#2 Issues in OUT2.sam

[Anupam Singh]

Hi, team,

I am running MToolBox Pipeline to analayze pair end data of fastq file, I have checked the shell script, the OUT2.sam file generated, with zero size, and so tha further process given NULL output.

the file OUT.sam.bam.marked.bam.marked.sam have some data, and upon running grep -v '^@' .... command OUT2.sam is generated with zero contents.

contents of OUT.sam.bam.marked.bam.marked.sam

@HD VN:1.4 SO:coordinate
@SQ SN:chrRSRS LN:16569
@RG ID:sample PL:sample PU:sample LB:sample SM:sample

I am running MToolBox.sh

MToolBox.sh -i fastq -M -m "-g /usr/local/bin/gsnap -D /mnt/analysis/MToolBox/genome_index/ -M chrRSRS -H hg19RSRS -t 8" -a "-o AT3023 -r /mnt/analysis/MToolBox/genome_fasta/ -s /usr/bin/samtools -FCP" -c "-i /home/varun/Illumina/OUT_AT3023/AT3023-contigs.fasta -m /usr/bin/muscle -b mt-class -s mt_classification_best_results.csv"

........................................
OUTPUT , where something seems, wrong
.......................................

ASSEMBLING MT GENOMES WITH ASSEMBLEMTGENOME...

WARNING: values of tail < 5 are deprecated and will be replaced with 5

samtools path is /usr/bin/samtools
for i in OUT_AT3023; do outhandle=AT3023; cd OUT_AT3023; assembleMTgenome.py -i OUT2.sam -o -r /mnt/analysis/MToolBox/genome_index/ -f chrRSRS.fa -a hg19RSRS.fa -s /usr/bin/samtools -FCP -o AT3023 -r /mnt/analysis/MToolBox/genome_fasta/ -s /usr/bin/samtools -FCP; cd ..; done > logassemble.txt
[sam_header_read2] 93 sequences loaded.
[sam_read1] reference '' is recognized as '*'.
[main_samview] truncated file.
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000

GENERATING VCF OUTPUT...

Reference sequence used for VCF: RSRS

PREDICTING HAPLOGROUPS AND ANNOTATING/PRIORITIZING VARIANTS...

Haplogroup predictions based on RSRS Phylotree build 16
Your best results file is mt_classification_best_results.csv

Loading contig sequences from file /home/varun/Illumina/OUT_AT3023/AT3023-contigs.fasta
Unable to compute haplogroup. ExitParsing pathogenicity table...
Parsing variability data...
Parsing info about haplogroup-defining sites...
Parsing info about haplogroup assignments...
ls: cannot access OUT_/annotation.csv: No such file or directory
ls: cannot access OUT_/annotation.csv: No such file or directory
Traceback (most recent call last):
File "/mnt/analysis/MToolBox/prioritization.py", line 12, in <module>
raw_file=open(sys.argv[1],"r").readlines()
IOError: [Errno 2] No such file or directory: 'priority_tmp.txt'
rm: cannot remove ‘priority_tmp.txt’: No such file or directory

Prioritization analysis done.

awk: cannot open annotation.csv (No such file or directory)
awk: cannot open annotation.csv (No such file or directory)
awk: cannot open *annotation.csv (No such file or directory)
Traceback (most recent call last):
File "/mnt/analysis/MToolBox/summary.py", line 26, in <module>
var_number=open("variant_number.txt", "r").readlines()
IOError: [Errno 2] No such file or directory: 'variant_number.txt'
rm: cannot remove ‘variant_number.txt’: No such file or directory
awk: cannot open prioritized_variants.txt (No such file or directory)

Analysis completed!

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Please suggest to debug it, or where could be the problem.

Regard,
Anupam