Question: NGS: Variant identification at specific positions in an enzyme engineering approach
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gravatar for olivier.rousseau.1
23 months ago by
olivier.rousseau.10 wrote:

Hi,

I am wondering how to extract the data of my NGS analysis. In my experience, I did site-directed saturation mutagenesis at 49 positions in my enzyme. So theoratically, i have all the possible mutations at each chosen positions. Then I did my own PCR fragment of 250 bp to avoid random sequences and barcode them depending of the YES/NO results of a screening. Finally I did the NGS illumina sequencing. The goal is to identify every mutations that i have for each positions in my pool. I am a beginner at bioinformatics and I am learning to use the tools in Galaxy. I mapped the sequences (12 millions) with my reference genome of 1400 bp and i can visualize them in IGV. But i would like an output file giving me the mutations obtained at specific positions.

I have looked in the litterature, but there is a lot of different technique for SNP, variant calling but often on human genome for cancer mutations which does not seem to fit my purpose.

Could somebody enlighten me on the tools to use and the easiest way to achieve my goal?

Thank you

ADD COMMENTlink modified 23 months ago • written 23 months ago by olivier.rousseau.10
0
gravatar for Jennifer Hillman Jackson
23 months ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello,

The core tools of interest to call SNPs are in the group NGS: Variant Analysis at Galaxy Main (http://usegalaxy.org). These tools and more are also available in the Tool Shed for use in a local/cloud Galaxy.

For your project, I suggest trying the tool Naive Variant Caller followed by the tool Variant Annotator to parse out the results. Running a few of the others (Freebayes, etc) and comparing will help you to determine which tool produces the best results to suit your needs and desired output. Use the tools in the group NGS: VCF Manipulation to filter/compare/format VFC datasets.

Best, Jen, Galaxy team

ADD COMMENTlink written 23 months ago by Jennifer Hillman Jackson25k

Hi,

Thank you for your answer. The output gives me the variation for each nucleotide. Would it be possible to extract the information of mutation by residues. To look at the codon and the output would give me, for example, that the Leu75 was mutated to Lys75? and also the frequency of the residue mutation?

Thank you

Olivier

ADD REPLYlink written 23 months ago by olivier.rousseau.10
0
gravatar for olivier.rousseau.1
23 months ago by
olivier.rousseau.10 wrote:

Hi,

Thank you for your answer. The output gives me the variation for each nucleotide. Would it be possible to extract the information of mutation by residues. To look at the codon and the output would give me, for example, that the Leu75 was mutated to Lys75? and also the frequency of the residue mutation?

Thank you

Olivier

ADD COMMENTlink written 23 months ago by olivier.rousseau.10
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