Question: StringTie error input file not sorted, even though it is
gravatar for sg197
10 months ago by
sg19720 wrote:

I've been following the stringtie pipeline for differential expression. I'm up to the stage of stringtie -e -B where I'm using the merged transcript file with the alignment (.bam) files to get read coverage tables.


stringtie $file -p 8 -e -B --fr 
            -G $root/FPKM_count/stringtie_merged_samples.gtf

I keep getting the error "the input alignment file is not sorted". My bam alignment files are definitely co-ordinate sorted, and I used them in the first step of the pipeline to generate the transcript assemblies with no problems. Anyone had a similar problem?? Thanks

rna-seq stringtie • 508 views
ADD COMMENTlink modified 10 months ago by RamRS30k • written 10 months ago by sg19720

Did you try sorting (no matter if you think it is or is not) sorted using samtools a representative BAM file and then try stringtie again? Did that work? Also please show the command that was used for sorting in the first place.

ADD REPLYlink modified 10 months ago • written 10 months ago by ATpoint38k

Hi, I sorted the bam files using STAR output settings (used instead of HISAT). I've checked the bam file using samtools view -H $file | grep s0 and the output returned @HD VN:1.4 . s0:coordinate so it is definitely sorted. The gtf file is also sorted in the same order, chr1 onwards, so it can't be because of an incompatibility issue?

ADD REPLYlink written 10 months ago by sg19720

The header is no guarantee for anything. Did you try sorting manually with samtools and then rerun it?

ADD REPLYlink written 10 months ago by ATpoint38k

Just realised it's an issue with the reading in the file, not the file itself. Thanks for the suggestions anyway!

ADD REPLYlink written 10 months ago by sg19720
Please log in to add an answer.


Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 706 users visited in the last hour