Generating genes and chromosomes from GATK

[fishinvest]

Hello,

I am using the GATK best practices pipeline with a reference genome and paired end reads and use UnifiedGenotyper and VariantFiltration to get a vcf file with all of my SNPs.

What is the best way to convert this into actual new genes for my read genome?

Thanks!

[vdauwera]

Hi there, can you clarify what you mean by "convert this into actual new genes"? Are you trying to get the separate haplotype sequences for your sample output as sequence files, perhaps?

[fishinvest]

Sorry! I am very new to NGS analysis and my PI kind of said 'go and learn how to turn reads into genes using a reference genome.' I have gotten some good help and direction leading my to the GATK best practices pipeline, but a lot of my terminology is still very weak so feel free to correct me.

I have a VCF file full of putative SNPs and I would like to generate files for each gene from the reference replacing reference with the alternative. I don't think a script to do that would be particularly difficult, but if there are methods out there already then I have a real tool with documentation (hopefully) I can read to better understand the reasoning behind it.

I should clarify that my reference and reads are from very close strains from a species of apicomplexa

[Kennels]

Hi,

I think what you want is to generate a consensus sequence based on the SNP calls of your alternates. There is a package called 'Samtools', and in it there is a method to call consensus: http://samtools.sourceforge.net/mpileup.shtml

You'll find a lot of documentation on the web about samtools - try and use the latest version of samtools (using mpileup) instead of ones that still use 'pileup' (v0.1.16 and before).

I think there are utilities in the GATK pipeline to find a consensus too though I can't remember the tool name.

Note though there are caveats to trying to generate a consensus sequence, which can be badly influenced by indels, and as far as I know there isn't a standard, 'easy' method. You might find you'll have to manually look at the alignments and decide for yourself.

[fishinvest]

Hey thanks for the help. I know about MPileup and I can use it short term but I was also hoping to incorporate all the filters and information from my Picard and GATK steps. I will have to look and see what MPileup can do there. Thanks again!

[vdauwera]

The GATK tool you're thinking of is probably FastaAlternateReferenceMaker: http://www.broadinstitute.org/gatk/g...enceMaker.html

[fishinvest]

Thanks VD, I will look into that one