Seasonal changes in the microbial community of the Eastern Mediterranean Sea: 11 um DNA samples
Genomic archaeal and bacterial DNA of the SSU rRNA gene V4-5 hypervariable region was amplified using the forward primers CS1_515F-Y (ACACTGACGACATGGTTCTACA_GTGYCAGCMGCCGCGGTAA) and reverse primers CS2_926R (TACGGTAGCAGAGACTTGGTCT_CCGYCAATTYMTTTRAGTTT) primers. PCR amplicons were generated in triplicate 25 l volumes containing 0.5 ng of template, 12.5 l of MyTaq Red Mix (Bioline), 0.5 l of 10 M of each primer. All PCR products were validated on 1% agarose gels and then triplicates were pooled. Four negative controls without template were run and sequenced in parallel. A unique 10-base barcode, obtained from the Access Array Barcode Library for Illumina (Fluidigm, South San Francisco, CA; Item# 100-4876), were added to samples in a second PCR amplification using the MyTaq HS 2X mastermix and 10 l reaction volumes. Samples were pooled in equal volume. The pooled library was purified using an AMPure XP cleanup protocol (0.6X, vol/vol; Agencourt, Beckmann-Coulter) and sequenced on an Illumina MiniSeq mid-output flow cell (2x150 paired-end reads). Based on the distribution of reads per barcode, the amplicons (before purification) were re-pooled to generate a more balanced distribution of reads. The re-pooled library was purified using AMPure XP cleanup, loaded onto a MiSeq v2 flow cell, and sequenced on an an Illumina MiSeq sequencer (2x250 paired-end reads). Sequence library preparation, pooling, and MiniSeq sequencing were performed at the DNA Services (DNAS) facility, Research Resources Center (RRC), University of Illinois at Chicago (UIC). MiSeq sequencing was performed at the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign (UIUC).