PARE sequencing; single-read cluster generation

[hoytpr]

We got a request to do PARE sequencing (J. Zhai et al. / Methods 67 (2014) 84–90, http://dx.doi.org/10.1016/j.ymeth.2013.06.025) but someone correct me if I'm wrong.

The "single-read cluster generation" needed for this protocol can only be performed with a qBot... correct?

So our NextSeq, which does all clustering in the flowcell, can't sequence these libraries (but an HiSeq2500 could)?
Thanks,
Peter

[GW_OK]

You mean a cBot?

No, you can do single reads on a NextSeq. I skimmed through the protocol and didn't see anything that made it cBot/Hiseq specific. Maybe I missed something?

[hoytpr]

Hi GW,
Yes I mean cBot (). But after digging deep in the protocol I saw this:

# During cluster generation on the cBot, it is necessary to select
the ‘‘SR_TubeStripHyb’’ recipe because the PARE sequencing
primer is not compatible with the standard Illumina TruSeq
Sequencing primer.
# Load the template and primer tube strips into the appropriate
positions on the cBot. Follow Illumina standard procedures to
perform single-read cluster generation with tube-strip primer
hybridization.

The group followed the published protocol exactly. If they had used different PARE sequencing primer, then the libraries would have been able to cluster on the NextSeq flowcell (it doesn't have "single-read cluster generation" capabilities). I'm going to work with them to adjust the protocol for the NextSeq.

If I'm wrong please let me know...

-p

[GW_OK]

But you can spike in custom primers, yeah?

And single read or paired-end, it doesn't really matter on the NextSeq? If it'll cluster you can just set it to do read 1.

We do single reads all the time with the 75 cycle kit.

[hoytpr]

Hadn't thought of custom primer spike-ins. What a cool idea!

Just add in the same number of pMoles... as long as one end of the library attaches to the flowcell, and the custom primers make it to the clustering (PCR) step without being washed out.

Does Illumina just give you a modified (shortened) primer in the 1 X 75 Kit? (I have so much to learn)

I've gotta try that!

Thanks,
-p

[GW_OK]

Custom primers have nothing to do with generation of clusters. Both ends of the library adapters must have the requisite P5 and P7 sites for proper bridge amplification on the flowcell.

And you don't necessarily have to spike in primers, you can use the custom primer wells on the cartridge. The spike-ins do have the added advantage of allowing you to run the standard phix library in parallel, though.

As far as I'm aware the primer sets are the same across all NextSeq kits. You can tell the machine to sequence in whatever number of cycles you want in either direction or the index reads, so long as the total number of cycles you are requesting is equal to or less than the number of cycles on the box plus 16.

[hoytpr]

Just to put a tack in this thread.
Communication in cores is always a key to success. The client created a perfectly compatible small RNA Illumina library. They just didn't know it.

Edit: They referred us to the article, but apparently were using a protocol handed down from someone who left the lab. Once we asked for the exact library structure, and asked the correct person at Illumina, it all became clear. Thanks for your help GW.