full-length cDNA library synthesis for PacBio

[justinjun]

I am planing to do the trascriptome analysis using PacBio technology.
After searching and reading the protocols, I know there are two methods used by people to generate full-length cDNA library for PacBio sequencing: 1. using "SuperScript Full-Length cDNA Library Construction Kit" from Invitrogen. The advantage of this method is that in this kit, 5'Cap specific Ab is used for selection of full-length of cDNA. But, unfortunately, this kit is discontinued for unknown reason (Anyone know the reason?)
2. using Clontech SMART Kit, which is also recommended by PacBio company.

Now, my 1st question is: Are there any better methods for full-length cDNA synthesis available and compatible with PacBio besides those two I mentioned above?
Such as, dUTP method (http://nar.oxfordjournals.org/content/37/18/e123.long) which is almost standard method for Illumina sequencing.
Or, "first-strand cDNA library" method (Sequencing of first-strand cDNA library reveals full-length transcriptomes: http://www.nature.com/ncomms/2015/15...comms7002.html).
My 2nd Q: Whether these two methods also can be used for PacBio platform?

Any input/thought will be appreciated!

[Magdoll]

This is an old post but I'll respond anyway.

A while back I heard Invitrogen is discontinuing Invitrogen Superscript --- but it seems like a website search still finds them, so maybe they are still available. However even if it's available one of the drawback of the Invitrogen kit is the input demand is very high.

The Clontech SMARTer kit by comparison has a very low input amount (I think 1ng polyA RNA).

There are several PacBio customers who use their own full-length cDNA kit. In general as long as the kit truly preserves full-length cDNA (using template switching, cap trap, etc), theoretically they should all work well. There have not been extensive comparison between all the kits, however.

[luc]

We are using Clontech SMARTER, but another kit I would like to try is this:

https://www.lexogen.com/teloprime-full-length-cdna-amplification/

[Magdoll]

Let me preface this by saying: what I am about to say can be wrong (because it's been a while) and is not official.

I believe a while back we tested the TeloPrime kit in-house at PacBio. I never got to a conclusive answer because we only did a small scale test and I got side-tracked by other things. But looking at my notes back then, my impression was: the kit works, it does generate FL transcripts. What I believe was different was we were unable to get transcripts > 3 kb, they simply didn't amplify (no bands...). And we suspected it might be because long transcripts degrade faster and if TeloPrime is a true cap-trap method, then you might be sacrificing for not getting long transcripts because there's few of them that have the cap.

For the transcripts that we did get, the results do look consistent with the Clontech SMARTer kit on the same sample and cleaner --- as in it looks like it only had the FL transcripts and did not contain what might be the degraded forms of longer transcripts.

Again, this is a one-off experiment. Maybe the kit has improved. Maybe on a different sample it would work better. Maybe we just had to optimized lib prep conditions and we didn't.

But just giving you my very distant memory of what happened. And for the sake of argument, my thought (and this is my personal thought) was: if what you care is everything has to be absolutely correct on the 5' end and you would rather not see degraded forms of longer transcripts, then probably a true FL kit like TeloPrime works for you. On the other hand, if you want to get as much transcripts as possible, the Clontech SMARTer kit is really flexible in that sense.

Again, just because it didn't work perfectly in that one experiment doesn't mean it won't work for you. I encourage you to contact Lexogen for further information. And would love to hear from your experience!

[lexogen]

TeloPrime vs. SMARTer full-length cDNA for PacBio

Hi, while these are posts from 2015 the issues raised are still relevant. This new publication benefits from the performance of Lexogen's TeloPrime kit and compares it to the current Iso-Seq protocol. Please read for yourself:

Cartolano, M. et al: cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing

http://dx.doi.org/10.1371/journal.pone.0157779

Abstract

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.