16S Amplicon sequencing of a 12-months field trial with implementation of wheat straw as high carbon amendment in a winter wheat-oilseed rape- rotation system Raw sequence reads

We performed a 12 months field to study the development of microbial community composition after the incorporation of wheat straw as high carbon amendment in am oliseed rape winter wheat rotation system in an agricultural field in Duernast, Germany. The soil type was described as silty-loam soil. Ten point eight kg wheat straw per plot was incorporated into first 10 cm of soil directly after the harvest of oilseed rape in August 2022 using a rotary harrow in an randomized plot design. Plot size was fourteen point eight square meters. Six plots were mixed with wheat straw (HCA) and 6 plots without wheat straw as control (non HCA). In November 2022, winter wheat (T. aestivum), cultivar Reform, was sowed using a plant density of 60 seed per square meters. Topsoil samples (0 to10 cm) were collected 6, 9, 11 and 12 months after implementation using soil cores (diameter 1 cm,10 cm length). We applied DNA extraction and 16S rRNA gene metabarcoding using the primers of the earth microbiome project (EMP, 515F/806R , Parada et al., 2016, Apprill et al., 2015) . Amplification: 98C for 1 min; 98C for 10 s / 55C for 30 s / 72C for 30 s (23 cycles); 72C for 5 min. PCR reaction mixture (total 25 ul); 12.5 ul of NEB Next High Fidelity Master Mix, 0.3 ul of each primer(10 pmol), 2.5 ul of 3% BSA, 1 ul of template DNA (5ng/ul), an adequate amont of DPEC-treated water. Sample purification: purified with MagSi-NGS Prep Plus magnetic beads (Steinbrenner Laborsysteme GmbH, Mannheim, Germany) using a beads:DNA ratio of 0.8:1. Indexing PCR: Nextera XT Index Kit v2 set B,C,D Amplification: 98C for 30s; 98C for 10 s / 55C for 30 s / 72C for 30 s (10 cycles); 72C for 5 min. Sample purification: mentioned above. Products are pooled equimolar to a final concentration of 4 nM and sequenced using the MiSeq Reagent kit v3 (600 cycles) for paired end sequencing. Raw sequences were separated from adapters using AdapterRemoval v2.1.7, and reads were trimmed with a minimum read length of 20 bp.