Microbiome Analysis of Deceased Bed Bugs
To further characterize the composition and relative abundance of bacteria present in deceased bed bugs, DNA was extracted from bed bug slurry using the Qiagen DNeasy Blood and Tissue Kit (Cat. No. 69504, Qiagen, Hilden, Germany). After extraction, DNA was quantified on a Qubit Fluorometer (ThermoFisher, Waltham, MA). 50 ng of DNA were used to produce amplicons using a MetaVx library preparation kit (performed by Genewiz, LLC). In brief, proprietary primers were used to amplify V3-V5 hypervariable regions of the bacterial 16S rRNA. Two rounds of PCR were performed, with the 2nd round being used for enrichment. Indexes were added to the ends of the amplicons to create indexed libraries for downstream analysis. Libraries were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and quantified by Qubit Fluorometer (ThermoFisher). Libraries were multiplexed and loaded onto an Illumina Miseq (Illumina, San Diego, CA, USA) following the manufacturers instructions. 2x300/250 paired end sequencing configuration was used.