I am running pe-utils to determine the insert length for Miso. However, firstly I am getting messages like the following:
WARNING: NB501137:15:HHYT3BGXY:1:23310:15215:17414 left mate starts later than right mate WARNING: NB501137:15:HHYT3BGXY:4:11608:2223:10635 left mate starts later than right mate WARNING: NB501137:15:HHYT3BGXY:1:21306:5350:10090 left mate starts later than right mate WARNING: NB501137:15:HHYT3BGXY:1:12103:10191:8692 left mate starts later than right mate
The run then ends with the following error and the insert_len file is empty: Error: Could not find any properly mated pairs to compute insert length with. Are you sure your BAM reads are properly paired and map the chromosome headers in the constitutive exon file?
I don't really know what all this mean. Why would the left mate start later than the right mate in the first place?
The fastq files were aligned with star and sorted by coordinate. I could not find anything in the Miso manual about how the files bam files should be sorted. I might have missed it.
If someone could perhaps help me solve the problem it would be greatly appreciated.
Does anyone have a fix for this? My chromosome headers were different before (chr1 vs 1) which I changed all to chr1 but then I get this same error message with the left mate/right mate issue. I have tried to run the miso protocol up to this step with a sorted and an unsorted file bam file. Any suggestions? I will gladly provide more information if required.