title: Microbiome of Polyplax serrata sucking louse
design: The V3V4 region of the 16S rRNA gene was amplified from all samples using the QIAGEN Multiplex PCR Kit (Qiagen, Hilden, Germany). Amplifications included four blank (negative) PCR controls and two commercial genomic DNA (positive) controls (ATCC MSA-1000 and MSA-1001, each consisting of the same ten bacterial species in different proportions). A two-step PCR protocol was applied with primers 341F and 805R containing staggered spacers and Illumina overhang adapters, followed by an index PCR to add sample-specific barcodes (Illumina 16S Metagenomic Sequencing Library Preparation Guide). Purified amplicons were quantified, pooled equimolarly, and sequenced on an Illumina MiSeq platform using v2 chemistry with 500 cycles.