ECOPICS_MX Bulk soil and rhizosphere 16S amplicon sequences.

METHODSSoil samples for DNA extractions were collected as described in Collins et al. 2020.Soil DNA was extracted with the Qiagen PowerSoil DNA extraction kit, following the recommended protocol.16S amplicons were sent to Macrogen Inc. for sequencing.Type of Read: Paired-endRead Length: 301 bpNumber of Samples: 140Library Kit Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2Library Protocol 16S Metagenomic Sequencing Library Preparation Part #15044223 Rev. BType of Sequencer Illumina platform16s (V3-V4)Bakt_341F: CCTACGGGNGGCWGCAGBakt_805R: GACTACHVGGGTATCTAATCCMacrogen Inc. workflow:1) Sample PreparationFor library construction, DNA/RNA is extracted from a sample. After performing quality control(QC), qualified samples proceed to library construction.2) Library ConstructionThe sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followedby 5' and 3' adapter ligation. Alternatively, "tagmentation" combines the fragmentation and ligationreactions into a single step that greatly increases the efficiency of the library preparation process.Adapter-ligated fragments are then PCR amplified and gel purified.3) SequencingFor cluster generation, the library is loaded into a flow cell where fragments are captured on a lawnof surface-bound oligos complementary to the library adapters. Each fragment is then amplifiedinto distinct, clonal clusters through bridge amplification. When cluster generation is complete, thetemplates are ready for sequencing.Illumina SBS technology utilizes a proprietary reversible terminator-based method that detectssingle bases as they are incorporated into DNA template strands. As all 4 reversible,terminator-bound dNTPs are persent during each sequencing cycle, natural competition minimizesincorporation bias and greatly reduces raw error rates compared to other technologies. The resultis highly accurate base-by-base sequencing that virtually eliminates sequence-context-specificerrors, even within repetitive sequence regions and homopolymers.4) Raw dataSequencing data is converted into raw data for the analysis.2. 2. Generation of Raw DataThe Illumina sequencer generates raw images utilizing sequencing control software for systemcontrol and base calling through an integrated primary analysis software called RTA (Real TimeAnalysis). The BCL (base calls) binary is converted into FASTQ utilizing illumina package bcl2fastq.Adapters are not trimmed away from the reads.