bioinfo@bioinfo-nrcb:~$ tophat --output-dir Tophat/out --num-threads 6 --read-mismatches 2 --read-edit-dist 2 --read-realign-edit-dist 1000 -a 8 -m 0 -i 70 -I 500000 -g 20 --min-segment-intron 50 --max-segment-intron 500000 --segment-mismatches 2 --segment-length 25 --library-type fr-unstranded --max-insertion-length 3 --max-deletion-length 3 --b2-sensitive Tophat/musa_acuminata_v1_cds.fa Tophat/SRR912644.fastqsanger
[2018-02-21 17:08:36] Beginning TopHat run (v2.0.9)
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[2018-02-21 17:08:36] Checking for Bowtie
Bowtie version: 2.1.0.0
[2018-02-21 17:08:36] Checking for Samtools
Samtools version: 0.1.19.0
[2018-02-21 17:08:36] Checking for Bowtie index files (genome)..
[2018-02-21 17:08:36] Checking for reference FASTA file
[2018-02-21 17:08:36] Generating SAM header for Tophat/musa_acuminata_v1_cds.fa
format: fastq
quality scale: phred33 (default)
[2018-02-21 17:08:38] Preparing reads
left reads: min. length=49, max. length=49, 4910516 kept reads (517 discarded)
[2018-02-21 17:09:05] Mapping left_kept_reads to genome musa_acuminata_v1_cds.fa with Bowtie2
[2018-02-21 17:10:58] Mapping left_kept_reads_seg1 to genome musa_acuminata_v1_cds.fa with Bowtie2 (1/2)
[2018-02-21 17:11:28] Mapping left_kept_reads_seg2 to genome musa_acuminata_v1_cds.fa with Bowtie2 (2/2)
[2018-02-21 17:11:51] Searching for junctions via segment mapping
Coverage-search algorithm is turned on, making this step very slow
Please try running TopHat again with the option (--no-coverage-search) if this step takes too much time or memory.
[2018-02-21 17:12:48] Retrieving sequences for splices
[2018-02-21 17:12:49] Indexing splices
[2018-02-21 17:12:49] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/2)
[2018-02-21 17:12:58] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/2)
[2018-02-21 17:13:05] Joining segment hits
[2018-02-21 17:13:16] Reporting output tracks
[FAILED]
Error running /usr/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 70 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir Tophat/out/ --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 1000 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p6 --no-closure-search --no-microexon-search --library-type fr-unstranded --sam-header Tophat/out/tmp/musa_acuminata_v1_cds.fa_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 Tophat/musa_acuminata_v1_cds.fa.fa Tophat/out/junctions.bed Tophat/out/insertions.bed Tophat/out/deletions.bed Tophat/out/fusions.out Tophat/out/tmp/accepted_hits Tophat/out/tmp/left_kept_reads.bam
Loading ...done
Can anybody clarify and solve this error......................
Thankyou.....
I think it's Memory problem.
U can follow this link for alternate commands
doi:10.1038/nprot.2012.016
Have you tried to run TopHat with the following option --no-coverage-search, as suggested by the log file (to check if it is a problem of computation power)? Do you have any resulting file? As suggested by the log file, only "Reporting output tracks" is failing and "Loading ...done" after seems ok.
Also, I think that you should strongly consider using something else than TopHat that is an old algorithm that is not maintained anymore and that is not nearly as efficient as new algorithms like STAR or HISAT2. TopHat's website state this:
Please note that TopHat has entered a low maintenance, low support stage as it is now largely superseded by HISAT2 which provides the same core functionality (i.e. spliced alignment of RNA-Seq reads), in a more accurate and much more efficient way.
Source: https://ccb.jhu.edu/software/tophat/index.shtml
For RNA-seq experiments, I recommend using STAR for alignements, FeatureCounts for counting reads / feature, and then you can follow the steps described in this paper for DEG Analysis:
https://f1000research.com/articles/5-1408/v2
Alternatively, you can use the Snakemake Worflow described here to run a complete RNA-seq pipeline from FASTQ files to the DEG analysis result (up-regulated genes, down-regulated genes list, graphs, etc):
https://github.com/maxplanck-ie/snakemake_workflows
Documentation is available here for the RNA-seq pipeline:
https://snakepipes.readthedocs.io/en/latest/content/workflows/RNA-seq.html#rna-seq