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  • Illumina MiSeqFGx - fastq vs. StrResults.txt

    Hey,
    I'll write my Bachelor Thesis in a few weeks and I'm new to NGS, so maybe this is quite a noob question - however, these ar my problems:
    1)
    We are sequencing different markers like D3S1358, D22S1045 and so on with our new Illumina MiSeqFGx. Now I opened the Alignment/Report/StrResults.txt and found a result like the following for one sample:

    Marker Allele Depth Sequence
    D22S1045 16 23 ATTATTATTATTATTATTATTATTATTATTATTATTATTACTATTATT
    D22S1045 17 78 ATTATTATTATTATTATTATTATTATTATTATTATTATTATTACTATTATT

    OK, so this sample has Alleles 16,17 for D22S1045 marker.
    Now I thought that all of the primer matching Sequences are stored in the .fastq files, so I started searching for the sequences in the .fastq files but havent found them.
    (grep -c ATTATTATTATTATTATTATTATTATTATTATTATTATTATTACTATTATT Sample.fastq/data --> Result = 0)

    What am I misunderstanding, I thought the reported sequences must be found in the .fastq file? Can anybody help?

    2)
    We determined the alleles with capillary electrophoresis before and now with NGS we have some other alleles as result for some samples. Sometimes Alleles determined with capillary electrophoresis arent even found by NGS and sometimes NGS finds more alleles for the same marker and the same sample than capillary electrophoresis. The StrResults.txt for example shows up 4 possible alleles and takes those with the highest depth value which is OK but someimes it only takes the one allele with the highest depth value ignoring the ones with slightly lower depth... I just dont get it...


    Sorry for my bad english

  • #2
    1. Have you tried with the reverse-complement sequence of your target?
    2. It should be related with threshold setting of the UAS software suite on FGx, I highly recommend you to read its user guide through first (Universal Analysis
    Software Guide). You can easily download it on illumina's website or ask for help via tech support hotline.

    Comment


    • #3
      Thx for your quick reply :-)

      1. Thx. Haven't tried that so far. I'll try today.
      2. Yes... that's what I suggested... I have to speak with our Guy who sets up the FGx

      Comment


      • #4
        Originally posted by SOLiDance View Post
        1. Have you tried with the reverse-complement sequence of your target?
        I deleted all lines with @ and + and the quality line with sed, reversed the sequences with | rev and created the complementary sequene with sed. (so far nothing wrong?)
        Now my grep finds more than the StrResults.txt tells me...
        StrResults.txt says there are 23 times the sequence with depth=16 and 78 times the sequence with depth=17 but I found 114 times depth 16 and 89 tomes depth 17...

        Any further ideas?

        Comment


        • #5
          Another, but actually the main problem is:

          The DNA-Profile of a sample clearly shows that one sample has the alleles 17/19 (capillary electrophoresis) for the marker D22S1045 but NGS only reports 17/17. NGS doesn't find the 19 allele at all...

          The same for an other sample where 17/17.3 (capillary electrophoresis) are the alleles shown in the DNA-Profile for D1S1656 but only 16 and 17 is found by NGS.... Why???
          Last edited by iltisanni; 03-31-2017, 04:04 AM.

          Comment

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