Computational protocol: RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition

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[…] DNA extracts were submitted for 16S rRNA gene amplicon sequencing performed at the University of Michigan Medical School according to Kozich et al. []. This protocol uses dual index-labeled primers that target the V4 region of bacterial and archaeal 16S rRNA genes (515F/806R) []. Pooled and purified libraries were sequenced on an Illumina MiSeq sequencer, using v2 chemistry 2x250 (500 cycles) paired-end reads. RTA v1.17.28 and MCS v2.2.0 software were used to generate data from four separate runs (run 1: March 12, 2013; run 2: April 28, 2013, run 3: May 31, 2013, run 4 (for CARD-FISH comparison): Jun 13, 2014). The same three replicate Enz DNA extracts, stored at -20°C since their extraction on September 9, 2012 were included in each sequencing run. We analyzed the data using mothur v.1.30.1 based on the MiSeq standard operating protocol accessed on August 13, 2014 using SILVA release 102 for alignment and classification. All statistical analysis, including the Bray-Curtis dissimilarity matrix and non-metric multidimensional scaling (NMDS) ordination generation, as well as AMOVA (10,000 iterations) and Metastats analysis were performed in mothur according to the 454 standard operating protocol [] accessed on August 13, 2014. We rarefied the data at a subsampling level that allowed inclusion of all technical comparisons (820 subsamples) and a level that excluded only the Bead DNA extraction protocol (2,800). Sequencing data for the Muskegon Lake samples compared to CARD-FISH data were generated on a separate MiSeq sequencing run, similar to the method described above. Data analysis was performed as described above. Fastq files were submitted to NCBI sequence read archive under BioProject PRJNA271696, SRA accession number SRP051811. […]

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