I work with mouse genome and I have RNA-Seq data from a mouse strain. When I align the reads in the mouse genome, a gene is not quantified since this gene does not appear in the reference mouse genome. How can I quantify this gene?
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felipead6 •
Manually add it to the genome as its own contig and re-align!
Edit: To be a little more explicit, you would append your reference fasta with the gene sequence of interest using something like cat musmus.fa newgene.fa > mus_edited.fa then add an entry in the GFF file for the gene, then re-build the reference index and re-align your reads.
You don't have to realign. Just add the gene of interest informations in the gtf file and rerun featurecounts (to count reads per gene) and then use edgeR or DESeq2.
That sounds interesting. How do I add the gene information in the .gtf file considering that I have the position of the gene available?
look at : http://www.ensembl.org/info/website/upload/gff.html for gtf column description, and add corresponding informations to the gtf (so open it in a text editor and edit it).
Do not use excel please : https://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-1044-7 https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-5-80
If the sequence is present and the annotation doesn't exist then this is indeed the correct approach!
I have the .gb file from genbank, is there any way to transform this to .gtf?
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