Does salmon work with combined short and long read bam file?

Question

[shinyjj]

Hi all,

I know salmon works separately with short-read and long-read files for transcript quantification. Then is it possible to use salmon with combined short and long reads into one file for better quantification? Technically, doesn't this make a better quantification as you have more and longer reads? You can think it as a hybrid sequencing using salmon for better quantification.

[bk11]

You can check that it has been discussed very extensively in this POST.

[GenoMax]

FYI. bk11 you can paste biostars URL's directly in post. They will be parsed automatically e.g. Salmon Quantification for RNA-seq Read Pairs with Different Lengths

[shinyjj]

Hmm thanks but in my case it’s more like ont/pacbio + illumina so the length is 800-3000bp + 100bp

Answer 1

[majd.abdulghani]

I would guess not, because Salmon has a specific error correction model for short reads and one for long reads, so putting them both in the same file would mess with that.

I, too, have short- and long-read data from the same samples and here is how I approached analyzing them with Salmon:

I used FMLRC2 to correct the long reads with the short reads, improving the per-base accuracy of the long-read data. Then I mapped the corrected long reads with minimap2 and fed the mapped files to Salmon using the --ont flag.

If you don't want to correct with FMLRC2, and would rather use both datasets for quantification at the same time, you can use StringTie, which has a mixed reads assembly mode (--mix), that takes both long- and short-read files as input to calculate gene abundances, but this may or may not be compatible with downstream tools, depending on what you want to do with the data after (For example, I used Salmon because this approach was not compatible with IsoformSwitchAnalyzeR).

[callumjcparr]

Why would StringTie not be compatible with IsoformswitchAnalyzeR? I know there are specific R tools to import salmon data into IsoformSwitchAnalyzeR but I would think you just need a counts table at transcript resolution to build the R list for isoformSwitch. Either choose to import TPM or NumReads.

They may have updated the documentation because I can see a specific section on importing from StringTie Importing Data from Kallisto, Salmon, RSEM or StringTie.

https://bioconductor.org/packages/release/bioc/vignettes/IsoformSwitchAnalyzeR/inst/doc/IsoformSwitchAnalyzeR.html#importing-data-from-kallisto-salmon-rsem-or-stringtie