DOI: 10.5061/dryad.6q573n647
title: Calling structural variants with confidence from short-read data in wild bird populations

description: Comprehensive characterisation of structural variation in natural
 populations has only become feasible in the last decade. To investigate
 the population genomic nature of structural variation (SV), reproducible
 and high-confidence SV callsets are first required. We created a
 population-scale reference of the genome-wide landscape of structural
 variation across 33 Nordic house sparrows (Passer domesticus) individuals.
 To produce a consensus callset across all samples using short-read data,
 we compare heuristic-based quality filtering and visual curation
 (Samplot/PlotCritic and Samplot-ML) approaches. We demonstrate that
 curation of SVs is important for reducing putative false positives and
 that the time invested in this step outweighs the potential costs of
 analysing short-read discovered SV datasets that include many potential
 false positives. We find that even a lenient manual curation strategy
 (e.g. applied by a single curator) can reduce the proportion of putative
 false positives by up to 80%, thus enriching the proportion of
 high-confidence variants. Crucially, in applying a lenient manual curation
 strategy with a single curator, nearly all (&gt;99%) variants rejected
 as putative false positives were also classified as such by a more
 stringent curation strategy using three additional curators. Furthermore,
 variants rejected by manual curation failed to reflect the expected
 population structure from SNPs, whereas variants passing curation did.
 Combining heuristic-based quality-filtering with rapid manual curation of
 structural variants in short-read data can therefore become a time- and
 cost-effective first step for functional and population genomic studies
 requiring high-confidence SV callsets.
description: The raw Illumina reads and assembled reference genome from this
 article are also published and available at NCBI, Bioproject number
 PRJNA255814 (<em>Passer domesticus </em>reference accession
 number SAMN02929199). Trimmed reads were aligned with BWA-MEM (bwa
 v.0.7.17) to the short-read reference genome assembly for <em>Passer
 domesticus </em>(Elgvin et al. 2017), NCBI:
 GCA_001700915.1_Passer_domesticus-1.0), and then sorted and indexed with
 Samtools (samtools v. 1.9). All unplaced scaffolds were removed and thus
 only mapped chromosomal regions were included in downstream
 analyses. Larger (&gt;20bp) structural variants
 (deletions, duplications, and inversions) from the aligned .bam
 files<strong> </strong>using LUMPY (Layer et al. 2014) and
 genotyped the resulting calls with SVTyper (Chiang et al. 2015), via the
 smoove pipeline (Pedersen et al. 2020). The resulting VCF file of raw
 structural variant calls analysed in the study is included in the
 following file: sparrow_all.smoove.square.anno.vcf.gz
 Repetitive elements were identified using the Earl Grey TE
 annotation pipeline (version 1.2) (Baril et al. 2021, 2022), configured
 with RepBase (version 23.08) and Dfam (version 3.4) repeat libraries
 (Hubley et al. 2016; Jurka et al. 2005). Briefly, Earl Grey first
 annotated known repeats using the <em>Aves</em> repeat
 library. Following this, Earl Grey identified and refined novel TEs using
 an automated and iterative implementation of the “BLAST, Extract, Extend”
 process (Platt et al. 2016). Following the final TE annotation
 (passerDomesticusAnnotatedRepeats.gff), overlapping and fragmented
 annotations were resolved by Earl Grey before the final TE
 quantification.
description: # Calling structural variants with confidence from short-read data in wild
 bird populations --- Included files: *
 **sparrow_all.smoove.square.anno.vcf.gz** = VCF file of raw structural
 variant calls analysed in the study * Scripts and walk-throughs for
 generating the above VCF file, downstream filtering and plotting in
 Samplot, creation of a PlotCritic curation project *
 **passerDomesticusAnnotatedRepeats.gff** = TE annotation generated in this
 study ## Description of the data and file structure Larger (&gt;20bp)
 structural variants (deletions, duplications, and inversions) from the
 aligned .bam files using LUMPY (Layer et al. 2014) and genotyped the
 resulting calls with SVTyper (Chiang et al. 2015), via the smoove pipeline
 (Pedersen et al. 2020). The resulting VCF file of raw structural variant
 calls analysed in the study is included here: *
 sparrow_all.smoove.square.anno.vcf.gz A Unix or Linux command line
 terminal can be used to open the file. Functions such as "less"
 or "zcat" can open the gzipped VCF file. Features can be queried
 using, e.g., Bcftools. Snakemake pipeline for generating the .bam files
 from raw Illumina reads is provided as: * Snakefile_BWA_GATK Example
 scripts on how to generate and filter the above VCF-file are provided in:
 * Smoove_commands_to_generate_VCF_of_raw_SVs_and_example_filtering.txt
 Example scripts on how to generate the Samplot images and PlotCritic
 curation project from a filtered VCF file are provided in: *
 Samplot_and_PlotCritic_commands.txt The script to select only SVs with a
 specific number of individuals per genotype class, plus randomly select
 those individuals to generate a Samplot command to plot each image: *
 gen_samplot.py ## Sharing/Access information The raw Illumina reads and
 assembled reference genome from this article are published and available
 at NCBI, Bioproject number PRJNA255814 (*P. domesticus* reference
 accession number SAMN02929199).