The 20210901 release includes genotypes from whole-genome sequences. Genotypes are compared for concordance, and strains that are 99.985% identical to each other are grouped into isotypes. One strain within each isotype is the reference strain for that isotype. To look up isotype assignment, see Isotype List. All isotype reference strains are available on CeNDR.
- Strains: 681
- WGS strains: 681
- Isotypes: 509
- Genome: June2021
|Strain Data||Includes strain, isotype, location information, and more.||20210901_c_tropicalis_strain_data.csv|
|Strain Issues||This link contains all strain issues for this release|
|Alignment Data||This link contains all alignment data as BAM or BAI files.|
|Soft-Filtered Variants||The soft-filtered VCF includes all variants and annotations called by
the GATK pipeline. The QC status of each variant (INFO
WI.20210901.soft-filter.isotype.vcf.gz is not included in this release
WI.20210901.soft-filter.isotype.vcf.gz.tbi is not included in this release
|Hard-Filtered Variants||The hard-filtered VCF includes only high-quality variants after all
variants and genotypes with a failed QC status are removed. To obtain
vcf for a single or a subset of strains, use
|Imputed Variants||The imputed VCF includes all the variants from the hard-filtered Isotype VCF, but all missing genotypes have been imputed using Beagle v5.1.||
|Reference Genome FASTA (WS270)||The reference genome build from Wormbase used for alignment and annotation.||20210901_c_tropicalis_June2021.genome.fa|
|Transposon Calls||We have performed transposon calling for C. elegans isotype reference strains as a part of Laricchia et al.. For C. briggsae and C. tropicalis, these data will be deposited as soon as they are generated.||20210901_c_tropicalis_transposon_calls.bed is not included in this release|
|Tree||Tree generated using neighbour-joining algorithm as implemented in QuickTree in Newick and PDF format.||
|Haplotypes||Haplotypes for isotypes were calculated and plotted as described in Lee et al.||
20210901_c_tropicalis_haplotype.png is not included in this release
|Sweep Haplotypes||The most frequent haplotype that covers at least 30% of the chromosome and is found on chromosome centers was determined and classified as a selective sweep. For more details of C. elegans selective sweeps, see Andersen et al. and Lee et al.. The plot shows red (swept), gray (non-swept), and white (not classified) regions.||
20210901_c_tropicalis_sweep.pdf is not included in this release
|Hyper-Divergent Regions||The hyper-divergent regions are characterized by higher-than-average density of small variants and large genomic spans where short sequence reads fail to align to the reference genome. The C. elegans hyper-divergent regions were identified as described in Lee et al.. C. briggsae and C. tropicalis hyper-divergent regions will be released in the future.||20210901_c_tropicalis_divergent_regions_strain.bed is not included in this release|
|Download BAMs Script||You can batch download individual strain BAMs using this script.||20210901_c_tropicalis_bam_bai_download.sh|
Methods / Pipelines
This tab links to the nextflow pipelines used to process wild isolate sequence data.
FASTQ QC and Trimming
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.
Trimmed reads were aligned to C. tropicalis reference genome for NIC58 (NIC58_nanopore version June2021 published here) 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.
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 (126.96.36.199).
- 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
Heterozygous polarization and filtering thresholds were optimized for single nucleotide variants (SNVs).
Additionally, insertion or deletion (indel) variants less than 50 bp are more reliably called than indel variants greater than this size. In general, indel variants should be considered less reliable than SNVs.
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
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 (
1/1across 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 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:
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.
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.
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
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 XZ1516.
Divergent regions: Divergent regions were calculated as detailed in Lee et al. 2021
SnpEff Annotation: The predicted impact of each variant site was annotated with SnpEff (4.3.1t).
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 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.