Quantitation of Nextera XT library

[Cdiff]

I have searched the forum without success so apologies in advance if I have missed this elsewhere.

We are sequencing pooled Nextera XT preps of bacterial genomes, using the bead normalisation. Until now we have not carried out any post-normalisation quantitation and have been reasonably happy with clustering on the MiSeq V3 of between 1000K and 1600K/mm2

Recently, we have had a couple of failed runs with low clustering (<300K), possibly as a result of changes in staffing. We would like to use the Qubit ssDNA kit to quantify the pooled libraries. Does anyone have experience/suggestions/protocols?

[Buckethead84]

I think you'll need the dsDNA assay kit since these are libraries. It's fairly straightforward and you can just follow the manufacturer's protocol. Nothing particularly special needs to be done for library quantification.

[Cdiff]

We use the HS dsDNA kit for the Nextera XT input sample. But to measure the pooled normalised library, I think we need the ssDNA kit as the NaOH elution produces ss. Or am I wrong here?

[nucacidhunter]

Link to Qubit ssDNA manual below. You might need to use higher volume of pool for quantification. Start with 5 ul and if signal is below detection it can be increased up to 20 ul.

assets.thermofisher.com/TFS-Assets/LSG/manuals/Qubit_ssDNA_Assay_UG.pdf

You will need to set up a small volume PCR reaction to estimate pool size for calculating molar concentration. Primers should be complementary to P5 and P7 flow cell binding motives such as primer cocktail form a TruSeq PCR or KAPA qPCR kit.