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  • Tophat only maps a few reads

    Hello everyone,

    I use the data from the sra website.
    for executing Tophat I use the command:
    Code:
    tophat2 -a 5 -m 2 -N 10 -x 1 --read-edit-dist 12 -o <outDir> <reference genome> SRR493747.fastq
    Tophat gives no warnings:
    Code:
    2013-05-16 13:52:13] Beginning TopHat run (v2.0.6)
    -----------------------------------------------
    [2013-05-16 13:52:13] Checking for Bowtie
      Bowtie 2 not found, checking for older version..
                      Bowtie version:        0.12.7.0
    [2013-05-16 13:52:13] Checking for Samtools
                    Samtools version:        0.1.8.0
    [2013-05-16 13:52:13] Checking for Bowtie index files
    [2013-05-16 13:52:13] Checking for reference FASTA file
    [2013-05-16 13:52:13] Generating SAM header for /home/fedor/GENOMES/H_sapiens/H_sapiens_GRCh37
            format:          fastq
            quality scale:   phred33 (default)
    [2013-05-16 13:52:33] Preparing reads
             left reads: min. length=50, max. length=50, 999961 kept reads (39 discarded)
    [2013-05-16 13:52:46] Mapping left_kept_reads to genome H_sapiens_GRCh37 with Bowtie 
    [2013-05-16 14:03:40] Mapping left_kept_reads_seg1 to genome H_sapiens_GRCh37 with Bowtie (1/2)
    [2013-05-16 14:13:51] Mapping left_kept_reads_seg2 to genome H_sapiens_GRCh37 with Bowtie (2/2)
    [2013-05-16 14:22:17] Searching for junctions via segment mapping
    [2013-05-16 14:25:39] Retrieving sequences for splices
    [2013-05-16 14:28:49] Indexing splices
    [2013-05-16 14:28:49] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie (1/2)
    [2013-05-16 14:29:10] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie (2/2)
    [2013-05-16 14:29:33] Joining segment hits
    [2013-05-16 14:31:39] Reporting output tracks
    -----------------------------------------------
    [2013-05-16 14:33:46] Run complete: 00:41:32 elapsed
    The output of samtools flagstat on accepted_hits.bam:
    Code:
    5 in total
    0 QC failure
    0 duplicates
    25 mapped (100.00%)
    0 paired in sequencing
    0 read1
    0 read2
    0 properly paired (nan%)
    0 with itself and mate mapped
    0 singletons (nan%)
    0 with mate mapped to a different chr
    0 with mate mapped to a different chr (mapQ>=5)
    The output of unmapped.bam:
    Code:
    999993 in total
    39 QC failure
    0 duplicates
    0 mapped (0.00%)
    0 paired in sequencing
    0 read1
    0 read2
    0 properly paired (nan%)
    0 with itself and mate mapped
    0 singletons (nan%)
    0 with mate mapped to a different chr
    0 with mate mapped to a different chr (mapQ>=5)
    What did I do wrong, because this is a very low mapping percentage...

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