16S rRNA gene amplicon sequencing of gDNA from Lake 227 (IISD-Experimental Lakes Area), Jul 2018, 9 m depth
The 16S ribosomal RNA gene was amplified from genomic DNA using the universal prokaryotic primers 515F-Y (doi:10.1111/1462-2920.13023) and 926R (doi:10.1186/1471-2105-12-38). The primers had an attached Illumina sequencing adaptor and sequencing barcode in a configuration described previously (doi:10.1128/AEM.02772-10). Amplifications were done in triplicate using previously described reagents and thermocycler conditions (doi:10.1093/femsec/fiz161). Following amplification, 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, spiked with PhiX, and sequenced on the MiSeq platform (Illumina) with 2x250 base reads.