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GATK Paper

humaasifhumaasif University of SaoPauloMember

hi there
can any body help me with these lines in GATK Paper and how this problem is tackled in GATK
A framework for variation discovery and genotyping using next-generation DNA sequencing data
Previous approaches have relied on filtering SNP calls that have characteristics outside of their normal ranges, such as those occurring at sites with too much coverage, or by requiring non-reference bases to occur on at least three reads in both synthesis orientations

the main confusion is around
bases to occur on at least three reads in both synthesis orientations
i will appreciate if any image can help
Regards

Best Answer

Answers

  • SheilaSheila Broad InstituteMember, Broadie, Moderator admin

    @humaasif‌

    Hello,

    This means that previously, for a non-ref base to be taken seriously into consideration, the non-ref base had to be seen on at least 3 different reads on both the forward and reverse strands.

    I hope this helps.

    -Sheila

  • humaasifhumaasif University of SaoPauloMember

    thank you shella thats what i understand but the thing that is making me confuse is e.g. reads
    ..........................A.............................(REF)
    ..........................T.................................>
    ..........................T..................................> FWD
    ..........................T..................................>
    <........................A....................................
    <........................A.....................................
    <.........................A..................................... REV

    Am i right
    for i think REV strand should have the base complimentary to FWD
    Regards

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    @humaasif‌ Are you asking how to interpret the pileup format of this example? Or are you asking something else? Please clarify.

  • humaasifhumaasif University of SaoPauloMember
    edited August 2014

    no i just need to understand that is this statement means what i draw

    Previous approaches have relied on filtering SNP calls that have characteristics outside of their normal ranges, such as those occurring at sites with too much coverage, or by requiring non-reference bases to occur on at least three reads in both synthesis orientations.

    Reference GATK paper
    A framework for variation discovery and genotyping using next-generation DNA sequencing data

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    @humaasif, I'm sorry but I don't understand what you are asking. What are you trying to say with the read pileup example?

  • humaasifhumaasif University of SaoPauloMember

    ok leave that example :( may be it was not good enough.
    please can you just explain to me with any graphics or text how a mutation should be if you want to show it in both synthesis orientation i will be extremely obliged as it is bugging me

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    You can represent this in several different ways. Typically we would use an IGV screenshot. You can see some examples here: http://www.broadinstitute.org/software/igv/AlignmentData

    By default it does not show the base-by-base sequence of all reads, it only shows the bases that are different from the reference. When it does show the bases, it shows the forward-aligned transliteration (so for any reverse reads, the base displayed is the reverse complement). The orientation of the read is indicated by the arrow shape. You can also have the program emphasize orientation by coloring the reads differently if they are fwd or reverse.

    To satisfy the requirements explained in the paper, and assuming the reference base is A and the variant is C, you would have to see at least three forward reads and three reverse reads that have a C at the position of interest.

    Does that help?

  • humaasifhumaasif University of SaoPauloMember

    To satisfy the requirements explained in the paper, and assuming the reference base is A and the variant is C, you would have to see at least three forward reads and three reverse reads that have a C at the position of interest

    so the reverse stand will also have C
    (so for any reverse reads, the base displayed is the reverse complement

  • humaasifhumaasif University of SaoPauloMember

    Thank you Geraldine_VdAuwera you are a great help in making me go smoothly
    stay blessed

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