title: Environmental DNA reveals the structure of phytoplankton assemblages along a 2900-km transect in the Mississippi River
design: DNA from the two fractions for a site were combined. For some sites, DNA from the two fractions taken from the two replicate samples were combined. Phytoplankton sequences were amplified at the 23S rRNA gene region, which is located on the chloroplast and can amplify DNA from taxa such as cyanobacteria, green algae, and diatoms (Sherwood and Presting, 2007). Initial PCR amplification included Promega Mastermix, forward and reverse primers, gDNA, and DNase/RNase-free H2O. After an initial 3-minute period at 94 C, DNA was PCR amplified for 40 cycles at 94 C (30 seconds), 55 C (45 seconds) and 72 C (60 seconds), followed by 10 minutes at 72 C. Products were then visualized on an 2% agarose gel. 20 l of the PCR amplicon was used for PCR clean-up using ExoI/SAP reaction. In order to index the amplicons with a unique identifier sequence, the first PCR stage was followed by an indexing 8-cycle PCR reaction to attach 10-bp error-correcting barcodes unique to each sample to the pooled amplicons. These products were again visualized on a 2% agarose gel and checked for band intensity and that amplicons are the correct size. PCR products were purified and normalized using the Life Technologies SequalPrep Normalization kit and samples pooled together. Amplicons were sequenced on an Illumina MiSeq at the University of Colorado Boulder BioFrontiers Sequencing Center running the v2 500-cycle kit.