I was just wondering if when choosing the kit for my MiSeq run if there was a specific amount of overlap for my reads I should be aiming for. My current target amplicon is ~300bp, so a 500 kit should get me near 100% overlap. I had assumed this would be fine and dandy until someone else complained about issues with having too high an overlap, so now I’m a bit scared.
Are my fears sound, or are the issues he’s running into likely due to something entirely different?
Yes! You want full overlap of the two reads. The 250PE kit (500 cycles works great for v4, which is 250 bp. If your region is 300 bp, then you want the 300PE kit. However, we do use the 300PE kit on the v4 regions, but you’ll have to tell the sequencer you only want 2x250 reads. Although you won’t get the length advantage, you will get the increased number of reads.
pat