16S rRNA sequencing of the bacterial community associated with the saliva-secreting gland of Rhodnius milesi raised in Fiocruz Rio de Janeiro, Brazil.
The V3 and V4 variable region of the 16S rRNA was amplified by PCR, using the primers 27F and 519R with barcode on the forward primer, in a 28 cycle PCR  (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94¡C for 3 minutes, followed by 28 cycles of 94¡C for 30 seconds, 53¡C for 40 seconds and 72¡C for 1 minute, after which a final elongation step at 72¡C for 5 minutes was performed.  After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples are pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads. Then the pooled and purified PCR product was used to prepare DNA library by following Illumina TruSeq DNA library preparation protocol. Paired-end sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturerÕs guidelines. Each file name contain the barcode used in the sequencing. R1 and R2 sequences can have both ends of the sequences.
SUB3102100
mil.Fio.1