Quantification of a gene that is not in the reference genome
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6.7 years ago
felipead6 ▴ 10

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?

RNA-Seq sequencing • 2.0k views
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6.7 years ago
Jake Warner ▴ 830

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.

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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.

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That sounds interesting. How do I add the gene information in the .gtf file considering that I have the position of the gene available?

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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).

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just open the gtf with gedit or excel and fill

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If the sequence is present and the annotation doesn't exist then this is indeed the correct approach!

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I have the .gb file from genbank, is there any way to transform this to .gtf?

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That part is easily googled:

e.g:

https://github.com/riverlee/genbank2gtf

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