Question: Please adivse me on differential expression analysis on the STAR/Stringtie output
0
gravatar for seta
2.4 years ago by
seta1.2k
Sweden
seta1.2k wrote:

Hi all,

I about follow the “HISAT, StringTie and Ballgown” pipeline for RNA-seq analysis, but I used STAR (instead of HISAT) for mapping reads on the genome followed by Stringtie for genome-guided assembly. As you know “Ballgown” take the FPKM value (here, from stringtie) for doing differential expression analysis. But, for using DEseq or edgeR , we need raw count. As I know, the popular program for generating raw count are HTseq and RSEM, which HTseq is designed to work at the gene level (not transcript level) and RSEM accept the mapping file generated by aligning to transcriptome not genome. Could you please let me know how I should create raw count from bam file produced by STAR for further processing by edgeR analysis at the transcript level?

Thanks

ADD COMMENTlink modified 16 months ago by Biostar ♦♦ 20 • written 2.4 years ago by seta1.2k

String-Tie has a built-in script to address this issue <prepde.py>. It can be easy to overlook, so here's a direct link to their instructions: http://www.ccb.jhu.edu/software/stringtie/index.shtml?t=manual#deseq

ADD REPLYlink written 2.3 years ago by dunhamcg20
2
gravatar for dunhamcg
2.3 years ago by
dunhamcg20
dunhamcg20 wrote:

String-Tie has a built-in script to address this issue 'prepDE.py'. It can be easy to overlook, so here's a direct link to their instructions: http://www.ccb.jhu.edu/software/stringtie/index.shtml?t=manual#deseq

ADD COMMENTlink written 2.3 years ago by dunhamcg20

Is 'prepDE.py' reliable? I haven't still come across any paper using this.

ADD REPLYlink written 15 months ago by Arindam Ghosh160
0
gravatar for Sej Modha
2.4 years ago by
Sej Modha4.2k
Glasgow, UK
Sej Modha4.2k wrote:

You can use featureCounts from the subread package to calculate raw counts from STAR alignments.

ADD COMMENTlink written 2.4 years ago by Sej Modha4.2k

Thanks, just one thing. Please kindly tell me if the featureCount give the count per both gene and transcript?

ADD REPLYlink written 2.4 years ago by seta1.2k

You can define the feature type of interest using -t parameter.

 -t <string>         Specify feature type in GTF annotation. `exon' by
                      default. Features used for read counting will be
                      extracted from annotation using the provided value.

For more info: http://bioinf.wehi.edu.au/featureCounts/

ADD REPLYlink written 2.4 years ago by Sej Modha4.2k

Hi Sej

Thank you. For making sure, the count read per transcript is needed for doing differential expression analysis at the transcript level, yes?, Based on the manual, in default, featureCount give us the count per gene (-t exon -g gene_id), so for counting per transcript I just put -t transcript -g transcript_id, yes, is it right?

ADD REPLYlink written 2.4 years ago by seta1.2k
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