Please help,
I mapped my RNA-seq (Illumina) reads to E. coli index (index built from UCSC obtained FASTA file) using BowTie. Now I'm trying to assign gene annotation to the mapped reads. I decided to use HTSeq for this purpose. I use SAM file i obtained after running Bowtie (also used TopHat to get the SAM file) with GTF files from either UCSC Microbial or Ensembl Bacterial (I also tried converting them to GFF3 format). In either case, when I run HTseq-count using the command:
$ HTseq-count <file.sam> <file.gff (or .gtf)>
I get an error message such as:
Warning: Skipping read 'HWI-ST330_0103:1:1:19499:9909#CGATGT/1', because chromosome 'gi|49175990|ref|NC_000913.2|', to which it has been aligned, did not appear in the GFF file.
I also tried to provide GTF or GFF files while running TopHat, but it failed in that case also.
Can someone one make a suggestion or has previously encountered such problem and knows how to solve it? Please help?!
Also if anyone has done RNA-seq for bacteria, what is your workflow? My plan for now is BowTie (or TopHat) -> HTseq -> DEseq
Appreciate any feedback, thank you!
Max Bobrovskyy
University of Illinois
I mapped my RNA-seq (Illumina) reads to E. coli index (index built from UCSC obtained FASTA file) using BowTie. Now I'm trying to assign gene annotation to the mapped reads. I decided to use HTSeq for this purpose. I use SAM file i obtained after running Bowtie (also used TopHat to get the SAM file) with GTF files from either UCSC Microbial or Ensembl Bacterial (I also tried converting them to GFF3 format). In either case, when I run HTseq-count using the command:
$ HTseq-count <file.sam> <file.gff (or .gtf)>
I get an error message such as:
Warning: Skipping read 'HWI-ST330_0103:1:1:19499:9909#CGATGT/1', because chromosome 'gi|49175990|ref|NC_000913.2|', to which it has been aligned, did not appear in the GFF file.
I also tried to provide GTF or GFF files while running TopHat, but it failed in that case also.
Can someone one make a suggestion or has previously encountered such problem and knows how to solve it? Please help?!
Also if anyone has done RNA-seq for bacteria, what is your workflow? My plan for now is BowTie (or TopHat) -> HTseq -> DEseq
Appreciate any feedback, thank you!
Max Bobrovskyy
University of Illinois
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