Question: Variant calling with 5x coverage, human genome
gravatar for Daphnis
7 months ago by
Daphnis0 wrote:

I am new to analysing raw sequencing data - apologies in advance if I seem not to understand what I'm dealing with.

I have 6 human genome datasets (2 biological replicates, 3 time-points, 5x coverage each) and need a "simple" information about mutational frequency in each sample (#variants/1Mb). I understand most tools need a min coverage/base so won't be able to analyse this with samtools mpileup for example.

The hypothesis I am testing is that timepoint2 will have a larger number of variants compared to timepoint1 (the H0 being that there is no difference) so the total number of variants will include all the technical errors during sequencing, PCR etc. plus biological differences. Basically I am assuming that all samples will have similar numbers of baseline variants, all I care about is the difference between the totals. I do not need information about a specific base mutation or gene.

I have generated bam files with marked duplicates (each file is 10-13Gb). Can anyone help or give suggestions on how to proceed? Many thanks in advance.

variant snp alignment samtools bam • 215 views
ADD COMMENTlink modified 7 months ago by Jennifer Hillman Jackson25k • written 7 months ago by Daphnis0
gravatar for Jennifer Hillman Jackson
7 months ago by
United States
Jennifer Hillman Jackson25k wrote:


Minimum coverage is a parameter on several tools that call variants. The tools Naive Variant Caller followed by Variant Annotator will probably produce the data you want. Or at least enough that it can be parsed to produce the statistics you want using manipulation tools from these groups for the data reduction: Text manipulation; Datamash; Filter and Sort; Join, Subtract and Group.

Please see the Galaxy Variant analysis tutorials here for example workflows. These do not cover the NCV but do show the basic processes using Freebayes and related tools.


ADD COMMENTlink written 7 months ago by Jennifer Hillman Jackson25k
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