Cuffdiff contrast run error: "Malformed record in sample sheet" and "cuffdiff requires at least 2 SAM files"

Question

[jammydodger123456]

I have 7 different conditions; Egg, Met, NEJ1hr, NEJ3hr, NEJ24hr, Juvenile and Adult. I want to compare Egg to all other stages individually and each stage with the one that is immediately after it ie Met-NEJ1hr, NEJ1hr-NEJ3hr etc. Following suggestions on other posts, I have tried;

cuffdiff --use-sample-sheet -C contrasts.txt -p 10 ../PRJEBWB9PAULAnnotations.gff3 sample_sheet.txt

My sample sheet;

 sample_id       group_label
../EggB1/tophat_out/accepted_hits.bam   Egg
../EggB2/tophat_out/accepted_hits.bam   Egg
../MetB1/tophat_out/accepted_hits.bam   Met
../MetB2/tophat_out/accepted_hits.bam   Met
../MetB3/tophat_out/accepted_hits.bam   Met
../MetB4/tophat_out/accepted_hits.bam   Met
../NEJ1hrB1/tophat_out/accepted_hits.bam   NEJ1hr
../NEJ3hrB1/tophat_out/accepted_hits.bam   NEJ3hr
../NEJ3hrB2/tophat_out/accepted_hits.bam   NEJ3hr
../NEJ24hrB1/tophat_out/accepted_hits.bam   NEJ24hr
../NEJ24hrB2/tophat_out/accepted_hits.bam   NEJ24hr
../JuvenileB1/tophat_out/accepted_hits.bam   Juvenile
../JuvenileB2/tophat_out/accepted_hits.bam   Juvenile
../AdultB1/tophat_out/accepted_hits.bam   Adult
../AdultB2/tophat_out/accepted_hits.bam   Adult
../AdultB3/tophat_out/accepted_hits.bam   Adult

my contrasts.txt file;

condition_A     condition_B
Egg     Met
Egg     NEJ1hr
Egg     NEJ3hr
Egg     NEJ24hr
Egg     Juvenile
Egg     Adult
Met     NEJ1hr
NEJ1hr  NEJ3hr
NEJ3hr  NEJ24hr
NEJ24hr Juvenile
Juvenile        Adult

When I try to run this, I get the errors;

Malformed record in sample sheet:
   >  ../EggB2/tophat_out/accepted_hits.bam   Egg

For each of the reps. There is also the error;

Error: cuffdiff requires at least 2 SAM files

Has anyone come across these type of errors?

Answer 1

[Satyajeet Khare]

Can you just check that this samples is in correct format?

../EggB2/tophat_out/accepted_hits.bam   Egg

I mean, with single tab separation etc? Looks like this sample is not getting recognized because of which you are getting both errors.

[jammydodger123456]

Based on your suggestion, I renamed all of the bam files and moved them all into the same working directory and this has fixed it. My sample_sheet.txt now looks like this;

sample_id       group_label
EggB1.bam       Egg
EggB2.bam       Egg
MetB1.bam       Met
MetB2.bam       Met
MetB3.bam       Met
MetB4.bam       Met
NEJ1hrB1.bam    NEJ1hr
NEJ3hrB1.bam    NEJ3hr
NEJ3hrB2.bam    NEJ3hr
NEJ24hrB1.bam   NEJ24hr
NEJ24hrB2.bam   NEJ24hr
JuvenileB1.bam  Juvenile
JuvenileB2.bam  Juvenile
AdultB1.bam     Adult
AdultB2.bam     Adult
AdultB3.bam     Adult