Problems Using Htseq-Count And Samtools When Processing Rna-Seq Data

Question

[camelbbs]

HI, While I analyzed RNAseq data, I have some questions in using samtools and htseq-count. Can anyone help?

1. I want to use htseq-count to get all counts on known genes, so i run:

htseq-count brain_fetus1.sam ~/knowngene_hg19.gff

Error occured in line 3 of file /cchome/che/knowngene_hg19.gff.

Error: Feature uc001aaa.3. does not contain a 'gene_id' attribute    

  [Exception type: SystemExit, raised in count.py:55]

The gff file like this:

chr1    hg19_knownGene    gene    11874    14409    0.000000    +    .    ID=uc001aaa.3;Name=
chr1    hg19_knownGene    mRNA    11874    14409    0.000000    +    .    ID=uc001aaa.3;Name=;Parent=uc001aaa.3
chr1    hg19_knownGene    exon    11874    12227    0.000000    +    .    ID=uc001aaa.3.;Name=;Parent=uc001aaa.3
chr1    hg19_knownGene    exon    12613    12721    0.000000    +    .    ID=uc001aaa.3.;Name=;Parent=uc001aaa.3
chr1    hg19_knownGene    exon    13221    14409    0.000000    +    .    ID=uc001aaa.3.;Name=;Parent=uc001aaa.3

Is there an appropriate tools to convert gtf to gff?

1. another question is in samtools:

while i use samtools to figure out the counts in specified region, i run like this:

samtools mpileup -l test.bed brain.bam > test.txt

the test.bed file:

chr1    11873   14409   uc001aaa.3      0       +       11873   11873   0       3       354,109,1189,   0,739,1347,
chr1    11873   14409   uc010nxr.1      0       +       11873   11873   0       3       354,52,1189,    0,772,1347,
chr1    11873   14409   uc010nxq.1      0       +       12189   13639   0       3       354,127,1007,   0,721,1529,

It seems the -l option doesn't work. the result test.txt still contain the counts from the whole genome.

Thanks,

Che

Answer 1

[Istvan Albert]

1. make sure to set the -id attribute, by default your tool will use the gene_id See this: Counting reads in features with htseq-count
2. I tried your samtools example and it worked as expected. Make sure your bamfile is sorted and indexed.

[camelbbs]

Thanks. I think I got it.

Answer 2

[Arun]

Is there an appropriate tools to convert gtf to gff?

Try Genometools. After unzipping/installing, navigate to bin folder and type:

./gt gtf_to_gff3 -help

[camelbbs]

This tool cannot be compiled properly in my server.

[Arun]

In that case, I think you should ask your administrator to do it / help you with it or you should post here (in context of bioinformatics) or on stackoverflow explaining the output of the compilation error.