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Whole Exome Sequence Data Analysis, VCF file, help??

Hello!

This is my first post here.

I am attempting to run some analyses on some Human whole exome germ line sequence data, there are 120 samples split into two groups (two different conditions) and I would like to run them to detect variants. This is my first time working with exome data so sorry if this is a noob question.

I was given the files in VCF.gz format, and I uploaded them into galaxy with the intention of running an exome seq pipeline. However, I am unable to do so, as the files have been uploaded into galaxy in tabular format and the pipeline requires fastq. I tried to convert the file format but couldn't do so.

I previewed the VCF file to see what was inside and it looks like this: (first two lines pasted)

1   2   3   4   5   6   7   8   9   10
chr1    861368  .   CG  C   1020.73 .   AC=1;AF=0.500;AN=2;BaseQRankSum=3.298;DP=146;FS=165.905;MLEAC=1;MLEAF=0.500;MQ=86.82;MQ0=0;MQRankSum=-1.499;QD=6.99;RPA=3,2;RU=G;ReadPosRankSum=2.010;SOR=5.577;STR GT:AD:DP:GQ:PL  0/1:54,67:145:99:1058,0,653
chr1    874544  .   AG  A   971.73  .   AC=1;AF=0.500;AN=2;BaseQRankSum=1.050;DP=60;FS=46.340;MLEAC=1;MLEAF=0.500;MQ=89.05;MQ0=0;MQRankSum=-2.496;QD=16.20;RPA=4,3;RU=G;ReadPosRankSum=-0.361;SOR=2.332;STR GT:AD:DP:GQ:PL  0/1:18,40:59:99:1009,0,175
...

Are exome VCF files normally supposed to look like this?

If I need to run this file, can I skip processing steps and just skip to something like GatK since the file is already in VCF format?

Thanks! Sorry for all the questions!

exome sequencing
• 256 views • link
updated by Tm Tm • written by kelseyca • kelseyca

Doesn't the VCF file have an header ? Headers are usually represented with "#" and they go about 10-15 lines before your actual content starts.

• 1 views • link
written by Inquisitive8995 Inquisitive8995

It seems variant records are fine, but not normalized/left aligned. But VCF headers are missing. In general, most of the vcf analyzers/annotators would not work without headers. kelseyca

• 1 views • link
written by cpad0112 cpad0112

I believe that something went wrong with my import into Galaxy. I unzipped the VCF file in terminal and opened it and I can see the header now:

#CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  AML_001
• 1 views • link
updated by finswimmer finswimmer • written by kelseyca • kelseyca

Hello and welcome again,

Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time.

code_formatting

Thank you!

• 1 views • link
written by finswimmer finswimmer

2 answers

Hello and welcome kelseyca ,

I would recommend to you to first read something about the most important file formats in NGS. Those are

  • fasta
  • fastq
  • sam/bam
  • vcf
  • (bed)

In a very basic pipeline this is how these files are used or generated:

  1. Alignment of raw sequencing reads (fastq) against a given reference (fasta) produces a text file with alignment information (sam) which should be converted to its binary version (bam)
  2. VariantCalling on the alignment file (bam) [restricted to given regions (bed)] results in the vcf file.

You see that your vcf file contains variant information about one or more samples. Saying this you will be not able to start the analysis from the beginning. So just start interpreting your variants if there is no reason to do the alignment again.

fin swimmer

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written by finswimmer finswimmer

Hello kelseyca,

You already have a .vcf file of variants, so there is no need to process any data by GATK. But, still I would recommend you to understand the way your vcf files has been prepared. As it will give idea of the different software used, related parameters and processing steps used to generate vcf files from raw data in fastq files. Having this information, in future you can modify the final vcf file or other intermediate files based on your need.

As you are newbie in bioinformatics, you can refer bio-protocol link to get overview of the steps followed for exome data analysis. However, please take a note that software and parameters can greatly vary based on individual need.

• 1 views • link
written by Tm Tm

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