Question: Cuffdiff Problem
0
gravatar for abhi4.amity
5.8 years ago by
abhi4.amity0 wrote:
cuffdiff -o /storage/shared/abhishek/cuff_diff_24hr_out /storage/home/abhishek/rough_rice/Oryza_sativa.IRGSP-1.0.20.gtf /storage/shared/abhishek/HC24_out.sam /storage/shared/abhishek/HT24_out.sam

Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File /storage/shared/abhishek/HC24_out.sam doesn't appear to be a valid BAM file, trying SAM...
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File /storage/shared/abhishek/HT24_out.sam doesn't appear to be a valid BAM file, trying SAM...
[10:19:01] Loading reference annotation.
Warning: No conditions are replicated, switching to 'blind' dispersion method
[10:19:07] Inspecting maps and determining fragment length distributions.

Error: this SAM file doesn't appear to be correctly sorted!
current hit is at 3:28691269, last one was at 3:28691315
Cufflinks requires that if your file has SQ records in the SAM header that they appear in the same order as the chromosomes names in the alignments.
If there are no SQ records in the header, or if the header is missing, the alignments must be sorted lexicographically by chromosome name and by position.
error • 4.3k views
ADD COMMENTlink modified 5.8 years ago by Fabio Marroni2.3k • written 5.8 years ago by abhi4.amity0
2

There is something wrong with your input BAM file as mentioned in the error. Make sure the chromosome order in the SAM header follows the same order as chromosome appear in rest of your SAM file or vice-versa. I would just use Picard to sort my SAM file. Additionally, you can use Picard to validate your SAM (http://picard.sourceforge.net/command-line-overview.shtml#ValidateSamFile) (Set Validation_strinegncy =LENIENT while checking).

ADD REPLYlink written 5.8 years ago by Ashutosh Pandey11k
0
gravatar for Fabio Marroni
5.8 years ago by
Fabio Marroni2.3k
Italy
Fabio Marroni2.3k wrote:

Also, the first error you get seems to be "EOF marker is absent. The input is probably truncated.". Maybe your .bam/.sam file is truncated. You need to recreate it. Also see: error: EOF marker is absent when processing a BAM file

ADD COMMENTlink modified 5.8 years ago • written 5.8 years ago by Fabio Marroni2.3k
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