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  • Bowtie2 alignment problem

    Hi,

    when I align the following four identical Fastq entries (identical except for identifiers):

    @C003-U0418-F000014195/1
    GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA
    +
    -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
    @U0418-F000014195:1
    GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA
    +
    -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
    @U0418-F000014195/1
    GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA
    +
    -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
    @U0418-F000014195:2
    GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA
    +
    -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
    @C003-U0418-F000014195-2
    GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA
    +
    -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG


    against Ensembl transcripts I get the following result:

    C003-U0418-F000014195/1 4 * 0 0 * * 0 0 GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG YT:Z:UU
    U0418-F000014195:1 0 ENST00000369815 549 255 101M * 0 0 GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG AS:i:-10 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:72G0G27 YT:Z:UU
    U0418-F000014195/1 0 ENST00000337003 483 255 101M * 0 0 GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG AS:i:-10 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:72G0G27 YT:Z:UU
    U0418-F000014195:2 0 ENST00000369811 558 255 101M * 0 0 GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG AS:i:-10 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:72G0G27 YT:Z:UU
    C003-U0418-F000014195-2 0 ENST00000337003 483 255 101M * 0 0 GTTCCCATGCCTGGAGAAGCTAATGCCAACTCATCATGTGATAATTCAATTTGTACAATAAATTATGAACCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA -@1ACGDF=88D9@ECGGCGCGGFFG<FGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG AS:i:-10 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:72G0G27 YT:Z:UU


    So one Fastq entry does not align and the other four entries align uniquely against three different transcripts although I explicitly allow multiple alignments. The Bowtie2 call is as follows:

    @PG ID:bowtie2 PN:bowtie2 VN:2.2.6 CL:"/home/schuisv1/ngs/scRNA-seq/K562-Fluidigm-test/exon-pipeline-scripts/tools/bowtie2-align-s --wrapper basic-0 -p 4 --reorder -k 2000 --phred33 --score-min L,0,-0.1 -x /home/schuisv1/ngs/scRNA-seq/K562-Fluidigm-test/exon-pipeline-files/fasta-files/ensembl_rna_hs-bowtie2Index/bowtie2Index -U C003-U0418-F000014195.fastq"

    Has anybody seen such a behavior of Bowtie2 before?

    Best regards,

    Sven
    Last edited by sven.schuierer; 03-22-2016, 02:14 AM.

  • #2
    You seem to have polyA tails on your reads...perhaps trimming those first might help.

    Comment


    • #3
      Yes, you are right. If I remove the polyA tails, then I get the same five alignments for each read.

      Still I think this is a strange behavior - in particular, the fact that the alignment depends on the identifier (with the polyA tails).

      Comment

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