Hello, I am analyzing a sequencing experiment that used the V3-4 region using a functioning pipeline that (I think?) mostly follows the MiSeq SOP. Originally when I ran the pipeline everything seemed okay, but now I’m rerunning it and there is an issue with classify.seqs() step in which almost all of my sequences are left as unclassified and subsequently removed with remove.lineage(). I am going crazy trying to figure out what is going on, and why the two runs have such different behaviors. I don’t think the runs are using any different commands or reference files, and my best guess is that there is something funky going on during alignment as the first run had a final filtered alignment length of 818 whereas the second run has a length of 809.
I have tried identifying the issue, but it looks to me like identical commands are being used in both runs. Apologies if I am mistaken, but right now I would greatly appreciate a second set of eyes as a sanity check. 
Here is the logfile for the first run (that worked):
And here is the logfile for the second run (that didn’t work):
Any suggestions would be very much appreciated!