Bowtie error

[sanatbhadsavle]

Hi, on running miRge on one sample, I get the following error - `bowtie version: 1.2.3 cutadapt version: 3.1 Samtools error!: samtools, command not found. Type "samtools --version" and check the version Use argument -psam NOTE: Will not exit unless you encounter this error otherwise Collecting and validating input files...

miRge3.0 will process 1 out of 1 input file(s).

Cutadapt finished for file QD1065-01_S1_L003_R1_001 in 229.2512 second(s) Collapsing finished for file QD1065-01_S1_L003_R1_001 in 2.7011 second(s)

Matrix creation finished in 3.4465 second(s)

Data pre-processing completed in 247.1582 second(s)`

After this I get traceback calls like so - Alignment in progress ... Traceback (most recent call last): File "/sw/eb/sw/miRge3/0.0.9-foss-2020a-Python-3.8.2/bin/miRge3.0", line 8, in <module> sys.exit(main()) File "/sw/eb/sw/miRge3/0.0.9-foss-2020a-Python-3.8.2/lib/python3.8/site-packages/mirge/__main__.py", line 105, in main pdDataFrame = bwtAlign(args,pdDataFrame,workDir,ref_db) File "/sw/eb/sw/miRge3/0.0.9-foss-2020a-Python-3.8.2/lib/python3.8/site-packages/mirge/libs/manifoldAlign.py", line 100, in bwtAlign alignPlusParse(bwtExec, bwt_iter, pdDataFrame, args, workDir) File "/sw/eb/sw/miRge3/0.0.9-foss-2020a-Python-3.8.2/lib/python3.8/site-packages/mirge/libs/manifoldAlign.py", line 19, in alignPlusParse bowtie = subprocess.run(str(bwtExec), shell=True, check=True, stdout=subprocess.PIPE, text=True, stderr=subprocess.PIPE, universal_newlines=True) File "/sw/eb/sw/Python/3.8.2-GCCcore-9.3.0/lib/python3.8/subprocess.py", line 512, in run raise CalledProcessError(retcode, process.args, subprocess.CalledProcessError: Command 'bowtie /scratch/user/sanatbhadsavle/spermRNAseq/mouse/mouse/index.Libs/mouse_mirna_miRBase -n 0 -f --norc -S --threads 48 output_dir/miRge.2022-08-04_15-02-15/bwtInput.fasta' returned non-zero exit status 1.

What could be the problem? Thanks.

[arunhpatil]

Hi @sanatbhadsavle ,

It seems like the library is not accessible, can you check the path? On the other hand, try executing the following command (taken from the error above):

bowtie /scratch/user/sanatbhadsavle/spermRNAseq/mouse/mouse/index.Libs/mouse_mirna_miRBase -n 0 -f --norc -S --threads 48 output_dir/miRge.2022-08-04_15-02-15/bwtInput.fasta > test_output.sam

Basically, the following path should exist and if you say ls, it should list several files one of which should be mouse_mirna_miRBase with ebwt extensions: ls /scratch/user/sanatbhadsavle/spermRNAseq/mouse/mouse/index.Libs/

Let me know if this helps.

Thanks, Arun.

[sanatbhadsavle]

Hi Arun, My bowtie is working fine, bowtie -h spits the usual help message. I also ran the command you mentioned and it gave out this - `# reads processed: 757333

reads with at least one reported alignment: 3210 (0.42%)

reads that failed to align: 754123 (99.58%)

Reported 3210 alignments`

I should mention that I am running miRge with the Qiagen UMI mode, my original input command was - miRge3.0 -s QD1065-01_S1_L003_R1_001.fastq.gz -lib /scratch/user/sanatbhadsavle/spermRNAseq/mouse/ -db miRBase -on mouse -a AACTGTAGGCACCATCAAT --qiagenumi -umi 0,12 -o output_dir -gff -udd

[arunhpatil]

@sanatbhadsavle,

The command looks fine, the path to -lib seems incorrect.

For example in my laptop: ls /mnt/d/Halushka_lab/Arun/GTF_Repeats_miRge2to3/miRge3_Lib I should see the folder named mouse and then

ls /mnt/d/Halushka_lab/Arun/GTF_Repeats_miRge2to3/miRge3_Lib/mouse Should show the following sub-folders: annotation.Libs, fasta.Libs, index.Libs

Similarly, when you list the folder by the following command: ls /scratch/user/sanatbhadsavle/spermRNAseq/mouse/ you should see the folder named mouse and then

ls /scratch/user/sanatbhadsavle/spermRNAseq/mouse/mouse/ Should show the following sub-folders: annotation.Libs, fasta.Libs, index.Libs

Please let me know if the above paths exist. In case if you have doubts, then try to execute the following command: miRge3.0 -s QD1065-01_S1_L003_R1_001.fastq.gz -lib /scratch/user/sanatbhadsavle/spermRNAseq -db miRBase -on mouse -a AACTGTAGGCACCATCAAT --qiagenumi -umi 0,12 -o output_dir -gff -udd Here I have tweaked your absolute path.

Thanks, Arun

[sanatbhadsavle]

@arunhpatil Hey, Thanks for the help, that worked. I just had to take out /mouse/ from my path.

[arunhpatil]

@sanatbhadsavle,

Yay. I am glad it worked out for you.

Thanks, Arun.