stringtie merge error bam file not sorted

Question

[paumarc]

Hello

I am having an error using the following command line:

stringtie --merge -p 8 -G /home/ltr2/Genomes/GTF/hg19.gtf -o onwork/hisat2_test/gtf/stringtie_marged.gtf onwork/hisat2_test/margelist.txt

bam files has been sorted and indexed with samtools (so they are sorted) and hg19-fasta and hg19.gtf have been download from ucsc web page

magelist has one file per line with its full path

any ideas?

pau

[Nitin Narwade]

If you could post the output of the above-mentioned command, it would be really helpful to understand the error.

[paumarc]

Ok, I am sorry, i just forget to paste it

the error says : the input alignment file is not sorted!

the strange thing about the error is that it happens always with the last file (it do not matter if margelist has 2 or 10 bam files), it works normally until it is going to end.

It also occurs that files processed normally when they are not at the last place of the list are found as not sorted if they are at the last last place

thanks

[lakhujanivijay]

Add the output of following command for all your BAM files

samtools view -H your_bam_file.bam | grep "HD"

You should see something like this

@HD VN:1.0  SO:coordinate

[paumarc]

Thank you for your replay,

as i told you all files are sorted properly, and do not mather if i use two or more files at the marge list, it always gives the error at the last bam file

/hisat2_test/bam $ ls |xargs -i samtools view -H {} | grep "HD"

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

@HD VN:1.0  SO:coordinate

[lakhujanivijay]

Check your margelist.txt file once again and make sure it does not have a blank line in the end. Open it in a textpad or notepad.

[paumarc]

no spaces at margelist.txt file.

[lakhujanivijay]

Can you paste the contents of this file?

[paumarc]

i post it separatly by mistake, but you can see the margelist.txt

[paumarc]

Yes, of course. Notice that at the original file there not exist a blank line between lines with the path. I don't know why (maybe because I am in Linux), when I copy paste, the post at the forum does not recognize the 'new line'

/home/ltr2/onwork/hisat2_test/bam/2008-7981.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2011-11198.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2011-11211.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2017-21131.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2006-5237.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2017-20860.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2017-21260.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2017-20863.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2017-20880.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2017-21081.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2017-21080.sorted.bam
/home/ltr2/onwork/hisat2_test/bam/2011-11087.sorted.bam

thanks

[finswimmer]

Hello paumarc ,

• Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time.
  
• When answering to a question please use the ADD REPLY button below the post.

Thank you!

[paumarc]

ok, thank you. i was becoming crazy with the output

Answer 1

[paumarc]

Solved: margelist.txt should have the path of gtf files generated by stringtie in the previous step of the pipeline, not the path of bam files