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StringTie error input file not sorted, even though it is

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.

root="/mnt/fls01-home01/mfbx3sgh/scratch"   
file=$root/Genome/*.bam  

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
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updated by Ram Ram • written by sg197 • sg197

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.

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written by ATpoint ATpoint

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?

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written by sg197 • sg197

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

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written by ATpoint ATpoint

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

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written by sg197 • sg197

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