Index hopping Hiseq4000 and single cell

[Bidfudge]

Hi everyone,

We are currently working on single cell experiment with the C1 platform from fluidigm. We plan to sequence 384 single cells with the "SMART-Seq® v4 Ultra® Low Input RNA Kit for the Fluidigm® C1™ System".

In this kit, they used a dual index for the single cell multiplexing.The problem is that we have to sequence these transcriptomes on an Hiseq4000, and It is well know now that some contamination with excess index-primer will lead to an index swapping after demultiplexing :/.

So my question is, does-anybody have an experience to limit this biais? I read some blog, were people used a size-selection of the libraries to remove excess of free primers, or the use the ExonucleaseI enzyme to remove it...

Thanks for the help,
nicolas

[GenoMax]

As long as you are using dual indexing chances of mis-assignment of indexes is significantly reduced. See this white paper from Illumina for additional ideas to reduce this effect.

[Bidfudge]

Hello,

thanks for the answer, yeah I will use dual index on my sample, but as I am suppose to pass 384 cells on the same flow cells, I will be in the same position as this paper https://www.biorxiv.org/content/bior...25724.full.pdf

I heard that there is a Pippin Prep in my institue, maybe It's a good way to remove index primer from my libraries after pooling sample which have to pass on the same lane?

nicolas

[GenoMax]

Bead washing the pool may also help remove excess primers etc. There are other threads on here that go into specifics of how you would use that or someone else with experimental expertise will post in this thread later.