Hello!
I am really new to bioinformatics and am trying to get some help with HT-Seq analyses to count and get gene annotations of the mapped sequences obtained from Bowtie. What I am mostly having is formatting issues. I downloaded the Salmonella Typhimurium LT2 gtf file from the UCSC web page, and am using the SAM file obtained from Bowtie in the following command in HT-Seq:
htseq-count <sam_file> <gff_file>
The problem I am running into is that I can't locate fasta files (from which i built the Salmonella index) and gtf/gff files (with gene ids) from the same source. For eg., I downloaded the gtf file from the UCSC web browser, but cannot find sequences in the FASTA format (for BOWTIE alignment). So, when I try to use HT-Seq, I get this error:
Warning: Skipping read 'HWI-ST330_0109:1:1:7470:10545#CTTGTA/1', because chromosome 'gi|16763390|ref|NC_003197.1|', to which it has been aligned, did not appear in the GFF file.
When I tried using Refseq to get gff files (because I got my FASTA sequence from NCBI to build the Salmonella index), the formatting is once again not correct in the Refseq gff file because I get this error:
vanderpoolmbp:HTSeq-0.5.0p4 vanderpoollab$ htseq-count sequence1aligned.sam NC_003197.gff
Error occured in line 3436 of file NC_003197.gff.
Error: Feature NC_003197.1:rrsH:unknown_transcript_1 does not contain a 'gene_id' attribute
[Exception type: SystemExit, raised in count.py:55]
And lastly, I cannot find the Salmonella genome in ENSEMBLE bacteria!
Is there a commonly used genome browser that I can download both my FASTA and gtf files from?
Thank you very much for all your help in advance!!!!
Divya
UIUC
I am really new to bioinformatics and am trying to get some help with HT-Seq analyses to count and get gene annotations of the mapped sequences obtained from Bowtie. What I am mostly having is formatting issues. I downloaded the Salmonella Typhimurium LT2 gtf file from the UCSC web page, and am using the SAM file obtained from Bowtie in the following command in HT-Seq:
htseq-count <sam_file> <gff_file>
The problem I am running into is that I can't locate fasta files (from which i built the Salmonella index) and gtf/gff files (with gene ids) from the same source. For eg., I downloaded the gtf file from the UCSC web browser, but cannot find sequences in the FASTA format (for BOWTIE alignment). So, when I try to use HT-Seq, I get this error:
Warning: Skipping read 'HWI-ST330_0109:1:1:7470:10545#CTTGTA/1', because chromosome 'gi|16763390|ref|NC_003197.1|', to which it has been aligned, did not appear in the GFF file.
When I tried using Refseq to get gff files (because I got my FASTA sequence from NCBI to build the Salmonella index), the formatting is once again not correct in the Refseq gff file because I get this error:
vanderpoolmbp:HTSeq-0.5.0p4 vanderpoollab$ htseq-count sequence1aligned.sam NC_003197.gff
Error occured in line 3436 of file NC_003197.gff.
Error: Feature NC_003197.1:rrsH:unknown_transcript_1 does not contain a 'gene_id' attribute
[Exception type: SystemExit, raised in count.py:55]
And lastly, I cannot find the Salmonella genome in ENSEMBLE bacteria!
Is there a commonly used genome browser that I can download both my FASTA and gtf files from?
Thank you very much for all your help in advance!!!!
Divya
UIUC