Reduced sequences after joining paired ends

[Nicholas_Bokulich]

@rlhughes

I also demultiplexed using cutadapt without trimming barcodes/primers, imported, and joined, and did not observe this big decrease in sequence number

@rlhughes you are using vsearch to join paired-end reads, not dada2, but this post might explain the phenomenon you are seeing:

DADA2 paired-end (EMP): All sequences of same length

Ah I see what happened here. The way you trimmed your reads initially resulted in the reverse read always completely overlapping the forward read. That is, the full amplicon length varies (tightly) between about 251-255 nts. But since you cut off the first 5 nts of the reverse read, it always started at a position within the forward read, and because it was truncated at 203 nts, it never extended past the other end of the forward read. So every merged read is just the length of the forward read…