How to isolate gene from a single chromosome, RNA Seq experiment, using R package ?
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@guillaumedachy-11994
Last seen 2.2 years ago
Brussels

Hi everyone 

In a RNA Seq experiment where we put a transgene in mice cells, my gene of interest (that transgene) is on an "artificial" chromosome which contains only this gene. I'm trying desesperately to isolate that chromosome from the data, using R package cummeRbund (DESeq2 and edgeR are options too...). Does someone has an idea how to do that trick ?

Thank you so much for helping me... 

Cheers

Guillaume 

rnaseq r cummerbund • 942 views
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Dead Hans-Rudolf, 

Thank you for your quick answer ! 

my problem is the following : my reference genome (GTF) does not contain any information on my transgene, which is referenced as an extra chromosome. (the experiment was a transfection of a human gene in a mouse cell line)

My question is then : how can I get the informations (quantification of expression, isoforms) about that precise chromosome (which contains only one gene, the one we transfected) ? 

Does this question sound a bit more clear ? I didn't see how to do this in quasR , but maybe I didn't get a part of the package possibilities... 

Once again, thank you. 

Kind regards

Guillaume 

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Use the sequence of your transgene (ie the extra chromosome) as your 'auxiliary file'. After doing the alignment, you will get an extra BAM file for your transgene.

Hope this helps, Hans-Rudolf

 

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@gordon-smyth
Last seen 1 hour ago
WEHI, Melbourne, Australia

You add the human gene as an extra sequence (i.e., extra chromosome) to the FASTA file defining the mouse genome. (You do this yourself using unix tools rather than using an R package. You need to be familiar with FASTA, which is a simple text format.) Then align the reads to the augmented genome using an up-to-date aligner such as Subread or STAR. For example, using the Rsubread package, you would run buildindex() to build the index from the FASTA file then subjunc() to align the reads.

You will also need to add the transgene to the GFF file containing gene annotation. In the simplest case you might define a gene with a single exon being the whole of the extra chromosome. This is just a matter of adding a line to a GFF data.frame or file.

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@hotz-hans-rudolf-3951
Last seen 23 months ago
Switzerland

Hi Guillaume 

I am struggling to understand the actual problem you have, but have a look at the 'QuasR' package (https://bioconductor.org/packages/release/bioc/html/QuasR.html). There you have the possibility to align the reads in addition to the genome to auxiliary targets (i.e. your artificial chromosome).

 

Regards, Hans-Rudolf

 

 

 

 

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