title: Fleas from Household 30, Dog A, Pool 3
design: Samples were prepared in a similar process to the one described in Illuminas 16s Metagenomic Sequencing Library Preparation Protocol, Part #  15044223 Rev. B (Illumina Inc., San Diego, California, USA) with the following modifications:  The 16S rRNA gene V3/V4 variable region was amplified with fusion primers (forward primer 341f: 5-ACACTCTTTCCCTACACGACGCTCTTCCGATCT(N)0/6CCTACGGGNGGCWGCAG-3, reverse primer 805r: 5-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(N)0/6GACTACHVGGGTATCTAATCC-3). Region specific primers were previously described in Klindworth et al., 2013 (underlined sequences above), and were modified to add 0 or 6 random nucleotides ((N)0/6) and Illumina adapter overhang nucleotide sequences 5 of the genespecific sequences. Following initial amplification, reactions were cleaned using a 0.7x volume of AxyPrep Mag PCR clean-up beads (Axygen Biosciences, Union City, CA).  In a subsequent PCR, Illumina dual indexes and sequencing adapters were added using the following primers (forward primer: 5-ATGATACGGCGACCACCGAGATCTACAC[5555555555]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3, Reverse Primer:  5-CAAGCAGAAGACGGCATACGAGAT[7777777777]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3, where bracketed sequences are 10bp custom Unique Dual Indexes). Following PCR, reactions were cleaned using a 0.7x volume of AxyPrep Mag PCR clean-up beads (Axygen Biosciences). Quality and quantity of the finished libraries were assessed using an Agilent 4200 TapeStation DNA 1000 kit (Agilent Technologies, Santa Clara, CA) and Qubit dsDNA HS Assay Kit (ThermoFisher Scientific), respectively. Libraries were pooled in an equimolar fashion and appropriately diluted prior to sequencing. Paired end, 300 bp sequencing was performed using the Illumina MiSeq Sequencer and a MiSeq 600 bp (v3) sequencing cartridge.
identifiers.namespace: SUB14937090
identifiers.id: D30A3