Two-step PCR protocol for Illumina sequencing

[EmmaUoW]

Hi everyone,

This is my first post, so apologies if it is in the wrong place or if there is already a thread on the same topic!

I am currently trying to optimise a protocol to process 380 samples using the Illumina MiSeq platform. I am using the two-step PCR process to attach the necessary adapters to my samples in preparation for sequencing.

From doing a bit of reading around, there seems to be no standard protocol for doing this, everyone seems to adapt depending on the samples they are looking at. Am I right in saying this?

So far, I have used the same master mix components and concentrations previously used to successfully amplify my samples in the first step PCR. I am now trying to test different cycle numbers to see what the minimum number required is and trying a hot-start version of the master mix I use to reduce non-specific amplification. I was then going to clean-up and quantify before adding a set amount of DNA template for each sample to the second PCR (using same master mix and primer concentration etc as the first PCR) and then quantify again before pooling and denaturing to load onto the machine. I looked at the Nextera sample preparation guide and this states 12 cycles for the second PCR which seems a bit much as that kit amplifies sequences from transposon tagged genomic DNA, but this means I am unsure how many cycles is enough. I added the two quantification steps in as these samples have not been quantified before as they were so difficult to amplify in the first place.

I was just wondering if anyone had any suggestions or recommendations? Apologies for such a long post!

[microgirl123]

Are you using Nextera or Nextera XT adapters? I use the Nextera adapters, 5cycles (this is what the Nextera guide Rev. B calls for) with the conditions in the guide, and 50 ng of input DNA (quantified by Qubit for concentration and Nanodrop for 260/280 and 260/230). Afterwards, I quantify with the Qubit for pooling and then KAPA quant the final library.

[EmmaUoW]

Thanks for your reply. I am using Nextera XT adapters. The program in the guide says 12 cycles but I think this may be a little over the top. I was planning on doing the first PCR in as few cycles as possible, then purifying the products and quantifying them with Qubit and the Nanodrop and then using 50ng DNA of each sample in the second PCR beofre quantifying again and pooling. I think I just need to the cycle numbers for each PCR right!