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  • output for sequencing of reverse strand

    Hi Members,

    I've lost, yet again with direction, and sequencing. I'm working on 16s data. Paired end, Illumina reads. And since then I am confused over the output in read 2- fastq.gz

    5'------>primer SEQUENCE END 3'
    3' END SEQUENCE PRIMER<-------5'

    Paired end sequencing as per my understanding looks like above. Primer or index, or bar code, adapter, Pardon me for not using appropriate terms.

    My doubt:
    1) In the fastq of reverse strand, how do I have the output:
    END.............SEQUENCE
    OR
    SEQUENCE......END

    I'm confused over the direction in which I have the fastq output.
    Last edited by bio_informatics; 06-09-2015, 08:26 AM.
    Bioinformaticscally calm

  • #2
    DNA strands are anti-parallel. So R2 in your cartoon has the 5'-end to the right of that strand (and not 3' as you have drawn it).

    See this for a simple explanation: http://seqanswers.com/forums/showthread.php?t=42737

    Comment


    • #3
      Hi Genomax,

      Thanks for your reply and sharing the URL.
      I've edited my cartoon. Sorry.

      But then again, in which direction is the output present for reverse read in fastq?
      Bioinformaticscally calm

      Comment


      • #4
        DNA sequence is always represented as 5' --> 3'. So 5'--SEQUENCE ... END--3' for R2.

        Comment


        • #5
          Originally posted by GenoMax View Post
          DNA sequence is always represented as 5' --> 3'. So 5'--SEQUENCE ... END--3' for R2.
          Thanks for clearing clouds.

          That explains, why reverse read file has bar code/index at the starting of reads.

          Merci.!
          Bioinformaticscally calm

          Comment


          • #6
            Originally posted by bio_informatics View Post
            Thanks for clearing clouds.

            That explains, why reverse read file has bar code/index at the starting of reads.

            Merci.!
            You must be using a custom "inline" barcode/index. Illumina barcodes are never part of actual read.

            Comment


            • #7
              Originally posted by GenoMax View Post
              You must be using a custom "inline" barcode/index. Illumina barcodes are never part of actual read.
              Hi Genomax,

              Yes, I guess so. Thanks for sharing new term. Everyday a new learning.

              Data am having is based on entirely new chemistry/protocol. Here's the paper.
              Bioinformaticscally calm

              Comment

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