My ultimate question is whether my trimming and truncating provide for adequate overlap, and I would also like to verify my bp overlap.
So our paired-end reads covering the V3-V4 regions using 341F and 785R primers should yield a 444 bp (785 - 341) amplicon. In calculating the length of overlapping bases I get 156 bp (600 - 444) overlap.
If I truncate my forward and reverse at 260 and 240 respectively, my overlap would be 56 bp, correct? (260 + 240 - 444). Do I also need to include my left and right primer trims which are 17 and 21?
Please let me know if I can clarify any of the above.
Hi @microbiomeAnalyst,
Your calculations look right to me.
Assuming you are planning on using dada2, the required overlap you want to keep is 20nt+ natural variation in your target. For example if your expected target of 440bp can also be 430 bps, then you would need 20nt+10nt minimum overlap for proper merging, so your 56bp sounds pretty safe to me.
Not unless you were including these in your original calculations which I'm guessing you weren't. The trimming and truncating parameters refer to positions on your reads so your trimming (at 5' end) wouldn't change the position of your truncating (3' end) so you don't need to adjust your overlap calculation. But as you indicated, you should make sure your primers are removed prior to denoising.