SSU rRNA amlicon of soft scale insects microbiome
The symbiotic bacterial and fungal communities of the soft scale insect species were assessed using deep-sequencing of small subunit ribosomal RNA (rRNA) gene amplicons. Briefly, the widely used primer sets 341F/805R (with slight modifications), targeting the V3-V4 variable region of bacterial 16S rRNA was used for amplification (Bartram et al. 2011; Kennedy et al. 2014) A two-stage PCR (or targeted amplicon sequencing) approach was performed to generate amplicon libraries, as described previously (Bybee et al. 2011; Green et al. 2015). In the first of the two-stage amplification procedure, the templates were amplified (28 cycles) using the primers 341F and 805R containing 5 CS1 and CS2 linkers (Moonsamy et al. 2013). PCRs were performed in 10ľl reaction volumes using MyTaq HS 2X mastermix (Bioline, Taunton, MA). PCR conditions are specified in Table S1. Subsequently, a second PCR reaction was established, with 1ľl of amplification product from the first stage used as input to the second reaction. The primers for the second stage amplifications were the AccessArray barcoding system primers (Fluidigm, South San Francisco, CA), containing Illumina sequencing adapters, sample-specific barcodes (10 bases, with a minimum hamming distance of 3), and CS1 and CS2 linkers. The second stage PCRs were performed in 10ľl reaction volumes using MyTaq HS 2X mastermix (Bioline, Taunton, MA), and the PCR conditions were as follows: 5min initial denaturation at 95°C, followed by 8 cycles of: 95°C for 30s, 60°C for 30s, 72°C for 60s. Final PCR products were purified and equalized using SequalPrep Normalization Plate Kit (Thermo Fisher Scientific), according to the manufacturers instructions. Samples were pooled in equimolar ratio and quantified using a Qubit 2.0 fluorometer. Sequencing was performed on an Illumina MiSeq sequencer using standard V3 chemistry with paired-end, 300 base reads. Fluidigm sequencing primers, targeting the CS1 and CS2 linker regions, were used to initiate sequencing. Demultiplexing of reads was performed on instrument. Library preparation and sequencing was performed at the DNA Services Facility at the University of Illinois at Chicago.
run5_Elad15-Mm18_S160