Very happy to share our updated #Nano3P-seq protocol, adapted to #nanopore R10 chemistry. After a lot of troubleshooting both in the wet lab protocol but also the computational analysis pipeline, we finally made it! :-) www.biorxiv.org/content/10.1...

5) PolyTailor is a new tool that can predict tail length and content without relying on the annotation of polyA sites on Nano3P-seq data. It is a user-friendly and fast, code is already available to use! github.com/novoalab/pol...

So, what did we change? 1) TGIRT, a group II intron RT we used for template-switching reaction of Nano3P-seq library preparation had been depracated. Fortunately, @NEBiolabs had recently released Induro, a highly processive group II intron RT, so we adapted our method to use this enzyme

Nano3P-seq: charting the coding and non-coding transcriptome at single molecule resolution https://www.biorxiv.org/content/10.1101/2024.11.20.624491v1

I agree! But still good to know what output you’re getting… I am actually testing out the Nano3P-Seq protocol, which is an adaptation of direct cDNA Seq.. want to avoid PCR for this use case. Otherwise I am a fan of the cDNA-PCR kits. Which reminds me… I need to finish that manuscript 🤪

We would also thank the community for their interest in our work, which encouraged us to put the time and effort in upgrading Nano3P-seq to R10 chemistry. If you use the pipeline and/or code, please let us know how it goes - feedback very welcome! :)

New Article! Nano3P-seq: charting the coding and noncoding transcriptome at single-molecule resolution

4) Deprecation of the R9 chemistry also led to the deprecation of basecalled fast5 files, which were crucial for Nano3P-seq tail length prediction analysis. So we released a new in-house analysis tool called PolyTailor!

2) Template-switching oligos used for Nano3P-seq had a sequence context that was decreasing the tail prediction efficiency (due to TTC sequence at the adapter sequence end). We now changed our oligo sequence to improve tail prediction efficiency.

OB: Nano3p-seq can do all this!

Nano3P-seq: charting the coding and non-coding transcriptome at single molecule resolution https://www.biorxiv.org/content/10.1101/2024.11.20.624491v1