830.1(NM_000051.3): c.1803-270T > G. The variant is absent from all the population databases tested, including our in-house database, gnomAD (v2 and v3) and TopMed database freeze 8.…”

Different structural variant prediction tools yield considerably different results in Caenorhabditis elegans

“…BC4586 is a C. elegans strain containing experimentally validated structural variants (Maroilley et al 2021). Publicly available Illumina sequencing data allowed us to determine if the short-read SV callers in the SVE/FusorSV pipeline can resolve a 3910bp deletion (DEL-1; IV:9,853,675–9,857,585), a 552bp tandem duplication (DUP-1; IV:9,853,123– 9,853,675), and a 4812bp inversion (INV-1; IV:9,857,585–9,862,397).…”

Whole genome sequencing facilitates intragenic variant interpretation following modifier screening in C. elegans

“…Genomic DNA from suppressing strains was extracted using the Qiagen Blood and Tissue kit (Cat #: 13323) following standard procedure [ 22 ]. DNA was suspended in 10 mM Tris-HCl (pH 8.0), only samples that had a minimum A260/280 ratio of 1.8 were submitted for sequencing.…”

“…WGS datasets were analyzed with a custom pipeline. Read sequences were trimmed with Trimmomatic v0.39 and trimmed fastq sequences were aligned with BWA-MEM v0.7.17 using the Wormbase C. elegans genome version WS265 as the reference [ 22 – 24 ]. Variants were called with Varscan v2.3.9 called variants and annotated by CooVar [ 25 , 26 ].…”