Hello. I'm looking for suggestions on troubleshooting index (or barcode) PCR. Specifically, my problem is that some of my sample DNA concentrations dropped after adding the indices/barcodes. My ideal concentration is no lower than 9nM. One 96-well plate had 5 samples and another plate had 28 samples that were below 9nM.

For context, I'm adding Illumina indices to my mixed amplicon (combined ITS and 16S) sequences. After the index PCR, I cleaned up my samples and quantified them with Qubit. I had some samples that dropped significantly in their DNA concentration after this step (index PCR). For example, one sample had 133nM (measured after 16S amplicon PCR), but then dropped to 8nM (measured after index PCR).

Prior to indexing, I diluted the separate ITS and 16S plates to 20nm then combine 2uL of each of the 20nM diluted plates together. From the now mixed 4uL, I then use 1.5uL of each mixed amplicon sample for index PCR. For the index PCR, I typically use Kapa HotStart ReadyMix (7.5uL for 1 reaction), water (3uL for 1 reaction), and 1.5uL of each index (for each sample).

The thermocycler settings are:

I've already increased the DNA concentration input for the index PCR. Originally, it was 10nM, but 20nM worked better. I'm unsure about increasing the DNA concentration again as many of the samples lowest concentrations (prior to indexing) were around 20nM.

Another suggestion was to toggle with the thermocycler settings, but I'm unsure how to optimize these as other samples worked fine with these exact settings.

Thanks for reading this very long post. I'm open to any suggestions on how people have troubleshooted indexing!