title: Sample 27_HA_2, 16s V4 region. Barcode: CTAGTCTCGT
design: Samples were prepared from green leaves collected from four plant species: common blackberry (Rubus allegheniensis), Amur honeysuckle (Lonicera maackii), elderberry (Sambucus canadensis), and autumn olive (Elaeagnus umbellata). We established a completely randomized design composed of 15 treatments with five replicates per treatment. Four treatments consisted of leaves from a single plant species as a baseline for comparison of mixture treatments. Six treatments consisted of two-species mixtures, four treatments consisted of three-species mixtures, and one treatment was a mixture of all four species. Infusions were prepared by fermenting a total of 10 g green leaves in closed plastic 2 L buckets with tap water for 7 days. Thus, for the mixtures of two leaf species, 5 g of each species were infused together; for mixtures of three species, 3.3 g of each species were infused together; and for the mixture of four species, 2.5 g of each species were infused together. After 7 days, leaves and large particulates were strained out of the buckets by passing the infusion through cheesecloth.  To investigate differences in microbial communities among the leaf litter mixtures, 15 mL aliquots of 7-day-old whole infusion were taken from each container and stored at -80C until further processing. Genomic DNA was extracted from the samples using the UltraClean Soil DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad CA, Cat. No. 12800-50) according to the manufacturers instructions. The 75 DNA extracts were sequenced using Illumina MiSeq Bulk v3 analysis at the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign and bacterial operational taxonomic units (OTUs) were profiled across the 15 leaf litter mixtures. We targeted three hypervariable regions of the 16S rRNA gene using the following primer sets: V1-V3 forward 5GAGTTTGATCNTGGCTCAG3, reverse 5GTNTTACNGCGGCKGCTG3; V3-V5 forward 5CCTACGGGAGGCAGCAG3, reverse 5CCGTCAATTCMTTTRAGT3; V4 forward 5GTGCCAGCMGCCGCGGTAA3, reverse 5GGACTACHVGGGTWTCTAAT3. For Illumina library preparation by Fluidigm protocols, DNA from each water sample was amplified using the three primer sets added with Fluidigm Illumina linkers and unique barcodes (Muturi et al. 2017a). The final Fluidigm libraries were pooled for sequencing at the Keck Centers DNA Services laboratory. The V4 region yielded 8.5 million reads, more than two times the number of reads for the V3-V5 region (3.9 million reads) and more than three times the number of reads for the V1-V3 region (2.6 million reads); therefore the V4 region was used for subsequent analyses.