I've recently prepared an amplicon library using MiFish primers (Miya et al.) with adapter overhangs according to this protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). The annealing temperature I used for the amplicon PCR is 50, besides that all the conditions were the same as described in the protocol.

According to the publications, MiFish primers should yield an amplicon of the size of ~172 bp, which should result in a library size of ~322 bp. However, the fragment analysis results of the final libraries all show a sharp peak at around 460 bp, which makes my insert size around 300 bp.

Does anyone have an idea on what could have gone wrong?