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  • Problem somewhere during paired-end seq analysis -> no reads mapped

    Hello everybody,

    this is my fist time to try to handle to analyze paired-end sequencing files.
    Finally BamCoverage complained there are no mapped reads in my Bam file. Also IGV doesn't show any reads.
    I really don't know, where i lost my reads.
    Here is what i did:

    # Quality an adaptor trimming with trim galore:

    trim_galore ../paired_1.fastq ../paired_2.fastq -q 20 --paired --phred33

    # Mapping with bowtie

    bowtie_opts="-p 4 -m 3 -S"
    bowtie_index="path/6008.5_BAC.ebwt"
    bowtie $bowtie_opts $bowtie_index -I 0 -X 700 -1 ../paired_1 .fq -2 ../paired_2.fq > paired.SAM

    # Mapping report:
    # reads processed: 4743277
    # reads with at least one reported alignment: 1979307 (41.73%)
    # reads that failed to align: 2758162 (58.15%)
    # reads with alignments suppressed due to -m: 5808 (0.12%)
    Reported 1979307 paired-end alignments to 1 output stream(s)

    (-> 42 % of mapped reads might seem problematic. Since the sequenced plasmid was extracted from E.coli, there is a lot of contamination.)

    # SAMs to BAMs

    samtools view -bS ../paired.SAM > paired.BAM
    samtools index paired.BAM


    # BamCoverage
    BamCoverage -b paired.BAM -o BamCoverage/paired.bw

    This does not work and results in the following error message:

    "Samtools reports that the number of mapped reads is zero for the file paired.BAM. Please check that the file is properly indexed and that it contains mapped reads."

    When i google for this error message i cannot find a helpful answer. Maybe someone knows what is wrong. Since 1979307 reads could be mapped, i don't get why zero reads should be mapped.
    Thanks a lot, Alex

  • #2
    I got it on my own, after a short break. I forgot to sort the bam files...

    Comment


    • #3
      One point - it looks like you are using bowtie for alignment, which is obsolete for most purposes. Bowtie2 is a much more accurate aligner, and can detect indels. BWA is another great alternative, as is (commercial) Novoalign.

      Comment


      • #4
        Thanks for the hint. My adviser told me it is sufficient to use Bowtie 1.
        I tested Bowtie 2 now and i got 10 % more alligned reads. For another sample even 23 %, which is really a lot!
        Thanks again!

        Comment


        • #5
          Let's not forget BBMap as a good non-commercial alternative.

          Comment


          • #6
            @Alex852013: A large % of reads is still failing to align (if you are only getting 23% of reads to align) so you may want to investigate why that is so. Quick blasting @NCBI should give a clue as to whether you data has unexpected contamination.

            Comment


            • #7
              Originally posted by GenoMax View Post
              @Alex852013: A large % of reads is still failing to align (if you are only getting 23% of reads to align) so you may want to investigate why that is so. Quick blasting @NCBI should give a clue as to whether you data has unexpected contamination.
              Didn't he say 23% more not 23% total? In any case the original post talked about mapping a mixture of plasmid and E.coli reads against the plasmid and thus he expected a low percentage.
              Last edited by westerman; 01-07-2016, 10:13 AM. Reason: Added question mark.

              Comment


              • #8
                I missed that fact from the original post. @Rick: Thanks for pointing that out. If true #6 can be safely ignored.

                Comment


                • #9
                  Yes, it meant 23 % more, which makes 96 % in total. Perfect!
                  Nevertheless, thanks for caring!

                  Comment

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