featureCounts running error in metwibele preprocess

Thank you for developing this tool!

I have a problem with running metawibele preprocess (version 0.4.6) using shotgun metagenomic sequencing paired reads from human feces.

The input reads were QC-filtered by Kneaddata.

The code that I’ve run:
metawibele preprocess --input /data/data/GNC/HN00206838/0.RawData/Knead_outs/ --output /data/data/GNC/HN00206838/0.RawData/01_preprocess/ --output-basename demo --extension-paired "0041_kneaddata_paired_1.fastq,0041_kneaddata_paired_2.fastq" --extension ".fastq" --local-jobs 10 --threads 70

The error message I’ve got:

01/15/2024 12:18:38 PM - LoggerReporter - INFO: Executing with shell:  metawibele_gene_abundance -r /home/gihyeon/Pipelines/Metawibele/GEN001_201/01_preprocess/finalized/demo_genecatalogs.centroid.fna -u /data/data/GNC/HN00206838/0.RawData/Knead_outs/changed_2102420041_kneaddata_paired_1.fastq,/data/data/GNC/HN00206838/0.RawData/Knead_outs/changed_2102420041_kneaddata_paired_2.fastq -t 70 -s changed_210242 -w /home/gihyeon/Pipelines/Metawibele/GEN001_201/01_preprocess/mapping/changed_210242 > /home/gihyeon/Pipelines/Metawibele/GEN001_201/01_preprocess/mapping/changed_210242/changed_210242.mapping.stdout.log 2>&1 
01/15/2024 12:51:55 PM - anadama2.helpers - INFO: Execution complete. Stdout: 
01/15/2024 12:51:55 PM - LoggerReporter - ERROR: task 31, changed_210242__gene_abundance :  Failed! Error message : Failed to produce target `/home/gihyeon/Pipelines/Metawibele/GEN001_201/01_preprocess/mapping/changed_210242/changed_210242.sort.bed'. Original exception: Traceback (most recent call last):
  File "/home/gihyeon/.local/lib/python3.10/site-packages/anadama2/runners.py", line 219, in _get_task_result
  File "/home/gihyeon/.local/lib/python3.10/site-packages/anadama2/tracked.py", line 379, in compare
    stat = os.stat(self.name)
FileNotFoundError: [Errno 2] No such file or directory: '/home/gihyeon/Pipelines/Metawibele/GEN001_201/01_preprocess/mapping/changed_210242/changed_210242.sort.bed'

When I ran the demo version there is no error, and I figured out the demo.sort.bed file has

# Program:featureCounts v2.0.3; Command:"featureCounts" "-F" "SAF" "-T" "4" "-a" "/home/gihyeon/Pipelines/Metawibele/demo/01_preprocess/finalized/demo_genecatalogs.centroid.saf.gtf" "-o" "sample1.sort.bed" "sample1.sort.bam"

So, I applied that featureCounts command structure to my real sample as the below code and it was well done.

featureCounts -F SAF -T 4 -a ~/Pipelines/Metawibele/GEN001_201/01_preprocess/finalized/demo_genecatalogs.centroid.saf.gtf -o ./changed_210242.sort.bed  01_preprocess/mapping/changed_210242/changed_210242.sort.bam

It is difficult for me to find out what is the problem with metawibele preprocess.
Could you help me?

Thank you in advance.

Hi Gihyeon,

It looks like something was disordered during the metawibele_gene_abundance step. Could you check its log file (i.e. /home/gihyeon/Pipelines/Metawibele/GEN001_201/01_preprocess/mapping/changed_210242/changed_210242.mapping.stdout.log) and see if you can find more information about the error?


Hi Yancong,

I appreciate your valuable help…!
I have took a look into the log file and found out that

command:featureCounts -F SAF -T 70 -a  /home/gihyeon/Pipelines/Metawibele/GEN001_201/01>
Value for argumant -T is out of range: 1 to 64

The problem was due to the limitation of the thread of featureCounts, which is up to 64 (I set 70).

metawibele preprocess --input /data/data/GNC/HN00206838/0.RawData/Knead_outs/ --output /data/data/GNC/HN00206838/0.RawData/01_preprocess/ --output-basename demo --extension-paired "0041_kneaddata_paired_1.fastq,0041_kneaddata_paired_2.fastq" --extension ".fastq" --local-jobs 10 --threads 64 was well working.

Thank you for your help.