hybridization of custom low concentration ssDNA library

[pyridine]

I've been preparing libraries enriched for transposon-chromosome junctions using a sub-optimal non-kit procedure which gives me extremely low concentrations of ssDNA in the 200-1000 pM range, as determined by qPCR. To clarify, the final library is already in ssDNA form because there is an affinity capture step of biotinylated PCR product in the last part of the preparation on streptavidin beads, and denaturation is performed on the beads to elute the complementary strand. The final ssDNA product is then column-purified and eluted in 10 mM Tris-Cl pH 8.5.

The first time I performed this procedure, I had somewhat higher concentrations of each library (2.4 to 11.8 nM), and was able to successfully skip to pooling the 4 libraries directly in a 12 uL total volume, then take 3 uL of this, add 3 uL of 0.2 N NaOH, and proceed with the dilution to 10 pM in hybridization buffer as usual. This gave a good enough distribution of reads between each of the 4 indices, ranging between 16 and 24% of reads each on a MiSeq.

This time I'm trying to scale up the procedure and pool 8 libraries that are at a lower concentration, so it will not be possible for me to prepare the dilution that is denatured with an equal volume of 0.2 N NaOH. I can however, still dilute the libraries directly to a total pooled concentration of 8-10 pM in hybridization buffer (HT1), albeit with errors introduced from pipetting small volumes. My question is if the denaturation step is still necessary if the library is already ssDNA?

Also does anyone have experience reducing from 10 pM to 8 pM concentration in buffer HT1?

Thanks for any tips.

[kcchan]

You can always heat denature your samples just to be safe. Dilute your libraries in HT1 to the desired concentration and incubate it at 95°C for a minute or so and immediately place it on ice. Once the tube has cooled down you can load it on the MiSeq.

[pyridine]

Thanks for your reply. I'm glad to hear it suggested from someone else, since that was also what we decided to do. I'll know if it works later today. It will be a bit shy of a 1:100 dilution (8 uL library in 592 uL) but hopefully that will be too little difference to matter.

It seems that the low concentrations were low due to poor efficiency of the PCR enrichment this time around.