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Questions tagged [variant-calling]

The process of discovering variants in individuals starting from sequencing reads. Can be subdivided into germline variant calling and somatic variant calling. Can also be used for the more specific process of finding variants starting with reads aligned to a reference genome (SAM/BAM files).

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3 votes
3 answers
322 views

Non-ACGTN alternate alleles in VCF

I was trying to left normalize my vcf file using following command: bcftools norm -f reference.fasta -m -any input.vcf > output.norm.vcf However this gives the ...
4 votes
1 answer
69 views

Replicating VCF Filtering & Trait-Based SNP Extraction Workflow

I'm new to handling SNP genotyping data in VCF format. My goal is to identify significant SNPs linked to a specific trait (like "height") for specific samples in a multi-sample VCF file. My ...
3 votes
2 answers
1k views

How does DeepVariant construct RGB images from DNA sequences?

DeepVariant is a pipeline to call genetic variants from DNA sequencing data. A major step, before feeding the CNN, is to translate these DNA sequences into images. It's unclear why and how Google ...
2 votes
2 answers
27 views

true depth per gvcf location instead of min depth per gvcf block

I am using deepvariant to calls variants on my (WES) data. I am outputting a gvcf file. I am wondering (I cannot find anything in the documentation) how it would be possible to get the true depth per ...
4 votes
1 answer
66 views

Identifying somatic mutations in cell lines

I would like to identify the somatic mutations present in a cell line and characterise the genes that are potentially affected by those mutations. For example, are there oncogenes mutated in a ...
2 votes
0 answers
33 views

concatenating windowed VCF files, but quickly?

I am calling variants from WGS for a genetically diverse species which, with my sample size, produces a (bgzipped) VCF of around 500G. I am using a nextflow pipeline to chunk up the genome, call ...
2 votes
1 answer
87 views

How do I quantify a specific somatic variant?

I'm working with targeted Illumina sequencing data generated with DNA from diseased and healthy tissue (this is an age-related disease and is not cancer/neoplastic). My hypothesis is that the diseased ...
0 votes
1 answer
57 views

Variants from multiple tools: normalization before or after annotation with VEP?

We want to compare some variant calling tools and their calls on Whole Exome Sequencing data. We will have to normalize the variants that are called (.vcf format) before comparison using for instance ...
1 vote
0 answers
142 views

bwa mem hangs after a few thousand reads

I am trying to align a bunch of paired sample fastq files using bwa mem. My original command was: ...
1 vote
2 answers
94 views

How to run a GATK Docker Image with local files?

I'm trying to use the HaplotypeCaller from the GATK toolkit but I keep getting an error. I pulled GATK through Docker and am using this command: ...
0 votes
0 answers
66 views

ERROR IN FUNCOTATOR: [SQLITE_IOERR_LOCK] I/O error in the advisory file locking logic (disk I/O error)

I ran the "funcotator" step from the GATK somatic short variant discovery (snvs + indels) pipeline and used the output of filtermutectcalls as input, but ...
2 votes
1 answer
926 views

Compare VCF files

I have two strains, X and Y, and I would like to compare the variants found in both. I have my own parent strain, Z, that was sequenced and assembled to be used as the reference genome. I first ...
0 votes
2 answers
202 views

which SNP effect software, such as VEP, annovar or snpEff, allows the user to submit their own annotations in gtf format?

I have a use case where I want to annotate the variants from running Oxford Nanopore's Medaka on a very simple reference, a single sequence starting on an ATG and finishing on a stop codon (no introns)...
2 votes
0 answers
50 views

INDELS in PLINK files converted to VCF

I want to compare/validate variants called from sequencing data with array (plink format) variant data. I converted the plink files (.bim, .bed, and .fam files) with plink1 to vcf files. ...
1 vote
1 answer
70 views

Choosing the best number of species for assay

I am doing amplicon sequencing of a virus across many different regions. Lets say I have 20k unique variants of the same virus that I put into my pcr assay and after sequencing and amplifications I am ...
2 votes
0 answers
24 views

Feasible to find genetic variations of two samples using RNAseq data?

I have bulk RNAseq data from two strains of mice from Jackson Lab: C57BL/6 and B6.SJL. The former expresses a Ptprc-b allele and the latter expresses a ...
3 votes
0 answers
78 views

Different human reference genomes from NCBI and ENSEMBL, and variant annotation with Variant Effect Predictor (VEP)

Background: I have been working on a human variant calling pipeline, from whole exome reads to variant annotation. For variant annotation I used the Variant Effect predictor (VEP), which is Ensembl ...
4 votes
1 answer
133 views

Find SNPs in yeast genomes

I'm a new Bioinformatic scientist working for a yeast genetics company. Objective To create a database of yeast genomes from NCBI and identify SNP variants. In my pipeline FastQC, Trimmomatic, BWA ...
1 vote
0 answers
28 views

Which type of variant caller should I use in a WES normal cell line sample?

I have whole-exome sequencing data of an immortalised non-tumor (normal) cell line that I wish to assess for the presence/absence of APC/Wnt mutations. This is to double check that the cell line is ...
1 vote
1 answer
260 views

Getting VCF file that contain common SNPs from 6 VCF file using isec

I have 6 VCF files, where I would like to obtain the SNPs that are common (by position) in all the 6 files. I have tried this command ...
2 votes
1 answer
64 views

WES variant calling with DNBSEQ-T7: technical quality assessment

I recently received whole exome sequencing samples that were sequenced on an MGI sequencing instrument, DNBSEQ-T7. I am interested in somatic variant-calling on the paired tumor-normal samples. As ...
3 votes
1 answer
55 views

How do I generate a variant list using Illumina reads from a Salmonella genome?

I am planning on performing phylogenetic analysis of Salmonella specimens using WGS data from PulseNet and GenomeTrakr, for the purpose of public health surveillance & to provide context for ...
1 vote
1 answer
269 views

variant visualisation of VCF files for 30+ samples

I am relatively new to genome analysis and would like to compare 30+ vcf files, 1 sample per file. After filtering there are about ~20 variants per vcf file. I would like to evaluate to mutational ...
2 votes
1 answer
266 views

somatic SNV tool for ONT samples

I'm looking for a tool to call somatic single nucleotide variations on reads from an Oxford Nanopore Technologies (ONT) run. I have both tumor and normal samples and have already aligned them to a ...
0 votes
1 answer
43 views

Non-exome variants called from whole-exome sequencing data

I'm working on rather standard whole-exome sequencing data and treat it the same as whole-genome sequencing data (aligning it to the full GRCh38 reference assembly and calling variants with no exome-...
1 vote
0 answers
61 views

Benchmarking for variant identification using RNA-seq data

I am in need to benchmark the variant identification pipeline which uses RNA seq data alone without any matched-normal. I would like to know the reference dataset (and the pipeline on which the ...
1 vote
1 answer
60 views

Name this alternative INDEL format

I have recently been working with variants which are represented in a non-standard format. This alternative format uses a period instead of prefixing variants with a reference base. What is this ...
2 votes
0 answers
139 views

Difference between pileup and mpileup in VarScan and samtools

I'm going to call variants with VarScan from a pileup files created with samtools. I realized that there are in general two major possibilities to call variants, pileup as well as mpileup. The VarScan ...
9 votes
2 answers
3k views

How do I generate a variant list (i.e. VCF file) using Illumina reads from a human genome?

This is a problem I have to solve frequently, and I'd be interested in knowing what other methods people use to solve the same problem. About twice a year, I get asked to determine variants from ...
8 votes
1 answer
2k views

Merge 2 VCFs from different variant callers

I am working with WES data for detection of somatic variants and I have used two variant callers because no variant caller is complete in itself. I have used GATK Haplotypecaller for small variants ...
1 vote
1 answer
180 views

snakemake tutorial stuck in basics-step-5 (variant calling)

I am learning to use snakemake. I am following the tutorial (https://snakemake.readthedocs.io/en/stable/tutorial/tutorial.html). In the section called "Basics", steps 1 to 4 worked well, ...
0 votes
0 answers
17 views

How deep variant and others preprocessed insertion and deletion for extracting candidate variant?

I am trying to build a model for variant calling,but I am a bit confused how to pre-process the reads that has insertion and deletion and align them with reference? Should I add something to the ...
1 vote
1 answer
80 views

Reference variant detected as altered one in bam file

I received (from manufacturer) several .bam files and I used four callers (samtools, freebayes, haplotypecaller, deepvariant) to find some sequence variants. In obtained .vcf files, I took a closer ...
0 votes
0 answers
53 views

What is the best tools to find all the somatic mutations?

I am a beginner in this field. I would like to know which is the best way to get all the mutations from a certain sample. At this point, I am considering using GATK (here) and Maftools (here). I don't ...
0 votes
1 answer
2k views

Download or create VCF from Human Genome Diversity Project (HGDP)

I can't seem to find HGDP data in VCF format. Does anyone know where I can download it from? Perhaps there a Snakemake pipeline for calling VCFs from this data?
3 votes
2 answers
193 views

Detect mutation context in a read of a sam file

After sequencing (Illumina) of some DNA, I generated a sam file through alignment of a fastq file (using Bowtie2). Instead of using variant calling programs, I want to know the specific variances, ...
2 votes
0 answers
186 views

Assess ploidy estimation through SNV and CNV results

I want to assess ploidy estimation (i.e., diploid vs tetraploid) in a set of samples for which I have both results from the variant caller (i.e., SNVs) and from the copy number caller. What are the ...
0 votes
1 answer
324 views

Range of values for VAF in heterozygous genotype

(VAF = variant allele frequency) This question follows from this one. What is the expected range of values for VAF of mutations with heterozygous genotype (i.e., 0/1 or 0|1)? Is it possible to have 0% ...
1 vote
1 answer
683 views

GATK variant filtration using "SelectVariants" and use of JEXL queries

How to do variants selection in some corner cases using GATK and JEXL expressions? I am following the guidelines given in this links for variant selection for some specific cases are not as much ...
2 votes
1 answer
203 views

Consensus sequence from vcf file

I am generating a consensus sequence for SARS-Cov-2. I am visually inspecting every candidate SNP in a pileup. Sometimes I find that there are two consistent variants for the same position, but I can ...
2 votes
1 answer
57 views

Detecting SARS-CoV-2 variants from the mixed virus population

I have a fastq file from Nanopore sequencing data that contains reads from both the UK and South Africa variants of SARS-CoV-2. The variants are identified by three key mutations in the receptor-...
3 votes
2 answers
63 views

Calling for one specific SNP from multiple sequencing runs

Just learning the basics of bioinformatics and bash shell scripting. Is there a correct way to search for a specific allele with one or even multiple sequencing runs? I am searching for prevalence of ...
0 votes
0 answers
64 views

When can we have duplicated variants in a VCF file, excluding artifacts?

I read these two questions on duplicated VCF variants: 1, 2. However, it is not entirely clear to me when duplicated variants can arise (excluding merging artifacts). I want to understand if ...
2 votes
2 answers
125 views

Nanopore variant calling

So far I haven't done any variant calling as such. Nanopore I have used for 16s microbiome data. Now My question/doubt so how do I proceed for nano-pore virus sequencing data Steps: I get fast5 files ...
0 votes
1 answer
186 views

How should I deal with segmental duplications when aligning NGS reads to a reference genome?

This is a follow-up of my other question. I have been having trouble calling variants in the human SMN1 and SMN2 genes, because the human genome has a large segmental duplication there and these two ...
2 votes
2 answers
73 views

Detection of CNV(InDel) of intermediate size

My impression is that small InDel (a couple of bp) is identified through cigar string in BAM and typical CNV (at least thousands of bp) is detected through read depth. What about InDel or CNV with ...
1 vote
1 answer
193 views

Meaning of order of alleles in VCF GT field for unphased genotypes

Order of alleles in genotype field (on VCFs) can be used to infer haplotypes However, what about unphased genotypes? I have a VCF file where I only have unphased genotypes: ...
0 votes
1 answer
203 views

Convert VCF file to mpileup.txt

I am working on an iterative analysis that uses orthologous pipelines that require mpileup.txt files as input for a visualization step. This requires me to convert VCF files to mpileup.txt. This ...
1 vote
2 answers
655 views

How do I call variants using BCFTools without piping mpileup file?

I need to call variants on a large number of reads using both VarScan2 and BCFTools. Both of these variant calling tools use <...
0 votes
0 answers
36 views

Fastest way to count where two bam files are homozygote reference (inverted variant calling)

Maybe I'm having a brain-freeze so excuse me if this is a waste of everyone's time... I am working with an organism with ~700Mb genome. I have bam files of two individuals that are whole genome ...