Question: Tophat unable to produce accepted_hits.bam file for single end read data
gravatar for nikhilvgbt
4.4 years ago by
nikhilvgbt0 wrote:

I have been working on RNA-Seq data from last 6 months, and i am able to crate accepted_hits.bam file for paired end data, but i am not able to create the accepted_hits.bam file for single end data using Tophat.

The commad line i used for single end data is:

Tophat -p 12 -G hg19.gtf -o C1_R1_thout genome SRR154213.fastq

Tophat -p 12 -G hg19.gtf -o C1_R2_thout genome SRR154214.fastq

after running this command i got the accepted_hits.bam which is of 600 byte size file which is not correct aligned file..

please let me know if i have to change the command line arguments ..

Thank you



rna-seq tophat • 1.8k views
ADD COMMENTlink modified 4.4 years ago • written 4.4 years ago by nikhilvgbt0

You presumably received an error message at some point...

ADD REPLYlink written 4.4 years ago by Devon Ryan95k

There was no error while running tophat, the Tophat ran successfully for 2 hours using above command ..

ADD REPLYlink written 4.4 years ago by nikhilvgbt0

Even if the bam wasn't written, a log should have been, what was contained in that?

ADD REPLYlink modified 4.4 years ago • written 4.4 years ago by andrew.j.skelton735.9k

Hello andrew.j.skelton73 and Devon Ryan sir,

This is my log file content which tells about successfully completion of alignment, but still the accepted_hit.bam is of 1kb

[2016-01-22 00:57:24] Beginning TopHat run (v2.0.9)
[2016-01-22 00:57:24] Checking for Bowtie
          Bowtie version:
[2016-01-22 00:57:24] Checking for Samtools
        Samtools version:
[2016-01-22 00:57:24] Checking for Bowtie index files (genome)..
[2016-01-22 00:57:24] Checking for reference FASTA file
[2016-01-22 00:57:24] Generating SAM header for genome
    format:         fastq
    quality scale:     phred33 (default)
[2016-01-22 00:57:36] Reading known junctions from GTF file
[2016-01-22 00:57:40] Preparing reads
     left reads: min. length=49, max. length=49, 11503394 kept reads (146 discarded)
[2016-01-22 00:59:30] Building transcriptome data files..
[2016-01-22 00:59:50] Building Bowtie index from genes.fa
[2016-01-22 01:14:33] Mapping left_kept_reads to transcriptome genes with Bowtie2
[2016-01-22 01:16:33] Resuming TopHat pipeline with unmapped reads
[2016-01-22 01:16:33] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2
[2016-01-22 01:24:32] Mapping left_kept_reads.m2g_um_seg1 to genome genome with Bowtie2 (1/2)
[2016-01-22 01:28:55] Mapping left_kept_reads.m2g_um_seg2 to genome genome with Bowtie2 (2/2)
[2016-01-22 01:31:12] Searching for junctions via segment mapping
[2016-01-22 01:39:45] Retrieving sequences for splices
[2016-01-22 01:41:38] Indexing splices
[2016-01-22 01:42:18] Mapping left_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/2)
[2016-01-22 01:43:40] Mapping left_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/2)
[2016-01-22 01:45:57] Joining segment hits
[2016-01-22 01:49:19] Reporting output tracks
[2016-01-22 01:53:16] A summary of the alignment counts can be found in C1_R1_tophout/align_summary.txt
[2016-01-22 01:53:16] Run complete: 00:55:52 elapsed
ADD REPLYlink modified 5 months ago by RamRS27k • written 4.4 years ago by nikhilvgbt0

What are the contents of "C1_R1_tophout/align_summary.txt"? If it says that you should have alignments then try producing an unsorted SAM file rather than the sorted BAM file (i.e., use --no-convert-bam). That unsorted SAM file is actually produced first anyway, so this should be faster and allow you to start narrowing down where the error is occurring.

ADD REPLYlink written 4.4 years ago by Devon Ryan95k
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