Customizing pipeline for cDNA-PCR runs rather than gDNA amplicons.

[callumparr]

I wanted to ask if this pipeline could be altered slightly to run with cDNA-PCR libraries. We have custom UMIs in both forward and reverse linkers to create dsDNA from total RNA. I see that the first step is to bin reads to their respective location in the genome defined by the coordinates in the bed file. For a few amplicons in a run this seems practical but for the cDNA library, i.e. whole transcriptome RNA-seq this maybe not depend on how granular we get with the coordinates. I guess I could use the gene_id level coordinates from an annotation file. Do you know of another pipeline that is more suitable to run with cDNA-PCR rather than gDNA amplicons?

[rebeelouise]

Hi Callum - did you ever find anything? :)

I wanted to ask if this pipeline could be altered slightly to run with cDNA-PCR libraries. We have custom UMIs in both forward and reverse linkers to create dsDNA from total RNA. I see that the first step is to bin reads to their respective location in the genome defined by the coordinates in the bed file. For a few amplicons in a run this seems practical but for the cDNA library, i.e. whole transcriptome RNA-seq this maybe not depend on how granular we get with the coordinates. I guess I could use the gene_id level coordinates from an annotation file. Do you know of another pipeline that is more suitable to run with cDNA-PCR rather than gDNA amplicons?

[callumparr]

Hi Callum - did you ever find anything? :)

I wanted to ask if this pipeline could be altered slightly to run with cDNA-PCR libraries. We have custom UMIs in both forward and reverse linkers to create dsDNA from total RNA. I see that the first step is to bin reads to their respective location in the genome defined by the coordinates in the bed file. For a few amplicons in a run this seems practical but for the cDNA library, i.e. whole transcriptome RNA-seq this maybe not depend on how granular we get with the coordinates. I guess I could use the gene_id level coordinates from an annotation file. Do you know of another pipeline that is more suitable to run with cDNA-PCR rather than gDNA amplicons?

Hey, I did not find anything like a tool for handling custom library designs with ONT. There is some support for UMI in pychopper that is part of the epi2me workflow for cDNA libraries. For sure it supports the newer cDNA library kits and if your custom adapters/primers have UMI in the 5' then you could probably make it work.