Stringtie Error: Getting the input alignment file is not sorted! even though file is sorted

Question

[venura]

Hi,

I am getting the following error when I try to use the Stringtie merge function:

stringtie --merge -p $threads  -G DM_1-3_516_R44_potato.v6.1.working_models.gff3 -o stringtie_merged.gtf mergelist.txt

Error: the input alignment file is not sorted!
Alignments (7) already found for scaffold_128 !

As mentioned in previous posts I checked the indexed bam files and they look fine. Please see below.

  samtools view -H PVX_2d_1.bam | grep "HD"

Output:

@HD VN:1.0 SO:coordinate

I checked the mergelist.txt and there are no empty spaces nor empty lines. There I have twelve samples. Before merge step I assembled transcripts using indexed bam files:

stringtie --rf -p $threads -G DM_1-3_516_R44_potato.v6.1.working_models.gff3 -o ${SAMPLE}.gtf -l ${SAMPLE} ${SAMPLE}.bam

Can someone help me in this regard? Thanks in advance.

[Pierre Lindenbaum]

even though file is sorted

what is the output of a new indexing ?

 samtools index PVX_2d_1.bam

[venura]

I ran samtools index PVX_2d_1.bam followed by samtools view -H PVX_2d_1.bam | grep "HD" and got the following (same as earlier)

@HD VN:1.0 SO:coordinate

Hope I didn't misunderstand what you asked me to do.

[Pierre Lindenbaum]

yes, that's what I wanted.

Answer 1

[venura]

After communicating with the author, found out that the above error is a bug found in version 2.1.3. Problem solved after updating to ver 2.1.4.