Hello
I am getting lots of warning messages when I run HTSeq-count. I saw few other posts on this forum, but could not figure out the solution.
I am using paired-end illumina sequences. I have three replicates of one sample and each file has two pairs of fasta file (from two Lanes i.e. _L001_ _R1, _L001_ _R2, _L002_ _R1 and _L002_ _R2). I merged two _R1 files to make a single _R1 file and also did the same for _R2. I used BWA aligner and converted SAM to BAM, sorted the BAM file and converted the sorted BAM to SAM file. So the final SAM file is the sorted one.
Then I used the following command to run HTSeq-count
But the error log shows this
The last 10 lines of the output file is this
So I am not sure if I can ignore those warning messages. Is there any other way to get rid of those warning messages?
I am getting lots of warning messages when I run HTSeq-count. I saw few other posts on this forum, but could not figure out the solution.
I am using paired-end illumina sequences. I have three replicates of one sample and each file has two pairs of fasta file (from two Lanes i.e. _L001_ _R1, _L001_ _R2, _L002_ _R1 and _L002_ _R2). I merged two _R1 files to make a single _R1 file and also did the same for _R2. I used BWA aligner and converted SAM to BAM, sorted the BAM file and converted the sorted BAM to SAM file. So the final SAM file is the sorted one.
Then I used the following command to run HTSeq-count
HTML Code:
htseq-count -m intersection-strict –stranded=no Sorted_SAM_file GTF_file > OUTPUT_File
HTML Code:
100000 GFF lines processed. 200000 GFF lines processed. 300000 GFF lines processed. 400000 GFF lines processed. 500000 GFF lines processed. 600000 GFF lines processed. 700000 GFF lines processed. 800000 GFF lines processed. 861936 GFF lines processed. Warning: Read HWI-ST1023:203:H7NK5ADXX:2:1106:2557:97816 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?) … … Warning: Read HWI-ST1023:203:H7NK5ADXX:1:1205:10269:59178 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?) Warning: Read HWI-ST1023:203:H7NK5ADXX:1:1116:3922:31598 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?) Warning: Read HWI-ST1023:203:H7NK5ADXX:2:2116:4516:45139 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?) 72953003 sam line pairs processed.
HTML Code:
Traes_7DS_FF7C9C6FD 77 Traes_7DS_FF911FA4A 26 Traes_7DS_FF9F1CF23 0 Traes_7DS_FFA36F6DA 0 Traes_7DS_FFE9ACDAB 716 no_feature 15387432 ambiguous 249671 too_low_aQual 0 not_aligned 25066352 alignment_not_unique 0
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