sortmeRNA creating identical aligned and unaligned files?

[cottop]

Hello!

I am trying to process some microbial RNAseq data before importing counts into R studio.

I’ve been able to run falco, multi qc and trim galore! to remove adapter sequences. Since I have a large amount of duplication, I’ve been trying to remove rRNA sequences using sortmeRNA.

However, the unaligned and aligned output files are identical, so sortmeRNA doesn’t seem to be doing its job properly.

I’m wondering whether anyone could assist me in troubleshooting this problem? Thanks very much in advance for your help! I’ve just been using a single pair of sequences (R1/R2) to reduce computing time while I troubleshoot.

Here is my history: Galaxy | Australia

[jennaj]

Hi @cottop

Something went wrong with the implicit conversion to the uncompressed format (what this tool is expecting as input), then the tool couldn’t find the data, and all outputs are empty, and this message was reported for the reason:

You can purge all of the intermediate files since some have parts that are missing and those partial copies can’t be directly corrected.

Instead, start from the top, and try this:

1. Unzip collection on dataset collection 12 (output of Trim Galore)
2. Select the two collection outputs on the SortMeRNA tool form

Hope this helps and we can follow up more!

[cottop]

Hi @jennaj,

Thank you very much for taking the time to assist me, I really appreciate it. I uncompressed the output from trim galore! which created a 3 files: a reverse collection (compressed), a forward collection (compressed) and a single, uncompressed file which I assumed are the forward and reverse reads interleaved?

I then ran sortmerna using using the sequencing type: “interleaved paired end reads” using the uncompressed file. This created outputs containing aligned and unaligned reads with 0 bytes in each file.

I’m now attempting sortmerna again using sequencing type: “paired end reads” with the two compressed output files (above) as the inputs. I think this is what you advised me to in the first place so we’ll see how we go.

Thanks again!