Miseq dna

Tag pyrosequencing of 18S rDNA was performed using PCR amplification of the V2 and V3 region and by using the two eukaryotic primers 18S-82F (5′-GAAACTGCGAATGGCTC-3′) (Lopez-Garcia et al., 2003 (link)) and Euk-516r (5′-ACCAGACTTGCCCTCC-3′) (Amann et al., 1990 (link)). These primers were designed to amplify 470–480 bp in the region. Furthermore, cDNA (representing RNA) was amplified using the same primer pair targeting the same region. PCR reactions and barcode amplicon sequencing process described by Dowd et al. (2008) (link) was performed by the Mr. DNA Company (Shallowater, TX, United States)¹. Briefly, PCR conditions included a denaturation step at 95°C for 5 min, 30 cycles of amplification that were performed at 95°C for 30 s, 50°C for 30 s and 72°C for 1 min. A final extension step was performed for 7 min at 72°C. Subsequently, PCR products were purified using calibrated Ampure XP beads and the purified products were used to prepare the DNA libraries by following the Illumina MiSeq DNA high-throughput library preparation protocol. DNA library preparation and sequencing was performed at Mr. DNA (Shallowater, TX, United States)² on a MiSeq following the manufacturer’s guidelines. Sequences were submitted to GenBank-SRA under the accession number SRX3222420.