ONT long-read RNA sequencing - data analysis options
1
0
Entering edit mode
09191919 • 0
@9335d292
Last seen 6 months ago
Spain

Hi,

I am not a bioinformatician, completely new to RNAseq analysis, linux, R script and don't exactly trust myself when doing analysis of RNAseq data but no one else in my lab has experience either! I am doing my PhD and now find myself with 3 datasets to analyse and I would like to get as much out of it, as possible for my capabilities. I do not expect to get all suggestions done but would at least like to try (I do enjoy using R!) It is long-read RNAseq from cDNA, control vs disease, organs brain, lung(model1), lung(model2-reflects different phenotype of disease). I have removed adapters, then mapped reads to genome and transcriptome respectively using Minimap2, this gave me bam files, from this counted reads using R and then did DEseq2 analysis for DE. This worked really well for brain but has not work as well for lung as I get #N/A for many padj (this is a separate problem I am trying to solve..)

My question here is: can you recommend options for analysis I can look into to get the most out of long-read sequencing? For example isoformswitchanalzer. This is the first time long read sequencing is done on these organs in this disease so I don't have a particular question, I have been told that short-read sequencing is probably better if you are just looking at gene expression but we have done long-read so I am interested in what you can look at in long-reads that you can't in short-reads for sequencing.

If I can provide anymore information to help, please let me know. Thank you!!

DESeq2 LongRead DEXSeq IsoformSwitchAnalyzeR RNASeq • 619 views
ADD COMMENT
0
Entering edit mode
@mikelove
Last seen 1 day ago
United States

"This worked really well for brain but has not work as well for lung as I get #N/A for many padj"

See the vignette for the cause of this. You can look at the print out of summary(res) in the console.

"can you recommend options for analysis I can look into to get the most out of long-read sequencing? For example isoformswitchanalzer"

Yes, I would recommend looking at various workflows at the transcript level, including that package. We have a published workflow, where we use transcript counts (these can certainly be from long read):

https://bioconductor.org/packages/release/workflows/vignettes/rnaseqDTU/inst/doc/rnaseqDTU.html

There are other transcript level / splicing / isoform level software in Bioconductor:

https://bioconductor.org/packages/release/BiocViews.html#___DifferentialSplicing

ADD COMMENT

Login before adding your answer.

Traffic: 395 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6