Hl_larva_7
Microbiome screening targeting the hypervariable V4-V5 regions of the bacterial 16S rRNA gene was conducted according to published protocols (Caporaso et al., 2011; Walters et al., 2016). Primer pairs (515Fa and 926R) targeting the V4 and V5 loci, respectively, were used (Caporaso et al., 2011; Parada et al., 2016; Walters et al., 2016). The amplicons from all the samples and the negative DNA extraction controls were submitted to Sequencing Core of the University of Illinois at Chicago, USA, for sequencing. Amplicons were generated using a two-stage PCR amplification protocol as described previously (Naqib et al., 2018). The primers contained 5 common sequence tags (CS1 and CS2) as described previously (Moonsamy et al., 2013). PCR conditions were 95C for 5 minutes, followed by 28 cycles of 95C for 30, 55C for 45 and 72C for 30. Subsequently, a second PCR amplification was performed using Fluidigms Access Array Barcoded primers for Illumina (Fluidigm Cat#100-4876), and they contained both forward primer and reverse primer with barcode. The cycle conditions were 95C for 5 minutes, followed by 8 cycles of 95C for 30, 60C for 30 and 72C for 30. After sample amplification, purification and normalization were performed using SequalPrep plates (ThermoFisher Scientific, A1051001). Libraries were then pooled in equal volume and the pool was purified using an AMPure XP cleanup protocol (0.6X, vol/vol; Agencourt, Beckmann-Coulter) to remove fragments smaller than 300 bp. The final pooled libraries were quantified using Qubit quantification (Qubit 3.0 Fluorometer, ThermoFisher Cat#Q33217) and diluted to 2 nM.