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Old 06-04-2013, 01:32 AM   #1
Location: beijing,china

Join Date: May 2013
Posts: 11
Question How to deal with multiple accepted_hits.bam file as input into cuffdiff?

Hello All,

Because my data include paired-end reads and single-end reads, so i run the tophat twice as manual recommened. Aslo, two bam files result from only one sample, were produced by Tophat.
I use picard tools to sort two bam files and merge into one sorted_merged.bam file. Then , Cuffdiff was used to perform downstream DE genes analysis. error message appears like this,

File ./combined_thout/sorted_merged.sam doesn't appear to be a valid BAM file, trying SAM...
Error: sort order of reads in BAMs must be the same

The sorted bam files produced by picard were incompatible with other bam files provided by tophat.

Has anyone deal with this condition? Less information about data from different library type were mentioned in cufflink protocol.


Last edited by canhu; 06-04-2013 at 03:33 AM. Reason: lack of some important information
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Old 06-07-2015, 06:17 AM   #2
Location: Pakistan

Join Date: Apr 2015
Posts: 11
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Hi there, I hope you have resolved your issue.

I am using pair-end reads from four samples (one control, three treatments). I aligned both right and left files together. I managed through tophat2. Now I have four accepted_hits.bam files.

How do I proceed with my analysis?. Should I run cuffcompare or directly jump into cuffdiff2.

I need pair-wise Differential analysis, control vs m1, control vs m2, control vs m3, m1 vs m2, m1 vs m3, m2 vs m3.

I will use cummerbund for my analysis..
mfahim is offline   Reply With Quote

cuffdiff, multiple bam file, tophat

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