Nextera XT on HiSeq

[koadman]

Hi all, I've been looking at this as well. Reading through the Illumina protocol pdf, I noticed the following:

- Add 576 μl of HT1 to the DAL tube.
- Transfer 24 μl of PAL to the DAL tube containing HT1. Using the same tip, pipette up and down 3–5 times to rinse the tip and ensure complete transfer.


The recommended volumes for diluting PAL with HT1 represents a 25-fold
dilution. This dilution ratio was established by using the recommended
equipment (e.g. plate shaker calibrated for shaking speed) and following the
normalization procedure strictly under typical laboratory conditions (e.g. 20°–
25°C). If cluster density is found to be too high or too low, you may change
this dilution ratio to better suit the equipment, temperature, and user handling
in your laboratory after validation.

Which suggests that one might be able to play with the relative amounts of hybridization buffer and bead-normalized sample material. Would anybody care to speculate on whether this would or would not work for getting a library at the right loading concentration for the HiSeq? My understanding is that HiSeq loading concentration should be about 1.5x the miseq. The protocol calls for 24uL library in 600uL. What about 36uL library in 600uL?

[genomeseeker]

We're starting a HiSeq run next week on 2 pools of Nextera XT libraries. We quantified the final pools by qPCR (KAPA kit) and plan to treat them like all other libraries and load them 11pM.

Here's the instructions we received from tech support:
As long as you are not interested in following the Amplicon workflow, you can, in theory, sequence your Nextera XT libraries on a HiSeq. Currently Nextera XT is only supported on the MiSeq. If you want to sequence them on the Hiseq, here are some recommendations and guidelines. Please note that this is NOT supported or guaranteed.

1. Stop the Nextera XT protocol after PCR clean-up and don't proceed to Library Normalization. At this point, your library is already made.
2. Quantify each library using a dsDNA-specific fluorescent dye method, such as Qubit or picogreen.
3. Normalize and pool the libraries as needed.
4. Cluster on a cBot and use sequencing primers from the TruSeq Dual Index Sequencing Primer Box, Single Read or Paired End, depending on the type of the run.

[pmiguel]

Yeah, that is disappointing. Zymo has ssDNA/RNA clean-up/concentration columns. My guess is that they would work.

--
Phillip

[Christine]

Hello all,

I'm wondering if anyone has sequenced Nextera XT libraries on the HiSeq and what modifications were made to get the cluster density in range?

I've been sequencing the XT libraries on the MiSeq so far and am very pleased with the performance (and the normalization!). We're scaling up to a HiSeq run next, with up to 96 libraries per lane. Needless to say, I was looking forward to skipping all those individual library quantifications, but Illumina has advised me not to use the normalization beads when prepping for the HiSeq run. Their reasoning was that the concentration of the library pools will be too low to get a decent cluster density.

The yield from the beads is not sufficient for optimal cluster density on the HiSeq. This is why we recommend to skip the normalization step if you plan to sequence Nextera XT libraries on the HiSeq. At this step you can quantify your libraries using qPCR. It is a bit more work, but it is the only way to successfully sequence Nextera XT libraries on the HiSeq.

Surely there's a simple workaround. Can I concentrate the pools on some columns, followed by 8 qPCRs instead of ~800? The libraries will be single stranded, so the binding efficiency might not be great and the qPCR will need a correction factor, but I think it's worth a try. What do you think, seqanswers?