Hi all,
I've been using featureCounts to generate count tables out of my bam files. Previously, it worked fine with bam files which I generated with Subread.
Now, I'm using featureCounts with the bam files I generated with HiSAT2. However, non of the alignments were assigned to any genes, since the chromosome names in my gtf file and bam files were different. I changed the chromosome names in my bam files following the instructions in this post. However, the bam file I generate following this method turns out to be corrupted somehow. samtools view mybam.bam | head command does not give any output and when I run featureCounts, I receive "GZIP ERROR: -5" and still non of the alignments gets assigned to a gene. So I wonder how I can fix this discrepancy between my bam files and gtf file.
I used awk to format the header file and changed all chromosome names accordingly, but it didn't fix the issue. However, when I change chromosome names, blanks between columns change as well for some reason, meaning if there was a tab, it turns into a single space. So, I wonder if there is another way of solving this issue. I would be more than happy if you could help me out.
Here is how my gtf, header and old bam files look right now:
gtf:
NC_005943.1 RefSeq CDS 3259 4213 . + 0 transcript_id "gene34201"; gene_id "gene34201"; gene_name "ND1";
NC_005943.1 RefSeq CDS 4421 5462 . + 0 transcript_id "gene34202"; gene_id "gene34202"; gene_name "ND2";
NC_005943.1 RefSeq CDS 5850 7418 . + 0 transcript_id "gene34203"; gene_id "gene34203"; gene_name "COX1";
NC_005943.1 RefSeq CDS 7532 8215 . + 0 transcript_id "gene34204"; gene_id "gene34204"; gene_name "COX2";
NC_005943.1 RefSeq CDS 8357 8563 . + 0 transcript_id "gene34205"; gene_id "gene34205"; gene_name "ATP8";
NC_005943.1 RefSeq CDS 8518 9198 . + 0 transcript_id "gene34206"; gene_id "gene34206"; gene_name "ATP6";
old header file:
@HD VN:1.0 SO:coordinate
@SQ SN:chr1 LN:225584828
@SQ SN:chr10 LN:92844088
@SQ SN:chr11 LN:133663169
@SQ SN:chr12 LN:125506784
@SQ SN:chr13 LN:108979918
@SQ SN:chr14 LN:127894412
@SQ SN:chr15 LN:111343173
@SQ SN:chr16 LN:77216781
@SQ SN:chr17 LN:95684472
@SQ SN:chr18 LN:70235451
@SQ SN:chr19 LN:53671032
@SQ SN:chr2 LN:204787373
@SQ SN:chr20 LN:74971481
@SQ SN:chr3 LN:185818997
@SQ SN:chr4 LN:172585720
@SQ SN:chr5 LN:190429646
@SQ SN:chr6 LN:180051392
@SQ SN:chr7 LN:169600520
@SQ SN:chr8 LN:144306982
@SQ SN:chr9 LN:129882849
@SQ SN:chrM LN:16564
@SQ SN:chrUn_NW_014806053v1 LN:1653
fixed header file:
@HD VN:1.0 SO:coordinate
@SQ SN:NC_041754.1 LN:225584828
@SQ SN:NC_041763.1 LN:92844088
@SQ SN:NC_041764.1 LN:133663169
@SQ SN:NC_041765.1 LN:125506784
@SQ SN:NC_041766.1 LN:108979918
@SQ SN:NC_041767.1 LN:127894412
@SQ SN:NC_041768.1 LN:111343173
@SQ SN:NC_041769.1 LN:77216781
@SQ SN:NC_041770.1 LN:95684472
@SQ SN:NC_041771.1 LN:70235451
@SQ SN:NC_041772.1 LN:53671032
@SQ SN:NC_041755.1 LN:204787373
@SQ SN:NC_041773.1 LN:74971481
@SQ SN:NC_041756.1 LN:185818997
@SQ SN:NC_041757.1 LN:172585720
@SQ SN:NC_041758.1 LN:190429646
@SQ SN:NC_041759.1 LN:180051392
@SQ SN:NC_041760.1 LN:169600520
@SQ SN:NC_041761.1 LN:144306982
@SQ SN:NC_041762.1 LN:129882849
@SQ SN:NC_005943.1 LN:16564
@SQ SN:NW_014806053.1 LN:1653
@SQ SN:NW_014806054.1 LN:997
@SQ SN:NW_014806055.1 LN:843
old bam:
SRR5991098.18949202 0 chr1 42294 60 40M * 0 0 GCCCTGCCTCCCACAGCTTTATTTCTTTTGGTTTTGGATG HHHJJJJJJJJJJJJJJJJJIJJJIJJJJJIGIJJJIIJJ AS:i:0 XN:i:0 XM:i:0XO:i:0 XG:i:0 NM:i:0 MD:Z:40 YT:Z:UU NH:i:1
SRR5991098.2109594 0 chr1 42295 60 40M * 0 0 CCCTGCCTCCCACAGCTTTATTTCTTTTGGTTTTGGATGC HHGJJJJJJJJIJCHIIJJGIIJIJJJJIIFHHIGICHFH AS:i:0 XN:i:0 XM:i:0XO:i:0 XG:i:0 NM:i:0 MD:Z:40 YT:Z:UU NH:i:1
SRR5991098.16345197 0 chr1 42301 60 40M * 0 0 CTCCCACAGCTTTATTTCTTTTGGTTTTGGATGCAAAACA D:ADEDA;;=+AEEFFEECBEDI<D;>1B<>DD9DDEDAD AS:i:0 XN:i:0 XM:i:0XO:i:0 XG:i:0 NM:i:0 MD:Z:40 YT:Z:UU NH:i:1
SRR5991098.31884339 0 chr1 42303 60 40M * 0 0 CCCACAGCTTTATTTCTTTTGGTTTTGGATGCAAAACAAA HHHJJJJJJJJJJJJIJJJJJJHIIJIJIIJJJJJJJJJJ AS:i:0 XN:i:0 XM:i:
Cheers!
Thanks for the advice geek_y! While I was trying to do what you suggested, I realized that the chromosome names in my gtf file and the chromosome names that are given at NCBI's website that I downloaded this gtf file do not match. So, I found the correct chromosome name from the gft file itself and it fixed my problem. Thanks again!
Instead of closing the question, please mark the answer as accepted to indicate that it solved your problem. That will help others in the future.