V4-V5 16S rRNA tag seq of Trichodesmium spp.
Samples were filtered onto 5 um polycarbonate nucleopore filters. Extractions performed with MPBiomedical spin kit for soil. Extracts were sent for sequencing by a commercial vender (Molecular Research LP, MR DNA, Shallowater, TX, USA). Illumina MiSeq paired-end (2 x 300 bp) tag sequencing was performed with universal primers targeting ~400 base pairs spanning the V4V5 region of the 16S rRNA gene (515f: 5Ő-GTGCCAGCMGCCGCGGTAA-3Ő; 926r: 5Ő-CCGYCAATTYMTTTRAGTTT-3Ő)30. Library preparation and sequencing was carried at the facility. In brief, the 515f/929r primers with 8-bp barcodes on the forward primer were utilized in a PCR reaction with the HotStarTaq Plus Master Mix (QIAGEN:USA, Valencia, CA, USA) under the following conditions: 94_C for 3 min, followed by 28 cycles of 94_C for 30 s, 53_C for 40 s, and 72_C for 1 min, and a final elongation step at 72_C for 5min. PCR products were run on a 2% agarose gel to confirm amplification and check relative intensity of bands. Then, based on molecular weight and DNA concentrations, multiple samples were pooled together in equal proportions, purified with Ampure XP beads, and used to prepare the sequencing library following the Illumina TruSeq DNA library preparation protocol.