Is there a recommended/favorite protocol for performing template-switching cDNA synthesis where the final sequenced amplicon includes isotype-differentiating information? Something similar to Turchaninova et al but with validated primers that include isotype info. (It seems like that protocol may not be able to differentiate between IgG and IgE.)
An oligo-dT primed synthesis with Superscript-II, using an additional template switch oligo, should do it.
You just need to use constant region primers that bind to the appropriate isotypes during the subsequent PCR reaction.
In the Turchaninova protocol only the first 15 bps of _IGHG_x or IGHE are non-templated, which results in only 3 positions to distinguish the G and E isotype and none for reliable subtyping of _IGHG_x. This is an effect of primary PCR (not RT) primer location, which I assume was a trade-off between constant segment information and the requirement for proximal primer placement so that the CDR3 is still covered by the shorter one of the reads (100 bp). Two ideas on this:
- Have you looked at Cole et al.? Their protocol was specifically designed to provide constant region information.
- If your sequencing facility could do asymetric 400+160 bp reads (instead of the original 400+100 bp), you could move the constant primers further 3’. We now have a new (Illumina compatible) version of our human Ig single-cell PCR primer set, which I am happy to share. It would provide you with up to 70 bp of the IGH@ constant region, which should be enough for most applications (except IGHG2 vs IGHG4 identification).
Thanks, @bussec! I do recall the Cole protocol from the meeting in June, but I’m looking for something simpler. I would greatly appreciate it if you could share the latest primers you’re using for an Illumina workflow.
@bussec in asymmetric logic the short read is only used at the step demultiplexing step (MIGEC/Checkout) and is then discarded. The 400-bp reads are then assembled, merged and used for V/D/J/C mapping. The 100bp read is actually quite a mess, only first bases with the UMI tag get somewhat good quality. Extra 60 bases will likely have even worse quality.
But I’ll better ask Dr. Turchaninova before any further suggestions on isotype resolution…
Sorry, my bad! I should have looked at Fig. 2 of the paper again, which shows clearly that CDR3 is not involved during consensus building. So there should be no need to change the 400+100 setup.
Since the same figure also mentions that the raw amplicons can be up to 750 bps in length, what is the length of the overlap between the 5’- and 3’-end consensus that you get with the published protocol?
The length of the overlap actually depends on the length of product starting from C primer. So for 750-base long cDNA there will be ~50 overlapping bases. Distribution of overlapping bases is the derivative of the length of products.
As for the primers I’ve just got the reply:
We don’t have primers to reliably tell IGHGX, as the primers should be placed ~70 bases downstream of J and they are not so reliable (only 2 base difference between G2/4) so we decided not to focus on those in current protocol.