16S Amplicon library of Cimex vicarius
Using the QIAGEN Multiplex PCR Kit (Qiagen, Hilden, Germany), we amplified the V3V4 region of the 16S rRNA gene from all samples, as well as from four blank (negative) PCR controls and two commercial gDNA (positive) controls (ATCC MSA-1000 and MSA-1001, each containing the same ten bacterial species in different proportions).?Two-step PCR was performed with 341F and 805R primers, containing staggered spacer and Illumina overhang adapters, followed by 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 using v2 chemistry (2  250 bp paired-end reads).?