tophat2 alignment problems

[rakscalloni]

Hi!

I proceeded my RNA-seq sample alignment using tophat2. A folder was created containing some files, but the files "accepted_hits.bam" and "unmapped.bam" are missing. In my sample folder I only have:
two folders: "logs" and "tmp"
four files: "deletions.bed", "insertions.bed", "junctions.bed" "prep_reads.info".

I ran the alignment again and the same thing happened. Does anybody have the same problem or knows why this is happening?

The alignment process generated this message in the Terminal window:
[2014-05-26 10:38:52] Reporting output tracks
[FAILED]
Error running /usr/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir MSCd0Adip/ --max-multihits 20 --max-seg-multihits 40 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p5 --gtf-annotations Homo_sapiens.GRCh37.75.gtf --gtf-juncs MSCd0Adip/tmp/Homo_sapiens.juncs --no-closure-search --no-coverage-search --no-microexon-search --sam-header MSCd0Adip/tmp/Hsa_GRCh37_75_genome.bwt.samheader.sam --report-discordant-pair-alignments --report-mixed-alignments --samtools=/usr/bin/samtools --bowtie2-max-penalty 6 --bowtie2-min-penalty 2 --bowtie2-penalty-for-N 1 --bowtie2-read-gap-open 5 --bowtie2-read-gap-cont 3 --bowtie2-ref-gap-open 5 --bowtie2-ref-gap-cont 3 MSCd0Adip/tmp/Hsa_GRCh37_75.fa MSCd0Adip/junctions.bed MSCd0Adip/insertions.bed MSCd0Adip/deletions.bed MSCd0Adip/fusions.out MSCd0Adip/tmp/accepted_hits MSCd0Adip/tmp/left_kept_reads.bam
Loaded 375801 GFF junctions from MSCd0Adip/tmp/Homo_sapiens.juncs.


Thanks

[mastal]

Are you getting any error messages?

Where is the tophat stdout/stderr output going?

You should have a log file that says what the last command executed was.

[rakscalloni]

I took a look in the log file. Below I pasted its content. There is an error message at the end... However, I don't know what it means... I am a beginner in RNA-seq analysis...


[2014-05-26 10:11:36] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2014-05-26 10:11:36] Checking for Bowtie
Bowtie version: 2.1.0.0
[2014-05-26 10:11:36] Checking for Samtools
Samtools version: 0.1.19.0
[2014-05-26 10:11:36] Checking for Bowtie index files (genome)..
[2014-05-26 10:11:36] Checking for reference FASTA file
Warning: Could not find FASTA file Hsa_GRCh37_75.fa
[2014-05-26 10:11:36] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/bin/bowtie2-inspect Hsa_GRCh37_75 > MSCd0Adip/tmp/Hsa_GRCh37_75.fa
[2014-05-26 10:13:40] Generating SAM header for Hsa_GRCh37_75
format: fastq
quality scale: phred33 (default)
[2014-05-26 10:13:41] Reading known junctions from GTF file
[2014-05-26 10:13:56] Preparing reads
left reads: min. length=76, max. length=76, 8756 kept reads (616 discarded)
[2014-05-26 10:13:56] Building transcriptome data files..
[2014-05-26 10:14:29] Building Bowtie index from Homo_sapiens.GRCh37.75.fa
[2014-05-26 10:33:09] Mapping left_kept_reads to transcriptome Homo_sapiens.GRCh37.75 with Bowtie2
[2014-05-26 10:33:43] Resuming TopHat pipeline with unmapped reads
[2014-05-26 10:33:43] Mapping left_kept_reads.m2g_um to genome Hsa_GRCh37_75 with Bowtie2
[2014-05-26 10:33:48] Mapping left_kept_reads.m2g_um_seg1 to genome Hsa_GRCh37_75 with Bowtie2 (1/3)
[2014-05-26 10:33:49] Mapping left_kept_reads.m2g_um_seg2 to genome Hsa_GRCh37_75 with Bowtie2 (2/3)
[2014-05-26 10:33:50] Mapping left_kept_reads.m2g_um_seg3 to genome Hsa_GRCh37_75 with Bowtie2 (3/3)
[2014-05-26 10:33:51] Searching for junctions via segment mapping
[2014-05-26 10:35:04] Retrieving sequences for splices
[2014-05-26 10:36:16] Indexing splices
[2014-05-26 10:36:48] Mapping left_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/3)
[2014-05-26 10:37:05] Mapping left_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/3)
[2014-05-26 10:37:23] Mapping left_kept_reads.m2g_um_seg3 to genome segment_juncs with Bowtie2 (3/3)
[2014-05-26 10:37:40] Joining segment hits
[2014-05-26 10:38:52] Reporting output tracks
[FAILED]
Error running /usr/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir MSCd0Adip/ --max-multihits 20 --max-seg-multihits 40 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p5 --gtf-annotations Homo_sapiens.GRCh37.75.gtf --gtf-juncs MSCd0Adip/tmp/Homo_sapiens.juncs --no-closure-search --no-coverage-search --no-microexon-search --sam-header MSCd0Adip/tmp/Hsa_GRCh37_75_genome.bwt.samheader.sam --report-discordant-pair-alignments --report-mixed-alignments --samtools=/usr/bin/samtools --bowtie2-max-penalty 6 --bowtie2-min-penalty 2 --bowtie2-penalty-for-N 1 --bowtie2-read-gap-open 5 --bowtie2-read-gap-cont 3 --bowtie2-ref-gap-open 5 --bowtie2-ref-gap-cont 3 MSCd0Adip/tmp/Hsa_GRCh37_75.fa MSCd0Adip/junctions.bed MSCd0Adip/insertions.bed MSCd0Adip/deletions.bed MSCd0Adip/fusions.out MSCd0Adip/tmp/accepted_hits MSCd0Adip/tmp/left_kept_reads.bam
Loaded 375801 GFF junctions from MSCd0Adip/tmp/Homo_sapiens.juncs.