Need urgent help for Tophat + Cufflinks !!!

[fangquan]

Hi folks,

I'm running tophat (tophat -o output_dir a_ref a.fq) for alignment and cufflinks (cufflinks -o output_dir accepted_hits.bam) for assembles transcripts. The reads are single-end.

This is my command line:
tophat -o tophat_60PO ./hg19 ./60PO.fastq
cufflinks -o cufflinks_60PO tophat_60PO/accepted_hits.bam

The results:
Step 1:
$ ls -l tophat_60PO
-rw-rw-r-- 1 fangquan fangquan 1104315390 Aug 4 09:45 accepted_hits.bam
-rw-rw-r-- 1 fangquan fangquan 52 Aug 4 09:35 deletions.bed
-rw-rw-r-- 1 fangquan fangquan 54 Aug 4 09:35 insertions.bed
-rw-rw-r-- 1 fangquan fangquan 52 Aug 4 09:35 junctions.bed
-rw-rw-r-- 1 fangquan fangquan 70 Aug 4 05:54 left_kept_reads.info
drwxrwxr-x 2 fangquan fangquan 4096 Aug 4 09:04 logs


The results are weird, deletions.bed, insertions.bed, and junctions.bed are EMPTY files, with only track name.

#############################################
#############################################
Step 2:
$ls -l cufflinks_60PO
-rw-rw-r-- 1 fangquan fangquan 129 Aug 5 14:53 genes.fpkm_tracking
-rw-rw-r-- 1 fangquan fangquan 129 Aug 5 14:53 isoforms.fpkm_tracking
-rw-rw-r-- 1 fangquan fangquan 0 Aug 5 14:53 transcripts.gtf


The results are weird again, three files are all EMPTY. And I tried all ten samples but all end up weird results.



Can anyone help me figure out what's going wrong ? I have ten samples and all of them have the same weird results. Waiting for your replies !

Thanks,
Quan

[liuxq]

what is your reads length?
please try to set --segment-length parameter using half length of the reads

Hi folks,

I'm running tophat (tophat -o output_dir a_ref a.fq) for alignment and cufflinks (cufflinks -o output_dir accepted_hits.bam) for assembles transcripts. The reads are single-end.

This is my command line:
tophat -o tophat_60PO ./hg19 ./60PO.fastq
cufflinks -o cufflinks_60PO tophat_60PO/accepted_hits.bam

The results:
Step 1:
$ ls -l tophat_60PO
-rw-rw-r-- 1 fangquan fangquan 1104315390 Aug 4 09:45 accepted_hits.bam
-rw-rw-r-- 1 fangquan fangquan 52 Aug 4 09:35 deletions.bed
-rw-rw-r-- 1 fangquan fangquan 54 Aug 4 09:35 insertions.bed
-rw-rw-r-- 1 fangquan fangquan 52 Aug 4 09:35 junctions.bed
-rw-rw-r-- 1 fangquan fangquan 70 Aug 4 05:54 left_kept_reads.info
drwxrwxr-x 2 fangquan fangquan 4096 Aug 4 09:04 logs


The results are weird, deletions.bed, insertions.bed, and junctions.bed are EMPTY files, with only track name.

#############################################
#############################################
Step 2:
$ls -l cufflinks_60PO
-rw-rw-r-- 1 fangquan fangquan 129 Aug 5 14:53 genes.fpkm_tracking
-rw-rw-r-- 1 fangquan fangquan 129 Aug 5 14:53 isoforms.fpkm_tracking
-rw-rw-r-- 1 fangquan fangquan 0 Aug 5 14:53 transcripts.gtf


The results are weird again, three files are all EMPTY. And I tried all ten samples but all end up weird results.



Can anyone help me figure out what's going wrong ? I have ten samples and all of them have the same weird results. Waiting for your replies !

Thanks,
Quan

[fangquan]

Thanks,

The read length is 40 bp. I will try out your suggestion.

what is your reads length?
please try to set --segment-length parameter using half length of the reads

[fangquan]

Thanks,

The read length is 40 bp. I will try out your suggestion.

what is your reads length?
please try to set --segment-length parameter using half length of the reads

[chenyao]

Did you sort and index your "accepted_hits.bam" from Tophat?

[fangquan]

Hi, Thanks for your reply, but I thinks it's already sorted. This is what I saw from cufflinks manual.

"The SAM file supplied to Cufflinks must be sorted by reference position. If you aligned your reads with TopHat, your alignments will be properly sorted already. If you used another tool, you may want to make sure they are properly sorted as follows: sort -k 3,3 -k 4,4n hits.sam > hits.sam.sorted
"

Did you sort and index your "accepted_hits.bam" from Tophat?