[HeidelbergScience]

Can this be modified to work on the Ion Proton platform?

Yes, the CATS can be applied for any NGS platform, including Ion Torrent.
However, a platform-specific sequences has to be used.
Unfortunately we can not uncover Ion Torrent-specific primers here before our next publication is accepted.
However, one can easily derive whose sequences from the structure of the standard Ion Torrent library.

[HeidelbergScience]

This is consistent with my tests on a different template-switching protocol: I found that the LNA actually gets slightly lower yields, in every test I've run, except a much higher background of self-priming artifacts. It probably depends on the sequence of the TSO (the Illumina P1 adapter has a run of three cytosines that might hybridize to the G-overhang), but I wonder whether the extra yield reported in the Smart-seq2 paper is actually due to non-specific strand invasion rather than proper template switching (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853366/).

At any rate, this should go without saying, but just a public service announcement since I've met many people who don't seem to be aware: you can reduce your batch-to-batch variation in oligo effectiveness by measuring the true concentrations of your stocks by spectrophotometry, rather than just assuming the manufacturer correctly measured the amount that's in the tube and your resuspension efficiency was exactly 100%. I often find that my "100 μM" stocks are as little as 90 μM, or sometimes even above 100. But then I dilute accordingly before I aliquot.

We have made several furthers tests on LNA TSO (rGrG+G). The efficacy (of generating libraries from 5 pg 22 nt synthetic RNA or DNA) was similar to the rGrGrG. However, rGrG+G provided much higher incidence of "empty" libraries, probably due to higher Tm of the +G and the 3'-terminal dC in RT primer. We did not test it on longer templates though.

While rGrG+G was reported to work better on SMART-seq, we would not recommend it for CATS due to its high costs and by-products problem.

[HeidelbergScience]

At any rate, this should go without saying, but just a public service announcement since I've met many people who don't seem to be aware: you can reduce your batch-to-batch variation in oligo effectiveness by measuring the true concentrations of your stocks by spectrophotometry, rather than just assuming the manufacturer correctly measured the amount that's in the tube and your resuspension efficiency was exactly 100%. I often find that my "100 μM" stocks are as little as 90 μM, or sometimes even above 100. But then I dilute accordingly before I aliquot.

Very good point, thanks!

[Calixer]

Is there a publicly available human RNA-seq data set using CATS?

[HeidelbergScience]

Is there a publicly available human RNA-seq data set using CATS?

CATS publication data is now searchable on NCBI's Repository (SRA, Short Read Archive)

Project ID is SRP071638.

Here is the link to the project search results:

http://www.ncbi.nlm.nih.gov/sra/?term=SRP071638

Sample Names are as used in the publication.

[seq198]

Maybe I missed it, but is paired-end sequencing possible using CATS? Especially for longer ssDNA fragments?

[HeidelbergScience]

Maybe I missed it, but is paired-end sequencing possible using CATS? Especially for longer ssDNA fragments?

Yes, the paired-end sequencing is also possible, however, only in the commercially offered CATS kits:

Diagenode is now selling CATS kits for RNA-seq:

https://www.diagenode.com/categories/library-preparation

and they also offer a custom sequencing primer for read2 to be purchased separately:

https://www.diagenode.com/p/CATS-paired-end-sequencing-primer-50ul

CATS DNA-seq kits will be launched later, and will also include read2 custom sequencing primer.

[seq198]

Thank you for the information and congrats for finding a company for commercialisation! Do you know whether the custom sequencing primer does also work when CATS libs are multiplexed with other libs, i.e. when the standard Illumina read 2 primer is competing?

[HeidelbergScience]

Thank you for the information and congrats for finding a company for commercialisation! Do you know whether the custom sequencing primer does also work when CATS libs are multiplexed with other libs, i.e. when the standard Illumina read 2 primer is competing?

Thanks!
Yes, unfortunately, the standard Illumina primer for read2 will compete with the custom read2 primer and they can not be used together.