Killifish Hypoxia RRBS Part 6

Continuing sample alignment

I’m chugging along with the full sample alignment and mapping. I ran into a few issues with the SLURM scheduler timing out even though I modified the SLURM header the way IS told me to.

Errors in mapping code

/yaaminiv/ line 15: /vortexfs1/scratch/yaamini.venkataraman/03-mapping/stat/190626_I114_FCH7TVNBBXY_L2_20-N4.stat: No such file or directory

Since I’m running a script inside a singularity exec call, I realized I ran into the reverse of a previous problem. I needed to include the path to the output file using the path to the mounted directories, not the path on the host system!

#Get alignment statistics
for f in "${FASTQ[@]}"
  BAT_mapping_stat \
  --bam ${SINGMAPPED}/${f}.bam \
  --excluded ${SINGMAPPED}/${f}.excluded.bam \
  --fastq ${TRIMMED}/${f}_1_val_1.fq.gz \
  > /scratch/03-mapping/stat/${f}.stat

I obtained the mapping statistics for all 22 samples, but still needed to merge files before proceeding with the BAT_calling module. That’s when I ran into this error:

Error in tempfile() using template /XXXXXXXXXX: Could not create temp file /2rcjMIFtOS: Read-only file system at /usr/local/bin/BAT_merging line 220.

Since I hadn’t tried BAT_merging previously, I didn’t know what the error was. Looking at line 220 of BAT_merging didn’t give me any insight either. I decided to test out the code interactively before running the script through SLURM. I started a new container with singularity run and tried my BAT_merging code. I was able to make it past the header step before I ran into another error:

Singularity> BAT_merging \
> -o ${SINGMERGED}/05-N.bam \
> --bam ${SINGMAPPED}/190626_I114_FCH7TVNBBXY_L2_5-N1.bam,${SINGMAPPED}/190626_I114_FCH7TVNBBXY_L2_5-N2.bam,${SINGMAPPED}/190626_I114_FCH7TVNBBXY_L2_5-N3.bam
[INFO]	Tue Apr 12, 10:37:28, 2022	BAT_merging v0.1 started
[INFO]	Tue Apr 12, 10:37:28, 2022	Checking flags
[INFO]	Tue Apr 12, 10:37:28, 2022	Build header
Use of uninitialized value $readgroup in concatenation (.) or string at /usr/local/bin/BAT_merging line 231.
[INFO]	Tue Apr 12, 10:37:29, 2022	Merge BAMs
##### AN ERROR has occurred: Please view the log file

The command started to create 05-N.bam, so I know there are no issues with my paths. I’m not sure if the error in line 231 is related to the error in line 220. Out of curiosity, I tried running the command when only merging two files, since that’s what the example shows.

Singularity> BAT_merging \
> -o ${SINGMERGED}/05-N.bam \
> --bam ${SINGMAPPED}/190626_I114_FCH7TVNBBXY_L2_5-N1.bam,${SINGMAPPED}/190626_I114_FCH7TVNBBXY_L2_5-N2.bam

That command finished with a merged file and no errors. I referred to a BAT script Neel sent and didn’t see the merging step, and the website only refers to it being used when the same sample is sequenced multiple times. I was confused as to whether or not I even needed to merge the samples, so I emailed Neel to 1) clarify I should proceed with BAT_merging and 2) see if he had code that worked for merging more than two files.

Neel emailed me back said I need to use BAT_summarize to merge biological replicates, which falls in the BAT_calling module. So no need to use BAT_merging! I updated my SLURM script and variable file to reflect this change. Since I was done with the alignment module, I moved my output to my home directory on vortex.

Mapping results

I uploaded all mapping results here. I looked through the pdfs the BAT_mapping produced and it seems like most paired-end reads had < 25 hits, which could indicate that there wasn’t huge duplication in sequenced reads. To summarize the mapping statistics, I created this table. I should have created an R script to pull the results out but…oh well. Alignment ranged from 69-80% across all samples, with mean alignment at 74%. My quick scan didn’t see any batch or treatment impacts on alignment, but that can certainly be checked later.

Going forward

  1. Review next BAT module
  2. Run BAT_calling
Written on April 11, 2022