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  • Odd sequences in RNAseq fastq

    Hi all,

    Recently, I just ran iSeq for pair ended RNA sequencing and it gave me odd fastq results
    Some of Read1(i7) sequences have Adapter sequences with poly G at the end.

    @FS10000436:35:BPC29621-1807:1:1101:10010:1370 1:N:0:1
    GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCGCGAAATCTCGTATGCCGTCTTCTGCTTGAAAAAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
    +
    FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFF:FFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

    However paired Read2(i5) sequences gave me random and poor quality sequences
    @FS10000436:35:BPC29621-1807:1:1101:10010:1370 2:N:0:1
    GGGGGGGGGAATTTTTTTTTTTTAAAAAATATTTTTTTTTCTTTTTTTTTTTTTTTTTTCCCTTTTTTTTTTTATATTTTTTTTTTTTTTTTTATATTTTT
    +
    F:,,,,,,,,,,,F,,:,:FFF,,,::FF,,,,FFF,,,,,:,FFF::FF::,F::F,,,,,,,F,FF::::,,,,,,::F:FFFFFFF,F,,,,,,,,,:

    I guess this problem came from library length which is shorter than sequencing length. However, I can't understand why Read2 sequence doesn't show matched short sequences (like adapter with G sequences) but random sequences. Is it cluster problems by short sequences?

    Thanks in advance.
    Last edited by Kujin Kwon; 11-20-2019, 02:39 AM.

  • #2
    I take it these are NextSeq or NovaSeq?

    This is an adapter dimer that's read through the p7 sequence (ATCTCGTATGCCGTCTTCTGCTTG) into the FC and started spitting out Gs because no template = no signal = G in 2 color chemistry

    Comment


    • #3
      OP says these are from iSeq.

      Comment


      • #4
        Whoops. Same conclusion though. Gs are still the dark channel in the iSeq chemistry

        Comment


        • #5
          Thanks for the answer @cmbetts

          no template = no signal = G in 2 color chemistry
          Then, random sequence in read2 is also signal of no template?

          Comment

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