16S rRNA gene amplicon sequencing of a lake water column: 3.5 m depth
The 16S ribosomal RNA gene was amplified from genomic DNA using the universal bacterial primers 341f (5Ő CCTACGGGAGGCAGCAG 3Ő) and 806r (5Ő GGACTACHVGGGTWTCTAAT 3Ő). The primers had an attached Illumina sequencing adaptor and sequencing barcode in a configuration described previously [1]. Amplifications were done in triplicate PCRs using previously described reagents and thermocycler conditions [2]. Following amplification, triplicate PCR products were pooled, and the pooled product was normalized in concentration to other samples being sequenced before all samples were pooled into a final amplicon library. This library was gel purified using the Wizard SV Gel and PCR Clean-Up System (Promega), spiked with 8.5-10% PhiX, and sequenced on the MiSeq platform (Illumina) with 2x250 base reads. [1. Bartram AK et al. (2011), Appl. Environ. Microbiol. 77, 3846Đ3852, doi:10.1128/AEM.02772-10; 2. Kennedy K et al. (2014), Appl. Environ. Microbiol. 80, 5717Đ5722, doi:10.1128/AEM.01451-14.]
SUB2124897
ELA_L227_2011_3_5m