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Old 05-16-2013, 07:10 AM   #1
Jetse
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Location: The netherlands

Join Date: Nov 2012
Posts: 38
Default 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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