title: NoCal cat flea microbiome
design: The extraction of the genomic DNA (gDNA) from each flea pool was as previously described (Billeter, et al. 2016 - PMID:26824189). A locus comprising the variable regions V4-V5 from the 16S rRNA gene was chosen to be targeted as our PCR amplicon, using primers 519F (5-CAGCMGCCGCGGTAATWC-3) and 926R (5-CCGTCAATTCCTTTRAGGTT-3). A nested PCR reaction was performed for each one of the 19 flea samples, plus water as negative control (NTC), in order to double-tag the amplicons with both Illumina Nextera adapter sequences and barcodes (A-H forward and 1-12 reverse, see the manufacturer protocol for more details) (Faircloth and Glenn 2012 - PMID:22900027). Following PCR, amplicons were normalized with the Invitrogen SequalPrep kit and the library containing the double-tagged amplicons of each sample was submitted for paired-end 300bp sequencing on the Illumina MiSeq platform.