Rescuing a Nextera XT PAL?

[nickloman]

We regularly perform 96 barcode Nextera XT and the results are usually pretty good. However the last run on the MiSeq gave us a cluster density of a bit less than 200k, and less than half of the fragments were >200 bp. We think this may have been due to insufficient mixing of fragments with beads during the initial AMpure cleanup.

Rather than wasting about £2500 in consumables, we were wondering if it might be possible to salvage this library. One idea was to re-pool from the storage plate and do a clean-up of the new pooled amplicon library, probably with AMpure as that's what we have available (e-Gel would be another idea but we don't have that in the lab). Any ideas on whether this is a good idea?

Then we might do a KAPA qPCR on the final library to ensure we get the dilution right for the DAL to get the clusters up.

Possible? Pointless? Your opinions appreciated!

[mcnelson.phd]

Not sure what exactly you have left, but if you still have sufficient volume from your post-PCR AMPure cleanup plate, and you have a sufficient amount of DNA, then you can just treat the libraries like they're normal Illumina libraries and pool and denature them on your own.

This is how we normally treat our XT libraries because we've been bitten a few times with poor pooling or really high/low cluster densities. This assumes, of course, that you get enough yield after the post-PCR cleanup to go that route, but so far we've only had a few libraries that don't meet the required yield.

Only other option I can think of is if the PAL pool is still double stranded then you can just speedvac it to concentrate, do your KAPA qPCR to figure out the concentration, and then dilute and load like a normal library. You'd loose the ability to control the pooling, but at this point it seems your desperate to get something working with what you have.

[nickloman]

Thanks! I was beginning to think no-one was out there Unfortunately we binned the post-PCR plate rather hastily. Next time we will keep it.

We did try cleaning up the PAL using an AMPure protocol, but when we came to run the cleaned-up library on the Bioanalyzer, we didn't see any DNA, so we may have got it wrong.

We've given up on that plate and started again, but this time we will add some QC checkpoints after PCR to check things are progressing as expected.