16S rRNA soil bacteria crop fields in AR
Total metagenomic DNA from each freshly collected soil sample was extracted using PowerLyzer PowerSoil DNA isolation kit according to the manufacturer's instructions. Samples were sequenced at the UAMS Sequencing Core Facility using Illumina MiSeq platform. Primers specific to the V3 and V4 regions of the 16S rRNA gene were used. PCR amplification was performed on genomic DNA, which attaches Illumina adapters to the ends of the resulting amplicons.  These adapters contain sequences that allow attachment of unique barcodes sequences, which also allow the products to attach to the flow cell on the Illumina MiSeq system.  While attached to the flow cell, each amplicon was further amplified on the instrument, creating clusters of identical amplicons. Each amplicon cluster was sequenced using 600 cycles (300 cycles in each direction). Raw sequence data was processed in multiple steps using the QIIME pipeline. First, the 300 base pairs (bp) paired-end reads were joined using the fastq-join method with minimum allowed overlap of 120 bp and 15% maximum allowed difference within region of overlap. Second, reads with more than three consecutive base calls having Phred score <20 were truncated, reads with any ambiguous base call were discarded, and reads from different samples were tagged with sample identifiers and merged into a single FASTA file. Third, sequence reads were aligned against the core reference alignments of the greengenes database using pyNAST and operational taxonomic units (OTUs) were picked at 97% sequence similarity and assigned taxonomy using uclust. Sequences that failed the closed-reference alignment to the greengenes database were aligned de novo and OTUs with <2 aligned sequences were discarded.