minimap2 BAM compatibility? - Undefined value as an ARRAY reference : Line 614

[physnano]

Hi Danny,

New user to RNAFramework, thanks for developing. I am trying to run on (long) reads that have been aligned/sorted using minimap2 and am encountering the following issues:

[+] Making output directory... [+] Guessing file types:

Sample Type 5'-end trimming

1_align-sorted BAM Ignored 2_align-sorted BAM Ignored

[+] Getting transcripts from reference, and building count table base structure... [+] Inspecting SAM/BAM file headers... [+] Assuming that provided SAM/BAM files are already sorted. Skipping sorting... [+] Calculating per-base mutation counts and coverage. This may take a while...

[-] Processing sample "1_align-sorted" (PID: 9203) [-] Processing sample "2_align-sorted" (PID: 9205)

[+] Statistics:

[!] Error: Unable to copy counts table structure for sample "2_align-sorted" (No such file or directory)

It seems to be failing on line 614: Can't use an undefined value as an ARRAY reference at .../bin/RNAFramework/rf-count line 614.

My guess would be it doesn't like the BAM files from minimap2. Any assistance much appreciated.

Thanks Will

[dincarnato]

Hi Will,

Can you help me reproduce the issue by sharing the command line, BAM file (or part of it) and the reference FASTA file?

Thanks! Best,

Danny

[physnano]

Hi Danny,

I was able to narrow the issue down to a -om "T2C" option I was passing. Which is related to an additional issue I am now having. I now am running run rf-count with the -orc option however when I look at the raw_counts, I am only seeing insertions in the output. For context the data input is direct RNA nanopore reads, so I would expect many substitutions as well. I have attached the reference, and downsampled fastqs, and sam/bam files from minimap2. rf_test_data.zip

Thanks for your help

-Will

[dincarnato]

Can you give me the exact command you are executing?

[dincarnato]

Will,

I realized the issue. It looks like your BAM files miss one tag, the "MD" tag, which provides information on where the substitutions reside. You might want to run a samtools calmd --output-fmt=BAM 2ds_align-sorted.bam rep_mrnas.fasta > 2ds_align-sorted_reMD.bam prior to running rf-count. If you do that, the issue should be gone.

I have now also added to rf-count a warning to indicate whether the MD tag is missing. Please issue a git pull.

Let me know if this fixes. All the best,

Danny

[physnano]

Hi Danny,

Thanks so much everything looks great now!

Best,

Will