Cuffmerge and cuffdiff error

Question

[carrilloarzola3]

I am trying to use cuffmerge in order to merge 4 transcript assemblies that I have obtained through cufflinks. The data has worked through tophat, qualimap, and cufflinks but when using cuffmerge and cuffdiff I seem to run into these issues. I am obtaining these warning on cuffmerge and some of them mention .bam which is unusual considering I have no bam file in my input. After running cuffmerge I am able to obtain a merged_asm file but when I attempt to run cuffdiff with said file I get a segmentation error and no output this time. Would anyone possibly know how to fix these errors? Thanks in advance

mconesa:cufflinks-2.2.1.OSX_x86_64 mcs19$ ./cuffmerge -g /Users/mcs19/Desktop/Bowtie_IndexFiles/Enterobacter_cloacae_subsp_cloacae_atcc_13047.ASM2556v1.41.chromosome.Chromosome.gff3 /Users/mcs19/Documents/assemblies.txt 

[Fri Oct 19 14:18:31 2018] Beginning transcriptome assembly merge
-------------------------------------------

[Fri Oct 19 14:18:31 2018] Preparing output location ./merged_asm/
[Fri Oct 19 14:18:32 2018] Converting GTF files to SAM
[14:18:32] Loading reference annotation.
[14:18:32] Loading reference annotation.
[14:18:32] Loading reference annotation.
[14:18:32] Loading reference annotation.
[Fri Oct 19 14:18:32 2018] Quantitating transcripts
Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g /Users/mcs19/Desktop/Bowtie_IndexFiles/Enterobacter_cloacae_subsp_cloacae_atcc_13047.ASM2556v1.41.chromosome.Chromosome.gff3 -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 1 ./merged_asm/tmp/mergeSam_tmp.4.OI3WK5 
[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 ./merged_asm/tmp/mergeSam_tmp.4.OI3WK5 doesn't appear to be a valid BAM file, trying SAM...
[14:18:32] Loading reference annotation.
[14:18:33] Inspecting reads and determining fragment length distribution.
Processed 268 loci.                         
> Map Properties:
>   Normalized Map Mass: 5844.00
>   Raw Map Mass: 5844.00
>   Fragment Length Distribution: Truncated Gaussian (default)
>                 Default Mean: 200
>              Default Std Dev: 80
[14:18:33] Assembling transcripts and estimating abundances.
Processed 268 loci.                         
[Fri Oct 19 14:18:42 2018] Comparing against reference file /Users/mcs19/Desktop/Bowtie_IndexFiles/Enterobacter_cloacae_subsp_cloacae_atcc_13047.ASM2556v1.41.chromosome.Chromosome.gff3
Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).
[Fri Oct 19 14:18:44 2018] Comparing against reference file /Users/mcs19/Desktop/Bowtie_IndexFiles/Enterobacter_cloacae_subsp_cloacae_atcc_13047.ASM2556v1.41.chromosome.Chromosome.gff3
Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).

mconesa:cufflinks-2.2.1.OSX_x86_64 mcs19$ ./cuffdiff -o SV_Samples_Output /Users/mcs19/Bioinformatics_Project/cufflinks-2.2.1.OSX_x86_64/merged_asm/merged.gtf /Users/mcs19/Bioinformatics_Project/SV1_results/accepted_hits_SV1.bam /Users/mcs19/Bioinformatics_Project/SV2_results/accepted_hits_SV2.bam /Users/mcs19/Bioinformatics_Project/SV3_results/accepted_hits_SV3.bam /Users/mcs19/Bioinformatics_Project/SV4_results/accepted_hits_SV4.bam
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.
[13:39:46] Loading reference annotation.
Warning: No conditions are replicated, switching to 'blind' dispersion method
[13:39:47] Inspecting maps and determining fragment length distributions.
Segmentation fault: 11

Answer 1

[Philipp Bayer]

What's in your assemblies.txt? I have a hunch that your original RNASeq alignments never finished, as all of your software complains about missing end-of-file markers. You can check your input bam files using

 samtools view your_alignment.bam > /dev/null

if it still complains about EOF missing then your original RNASeq alignments never finished.

(You may also want to switch over to the newer replacement pipeline for tophat/cufflinks for the same authors' new pipeline, https://www.nature.com/articles/nprot.2016.095 )