Overview

The call command can be used to compare variants identified from Sanger sequencing with those present within a VCF. The call command uses BLAST to perform variant calling. This is a not a sophisticated method of variant calling, and is merely meant to facilitate in comparing Sanger sequencing with variants called in a VCF. Only homozygous calls are supported. Sanger sequences can be provided as AB1/ABI, FASTQ, or FASTA files. Sequences must be annotated with the sample name if you want to compare the genotype calls.

Annotating Samples

VCF-kit first tries to identify the sample from sequences using their filename. Because ABI/AB1 files can only contain one sequence, you must specify their sample using the filename. To identify the sample, VCF-kit searches for the sample name after splitting the string using the \W regular expression. For example:

The following Sanger sequence files (in AB1, fa, and fastq format) are all acceptable ways of specifying that the sample from the VCF is DL238:

DL238.AB1
DL238.fa
DL238.fastq.gz
DL238.20161205.fq.gz
DL238_20161205.fq.gz
DL238-20161205.fq.gz

In the first line, DL238.AB1 is split into DL238 and AB1. Among the split pieces, the name of a sample must match exactly.

The examples below do not work:

testDL238.AB1
testDL23820161205.AB1

You may have multiple samples within a single fastq or fasta file. In this case, you can use the description line to annotate each record:

> DL238
CGTCAAGGGGCACCGGATCG...

> @FCC1GJUACXX:6:1101:19686:24985#AAGGTCTA/2 |DL238|
CGTCAAGGGGCACCGGATCG...

And multiple samples may be present within the same file:

> DL238
CGTCAAGGGGCACCGGATCG...
> N2
CGTCAAGGCGCACCGGATCG...
> MY25
CGTCAAGGCGCACCGGATCG...

Usage

    vk call <seq> --ref=<reference> [--all-sites --vcf-targets <vcf>]

Perform variant calling using blast. Useful for validating variants using sanger sequencing.

  • <seq> - Fasta (fa), Fastq (fq, fastq), or ab1 format, determined by file extension containing sanger reads.
  • --ref - The reference genome used to perform calling.
  • --all-sites - Outputs comparison of every site that aligns within the alignment.
  • --vcf-sites - Only outputs sites present within the VCF.

Output

The following columns are output when using the call command:

  • CHROM - chromosome
  • POS - position
  • reference - The base at the genomic position within the reference genome. Should always be identical to REF, when REF is listed.
  • REF - The reference genotype within the VCF. Only listed when a variant is present.
  • ALT - The alternative genotype within the VCF. Only listed when a variant is present.
  • seq_gt - The Sanger genotype for the sample listed.
  • vcf_gt - The VCF genotype for the sample listed. Homozygous calls are listed as a single base.
  • sample - The sample being compared. Must be matched to the Sanger sequence and present within the VCF.
  • variant_type - The type of variant observed as compared to the Sanger sequence: SNV, Insertion, or Deletion.
  • classification - True/False Positive/Negative, assuming the Sanger sequence is correct.
  • index - The index of the Sanger sequence, as aligned.
  • alignment_start - The start of the blast alignment.
  • alignment_end - The end of the blast alignment.
  • strand - The strand aligned to (+ or -).
  • gaps - Total number of gaps in the alignment.
  • mismatch - Total number of mistmatches in the alignment.
  • bitscore - The bitscore of the alignment.

Examples

Note: Some columns removed from examples for conciseness.

--all-sites

Shows all calls from Sanger and VCF. Will output every position that was aligned to reference from Sanger sequence and corresponding VCF calls. Notice the absence of REF/ALT for sites not called in the VCF below.

vk call DL238_sanger.AB1 --ref=WBcel235 --all-sites illumina_sequencing.vcf.gz
CHROM POS reference REF ALT seq_gt vcf_gt sample variant_type classification index alignment_start alignment_end
X 14557504 C C DL238 545 14556961 14557595
X 14557505 T T DL238 546 14556961 14557595
X 14557506 G A G G G DL238 snp TP 547 14556961 14557595
X 14557507 C C DL238 548 14556961 14557595

--vcf-targets

Only show variants present in the VCF.

vk call DL238_sanger.AB1 --ref=WBcel235 --vcf-sites illumina_resequencing.vcf.gz
CHROM POS reference REF ALT seq_gt vcf_gt sample variant_type classification index alignment_start alignment_end
X 14557228 G G A G G DL238 snp TN 269 14556961 14557595
X 14557388 T T C T T DL238 snp TN 429 14556961 14557595
X 14557506 A A G G G DL238 snp TP 547 14556961 14557595
X 14557521 T T A T T DL238 snp TN 562 14556961 14557595