Methods / Pipelines

This tab links to the nextflow pipelines used to process wild isolate sequence data.

FASTQ QC and Trimming

andersenlab/trim-fq-nf -- (Latest 5fee6f7)

Adapters and low quality sequences were trimmed off of raw reads using fastp (0.20.0) and default parameters. Reads shorter than 20 bp after trimming were discarded.

Alignment

andersenlab/alignment-nf -- (d35c7)

Trimmed reads were aligned to C. tropicalis reference genome for NIC58 (NIC58_nanopore version June2021) using bwa mem BWA (0.7.17). Libraries of the same strain were merged together and indexed by sambamba (0.7.0). Duplicates were flagged with Picard (2.21.3).

Strains with less than 14x coverage were not included in the alignment report and subsequent analyses.

Variant Calling

andersenlab/wi-gatk -- (82ae132)

Variants for each strain were called using gatk HaplotypeCaller. After the initial variant calling, variants were combined and then recalled jointly using gatk GenomicsDBImport and gatk GenotypeGVCFs GATK (4.1.4.0).

The variants were further processed and filtered with custom-written scripts for heterozygous SNV polarization, GATK (4.1.4.0), and bcftools (1.10).

1. Heterozygous SNV polarization: Because C. tropicalis is a selfing species, heterozygous SNV sites are most likely errors. Biallelic heterozygous SNVs were converted to homozygous REF or ALT if we had sufficient evidence for conversion. Only biallelic SNVs that are not on mitochondria DNA were included in this step. Specifically, the SNV was converted if the normalized Phred-scaled likelihoods (PL) met the following criteria (a smaller PL means more confidence). Any heterozygous SNVs that did not meet these criteria were left unchanged.
   • If PL-ALT/PL-REF <= 0.5 and PL-ALT <= 200, convert to homozygous ALT
   • If PL-REF/PL-ALT <= 0.5 and PL-REF <= 200, convert to homozygous REF

1. Soft filtering: Low quality sites were flagged but not modified or removed.

   For the site-level soft filter, variant sites that meet the following conditions were flagged as PASS. These stats were computed across all samples for each site.

   • Variant quality (QUAL) > 30 (this filter is very lenient, only three sites failed)
   • Variant quality normalized by read depth (QD) > 20
   • Strand bias of ALT calls: strand odds ratio (SOR) < 5
   • Strand bias of ALT calls: Fisherstrand (FS) < 100
   • Fraction of samples with missing genotype < 95%
   • Fraction of samples with heterozygous genotype after heterozygous site polarization < 10%

   For the sample-level soft filter, genotypes that meet the following filters were flagged as PASS for each site in each sample:

   • Read depth (DP) > 5
   • Site is not heterozygous

2. Hard filtering: Low quality sites flagged in soft-filter are removed

   • For the site-level hard filter, variant sites not flagged as PASS were removed.
   • For the sample-level hard filter, genotypes not flagged as PASS were converted to missing (./.), with the exception that heterozygous sites on mitochondria where kept unchanged.

   After the steps above, sites that are invariant (0/0 or 1/1 across all samples, not counting missing ./.) were removed.

Determination of filter thresholds

We examined our filter thresholds for a previous C. elegans release. A variant simulation pipeline was used as part of this process:

• Variant Simulations - andersenlab/variant-simulations-nf

Please see the filter optimization report for further details.

Isotype Assignment

andersenlab/concordance-nf -- (8befe4c)

Isotype groups contain strains that are likely identical to each other and were sampled from the same isolation locations. For any phenotypic assay, only the isotype reference strain needs to be scored. Users interested in individual strain genotypes can use the strain-level data.

Strains were grouped into isotypes using the following steps:

1. Using all high quality variants (only SNPs from the hard-filtered VCF) and bcftools gtcheck, concordance for each pair of strains was calculated as a fraction of shared variants over the total variants in each pair.

2. Strain pairs with concordance > 0.99985 were grouped into the same isotype group. The threshold 0.99985 was determined by:

   • Examining the distribution of concordance scores.
   • Capturing similarity between strains to minimize the number of strains that get assigned to multiple isotype groups.

3. The following issues, which were rare, were resolved on a case-by-case basis:

   • If one strain was assigned to multiple isotypes.

When issues arose, the pairwise concordance between all strains within an isotype were examined manually. Strains and isotypes may be re-assigned with the goal that strains within the same isotype group should have high concordance with each other, and strains from different isotype groups should have lower concordance.

Site-level filtering and annotation

andersenlab/post-gatk-nf -- (07c87c44)

1. Tree generation: Trees were generated by converting the hard-filtered VCF to Phylip format using vcf2phylip (030b8d). Then, the Phylip format was converted to Stockholm format using Bioconvert (0.3.0), which was then used to construct a tree with QuickTree (2.5) using default settings. The trees were plotted with FigTree (1.4.4) rooting on the most diverse strain JU1374.

2. Divergent regions: Divergent regions were calculated as detailed in Lee et al. 2021

andersenlab/annotation-nf -- (ac804c5a)

1. BCSQ Annotation: Variant impacts were then annotated using bcftools csq (v1.14), which takes into consideration nearby variants and annotates variant impacts based on haplotypes.

Imputation

Imputation was done using Beagle (5.2) with the following parameters: window=5 overlap=2 impute=true ne=100000 imp-segment=0.5 imp-step=0.01 cluster=0.0005.