[Gonza]

Hi all, I have a cuffmerge problem. It says "EOF marker is absent. The input is probably truncated". I have run tophat and cufflinks successfully. What does this mean??
Thanks.
G

[gonzalo@localhost trimmed_reads]$ cuffmerge -g genes.gtf -s genome.fa -p 20 assemblies.txt

[Sat Sep 13 12:33:25 2014] Beginning transcriptome assembly merge
-------------------------------------------

[Sat Sep 13 12:33:25 2014] Preparing output location ./merged_asm/
[Sat Sep 13 12:33:35 2014] Converting GTF files to SAM
[12:33:35] Loading reference annotation.
[12:33:37] Loading reference annotation.
[12:33:40] Loading reference annotation.
[12:33:43] Loading reference annotation.
[12:33:45] Loading reference annotation.
[12:33:48] Loading reference annotation.
[Sat Sep 13 12:33:51 2014] Quantitating transcripts
You are using Cufflinks v2.2.1, which is the most recent release.
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g genes.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 20 ./merged_asm/tmp/mergeSam_fileJRPl29
[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_fileJRPl29 doesn't appear to be a valid BAM file, trying SAM...
[12:33:51] Loading reference annotation.
[12:33:57] Inspecting reads and determining fragment length distribution.
Processed 28060 loci.
Map Properties:
Normalized Map Mass: 154834.00
Raw Map Mass: 154834.00
Fragment Length Distribution: Truncated Gaussian (default)
Default Mean: 200
Default Std Dev: 80
[12:33:59] Assembling transcripts and estimating abundances.
Processed 28060 loci.
[Sat Sep 13 12:34:11 2014] Comparing against reference file genes.gtf
You are using Cufflinks v2.2.1, which is the most recent release.
[Sat Sep 13 12:34:34 2014] Comparing against reference file genes.gtf
You are using Cufflinks v2.2.1, which is the most recent release.

[Brian Bushnell]

That message is a bug in Samtools; don't worry about it.

[Gonza]

Thanks. I ignored it and successfully run tuxedo. However, when loading cuffdiff to R i keep getting this error. I have no idea what this is. Any thiughts on solutions? thanks...

> cuff_data <- readCufflinks()
Creating database /Users/gonzalovillarino/Desktop/05_cuffdiff/cuffData.db
Reading Run Info File /Users/gonzalovillarino/Desktop/05_cuffdiff/run.info
Writing runInfo Table
Reading Read Group Info /Users/gonzalovillarino/Desktop/05_cuffdiff/read_groups.info
Writing replicates Table
Reading /Users/gonzalovillarino/Desktop/05_cuffdiff/genes.fpkm_tracking
Error in scan(file, what, nmax, sep, dec, quote, skip, nlines, na.strings, :
line 1305 did not have 17 elements

> cuff_data
Error: object 'cuff_data' not found
CuffSet instance with:
0 samples
0 genes
0 isoforms
0 TSS
0 CDS
0 promoters
0 splicing
0 relCDS

[Nanu]

When I am running the cuffmerge then i got the following error. I mentioned the .gtf file name in it which was created by cufflinks

[Tue Oct 14 17:08:29 2014] Beginning transcriptome assembly merge
-------------------------------------------

[Tue Oct 14 17:08:29 2014] Preparing output location ./merged_asm/
Warning: no reference GTF provided!
[Tue Oct 14 17:08:31 2014] Converting GTF files to SAM
[FAILED]
Error: gtf_to_sam not found on this system. Did you forget to include it in your PATH?

[Rashedul Islam]

I have solved the problem by adding this line to the .hashrc file.

"export PATH=$PATH:/home/cufflinks-2.2.1.Linux_x86_64"

[aniruddha.otago]

Hi there,

I am running cuffmerge and having this problem:

My command:
cuffmerge -g /home/aniruddha/GTF_files/Human_genome/GRCh37_build_65/Homo_sapiens.GRCh37.65.gtf -p 10 /home/aniruddha/PDL1_RNA-Seq_8_AC/Cuffmerge_8PDcelllines/assembled_tc_list.txt

[Sun Jun 26 12:02:08 2016] Beginning transcriptome assembly merge
-------------------------------------------

[Sun Jun 26 12:02:08 2016] Preparing output location ./merged_asm/
[Sun Jun 26 12:02:23 2016] Converting GTF files to SAM
[FAILED]
Error: gtf_to_sam not found on this system. Did you forget to include it in your PATH?

When I see the cufflinks executables I dont see gtf_to_sam .
am I missing something ?


cufflinks-2.2.1.Linux_x86_64]$ ls
AUTHORS README cuffdiff cuffmerge cuffquant gffread isoforms.fpkm_tracking test_data.sam
LICENSE cuffcompare cufflinks cuffnorm genes.fpkm_tracking gtf_to_sam skipped.gtf transcripts.gtf


Thanks for your help.

[Brian Bushnell]

I recommend you avoid the Tuxedo tools. You can get much better results with something else. I won't give a specific alternative because I don't know the goal of your research, but I cannot imagine a question where the Tuxedo suite is the best answer. Or even a good answer.

[aniruddha.otago]

I think I have solved the problem now. For some reason gtf_to_sam tool was missing.
Anyways, for this particular analysis at this stage all I am wanting is to identify differential expression and differentially expressed isoforms. There is hardly any consensus of the best tools to be used. Taxedo suite is off course the kost used one. Anyone have any useful data driven opinion or comments on the use of tools for indetifying DE. ? Also if you have any comments on tools or approaches you think its good for comparing DE of genes with DNA methylation profiles of the same samples. ?
Thanks in advance.

[Brian Bushnell]

DESeq and edgeR are commonly used for differential expression analysis, and both are superior to Tuxedo tools, from what I understand. Cufflinks is incapable of determining that genes specific to the Y chromosome are differentially expressed between human males and females. If that's not sufficient evidence of it being a bad tool, I can't imagine what is. I don't currently have a link to that study, but I can probably get it in a few days when my co-worker who initially found it is available.