All code to take raw SSU rRNA gene sequencing reads to final publication figures and through all presented analyses is located at the following URL:
https://github.com/chuckpr/CSIP_succession_data_analysis.
DNA sequences are deposited on MG-RAST (Accession XXXXXXX).
Twelve soil cores (5 cm diameter x 10 cm depth) were collected from six sampling locations within an organically managed agricultural field in Penn Yan, New York. Soils were sieved (2 mm), homogenized, distributed into flasks (10 g in each 250 ml flask, n = 36) and equilibrated for 2 weeks. The soil type was Honeoye previously measured to be approximately neutral pH (for a thorough description of the site see \citet{Berthrong_2013}). We amended soils with a mixture containing 2.9 mg C g\(^{-1}\) soil dry weight (d.w.) and brought soil to 50% water holding capacity. By mass the amendment contained 38% cellulose, 23% lignin, 20% xylose, 3% arabinose, 1% galactose, 1% glucose, and 0.5% mannose. 10.6% amino acids (made in house based on Teknova C9795 formulation) and 2.9% Murashige Skoog basal salt mixture which contains macro and micro-nutrients that are associated with plant biomass (Sigma Aldrich M5524). This mixture approximates the molecular composition of switchgrass biomass with hemicellulose replaced by its constituent monomers \citep{Schneckenberger_2008}. We set up three parallel treatments varying the isotopically labeled component in each treatment. The treatments were (1) a control treatment with all unlabeled components, (2) a treatment with \(^{13}\)C-cellulose instead of unlabeled cellulose (synthesized as described in SI), and (3) a treatment with \(^{13}\)C-xylose (98 atom% \(^{13}\)C, Sigma Aldrich) instead of unlabeled xylose. Other details relating to substrate addition can be found in SI. Microcosms were sampled destructively at days 1 (control and xylose only), 3, 7, 14, and 30 and soils were stored at -80 \(^{\circ}\)C until nucleic acid extraction. The abbreviation “13CXPS” refers to the \(^{13}\)C-xylose treatment (\(^{13}\)C Xylose Plant Simulant), “13CCPS” refers to the \(^{13}\)C-cellulose treatment, and “12CCPS” refers to the control treatment.
We used DESeq2 (R package), an RNA-Seq differential expression statistical framework \citep{love2014}, to identify OTUs that were enriched in high density gradient fractions from \(^{13}\)C-treatments relative to corresponding gradient fractions from control treatments (for review of RNA-Seq differential expression statistics applied to microbiome OTU count data see \citep{McMurdie2014}). We define “high density gradient fractions” as gradient fractions whose density falls between 1.7125 and 1.755 g ml\(^{-1}\). For each OTU, we calculates logarithmic fold change (LFC) and corresponding standard error for enrichment in high density fractions of \(^{13}\)C treatments relative to control. Subsequently, a one-sided Wald test was used to assess the statistical significance of LFC values with the null hypothesis that LFC was less than one standard deviation above the mean of all LFC values. We independently filtered OTUs prior to multiple comparison corrections on the basis of sparsity eliminating OTUs that failed to appear in at least 45% of high density fractions for a given comparison. P-values were adjusted for multiple comparisons using the Benjamini and Hochberg method \citep{benjamini1995}. We selected a false discovery rate of 10% to denote statistical significance.
See SI for additional information on experimental and analytical methods.