Samtools sort error

Question

[ARich]

Dear Biostar users,

I have been working with metagenomics data.

In order to perform binning I need to create depth file.

• For this I first ran bbwrap with spades contigs as reference and samples as input reads.
  

for i in ${finallist}
  do
    bbwrap.sh \
    ref=spades/contigs.fasta \
    in=${i},${i%1*}u.final.clean.fq \
    in2=${i%1*}2.merged.final.clean.fq,NULL \
    t=24 \
    out= ${i%1*}sam.gz \
    covstats= ${i%1*}coverage
   done

• After this I did samtools view with follow command:

    samtools view \
    -S \
    -b \
    -u \
    ${i%1*}sam.gz > \
    ${i%1*}bam

• Followed by sorting the generated bam file using samtools sort function with following command:
  

   samtools sort \
    -m 24G \
    -@ 3 \
    ${i%1*}bam \
    -o  ${i%1*}sorted.bam

During the sorting I getting strange character error messages or terminal with empty file in the end
I tried to solve the problem by checking the header of my reference and bam file and it looks the same.

Here are first five lines of my bam file:

NS500633:12:H7JF2BGXX:1:11101:4115:8221 83      NODE_179_length_41253_cov_14.682023     5385    44      121=    =       5235    -271GTGGAAAATGAAAAATATATTGATATCCTATGGGAAGGCTATGGCAAGGGGGAAACGGTGACGATTGGCGAAGATAAGGAATTCCTCATGGCAGATGCAAAGTCAAAGGATGGAATGGAAC        FFFF<FFF.FF<F<F7<FFAFAAF<FFF.<.FF<FFFFAFFFFFF<FFF<FFF<FFFFFFFFFFFAF.FAFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFF<FFFFFFFFFFFFFAAAA7    NM:i:0  AM:i:44
NS500633:12:H7JF2BGXX:1:11101:4115:8221 163     NODE_179_length_41253_cov_14.682023     5235    45      148=    =       5385    271 GGATGGAAAAGAGGATGGGGGATAGATATTGCAGGATATAAAAAAATCAACATCCAGGCACAGTACGGCAATTGCGTGGTTATTTTGAAATATCCGGATGACATGACGGAATATGTGGTTGTGGATAAAGGGTTTGGTTTTTCCCTGC  AA<AAFAAFFFFFFFFFFAFFFFAFFFFFFFFAFFAF.F<AFFFAFFFF7FFFFFFF.FAFFFAFF<FFAFF<FFFF<AFA7FFFFF77FF<FF.FFF<<.F.F<.FFAF7F)7FFFFFFFFFAAF.7FFFFFFAF.A777A<7A<A< NM:i:0  AM:i:45
NS500633:12:H7JF2BGXX:1:11101:7872:8238 83      NODE_395_length_25884_cov_15.386465     4487    45      145=    =       4287    -345CACTAAAACGTCAGGCTTTAGAAAAGGCCAGCCCGGTAAACCAACAGATTCAATATCCGGCCTGGACGGATGATGATGACTTAATGAGCTGGCTCAGCTCATTTGGCAAACGATAATTATGTCAGCAACTCACATAGTCCTCCAA     FFF<FFAFF7FF<FFA7F7AAAAF<AFFFFF..7FFFFFF<F<FFFFFFAFFF.FFFFFF.AFFFFAFFF<F<FFFFFFFFFFFAFFFF.FFFFFFFA<FAFF.FFFFFFFFFFFFFAFFFFFFFFFFFAFFFFAFFFFFAAAAA    NM:i:0  AM:i:45
NS500633:12:H7JF2BGXX:1:11101:7872:8238 163     NODE_395_length_25884_cov_15.386465     4287    43      78=     =       4487    345 GCATATATGAAGGATTACAAACTCGAACAGGCTCCCAGCACACACCCATTGTTCTGCAACCGCAGCGGCGCAAAATTC   AAAAAFAFFFFFFFFFAFFF.FAFFA.FFFFFFFFFFFF.FFFAFFFFFFFFFFFFFF)AAFF7<F<FFFFFFFAFFF       NM:i:0  AM:i:43
NS500633:12:H7JF2BGXX:1:11101:8333:8251 77      *       0       0       *       *       0       0       CCTACACCGTGGAATGTGACTTCATAAGTAGCGGAACCCTTTCTGGAGCGAACTAATTCGCCGCCTGCACGAGCAGCTTCGAATACGAGTGCGCGTTTGGACTTTGCTGCC      AAAFFFFAFFFFFFFFFFFFFFFFF.AFFAFFAFFAFFFFAFFF<FAFFF<FF..<7A<<AFFFFA<F<FF<AFFFFFFFAFFFFFFFFFFAFAFF<FFFFFF7FFAA<FF

And below is the five lines of grep from my reference headers:

>NODE_1_length_199624_cov_13.770641
>NODE_2_length_193066_cov_15.912808
>NODE_3_length_183817_cov_14.803240
>NODE_4_length_163959_cov_11.179160
>NODE_5_length_128783_cov_13.807804

I am really unable to understand where is the problem coming from. It would really great it someone can help with this.

Thanks in advance!
Cheers,
AR

[Pierre Lindenbaum]

step 2: is it really ? ...}sam.gz) > \ ${AN...

[ARich]

Thank Pierre for the reply, Yes it is sam.gz. Nonetheless, I also tried without compression verison and I still got the error message during sorting

[Pierre Lindenbaum]

no, I meant that redirection > \

[ARich]

Yes its there. Isn't that correct?

Answer 1

[ARich]

The issue is resolved. I had installed the samtools through conda and it did not install HTSLIB and that was the issue. Now I have installed samtools locally and it ran normally.

Thank you helping me think beyond the script.

Cheers, AR