DOI: 10.5061/dryad.vq83bk3zj
title: Data from: A method for sampling the living wood microbiome

description: Efforts to characterize microbial life across diverse environments have
 progressed tremendously, yet the microbiome of Earth’s largest biomass
 reservoir—the wood of living trees has been largely unexplored. Current
 understanding of the tree microbiome is largely confined to roots and
 leaves, with little attention given to the endophytic microbiome of wood.
 Emergent studies have indicated this zone as a niche for unique taxa, of
 consequence for ecosystem health and global biogeochemical
 cycles. The lack of investigation derives partly from the
 physical recalcitrance of wood, which presents challenges during sampling,
 homogenization, and the extraction of nucleic acids. In response
 to these issues, we present an optimized method for processing wood for
 use in microbial analyses, from sampling through downstream PCR-based
 analyses. Using methane-cycling taxa as model endophytes, we assess losses
 in recovery during our method, and determine a limit-of-detection of
 approximately 500 cells per 100 mg of (dry) wood. For all six
 species evaluated—which represented genera of broad and needle-leaf
 angiosperms and gymnosperms—PCR inhibition proved minimal, and we expect
 this method to be applicable for a majority of tree species. The methods
 presented herein can facilitate future investigation into the wood
 microbiome and global microbial ecology of methane cycling.
description: # Data from: A Method for Sampling the Living Wood Microbiome --- Included
 herein is raw ddPCR (BioRad QX200 droplet reader; mix of EvaGreen and
 probe chemistries) data used in methods testing, broken down into the
 subsections from the manuscript and gene target. Some processed metadata
 (e.g., sample mass) is also included. Additionally, the raw sequencing
 files (paired end sequencing, Illumina MiSeq) for the 3 sequenced red
 maple heartwood samples is included (both forward and reverse reads).
 Empty entries later categorized as NA or NULL during R processing. ***Raw
 ddPCR Data from QX200***
 **zymo_1_24_test_core_pmoA_20220616_205334_819.csv,**
 **Tree_Methods_pmoA_Eva_20230226_210141_169.csv,**
 **Tree_Methods_mmoX_EvaGreen_20230301_194341_834.csv,**
 **Tree_Methods_mcrA_EvaGreen_20230228_173602_437.csv,**
 **Methods_Tree_Serial_Dil_mcrA_P_20230302_214357_917.csv,**
 **Methods_Plate_mcrA_Probe_20230223_182334_914.csv,**
 **Methods_plate_mcrA_FAM_redos_20230225_233035_079.csv,**
 **mcrA_probe_Stds_curve_20230305_223923_356.csv** Well: "Well
 ID" Sample description 1: "Sample Info 1" Sample
 description 2: "Sample Info 2" Sample description 3:
 "Sample Info 3" Sample description 4: "Sample Info 4"
 Target: "PCR Target Gene" Conc(copies/µL): "DNA
 Concentration (copies/µL)" Molecular Weight(pg/µL): "Molecular
 Weight (pg/µL)" Status: "Sample Status" Status Reason:
 "Status Explanation" Experiment: "Experiment ID"
 SampleType: "Sample Type" TargetType: "Target
 Molecule" Supermix: "PCR Mix Used" DyeName(s):
 "Fluorophore Used" Copies/20µLWell: "Copies per Well"
 TotalConfMax: "Confidence Interval Max" TotalConfMin:
 "Confidence Interval Min" PoissonConfMax: "Poisson CI
 Max" PoissonConfMin: "Poisson CI Min" Accepted Droplets:
 "Valid Droplet Count" Positives: "Positive Droplet
 Count" Negatives: "Negative Droplet Count" Ch1+Ch2+:
 "Both Channels Positive" Ch1+Ch2-: "Channel 1 Only
 Positive" Ch1-Ch2+: "Channel 2 Only Positive" Ch1-Ch2-:
 "Both Channels Negative" Linkage: "Genetic Linkage"
 CNV: "Copy Number Variation" TotalCNVMax: "Total CNV
 Max" TotalCNVMin: "Total CNV Min" PoissonCNVMax:
 "Poisson CNV Max" PoissonCNVMin: "Poisson CNV Min"
 ReferenceCopies: "Reference Gene Copies" UnknownCopies:
 "Unknown Gene Copies" Threshold1: "First Threshold"
 Threshold2: "Second Threshold" Threshold3: "Third
 Threshold" ThresholdSigmaAbove: "Threshold Sigma Above"
 ThresholdSigmaBelow: "Threshold Sigma Below" ReferenceUsed:
 "Reference Used" Ratio: "Copy Number Ratio"
 TotalRatioMax: "Max Total Ratio" TotalRatioMin: "Min Total
 Ratio" PoissonRatioMax: "Max Poisson Ratio"
 PoissonRatioMin: "Min Poisson Ratio" Fractional Abundance:
 "Fractional Abundance (FA)" TotalFractionalAbundanceMax:
 "Max FA Total" TotalFractionalAbundanceMin: "Min FA
 Total" PoissonFractionalAbundanceMax: "Max Poisson FA"
 PoissonFractionalAbundanceMin: "Min Poisson FA"
 MeanAmplitudeOfPositives: "Mean Positives Amplitude"
 MeanAmplitudeOfNegatives: "Mean Negatives Amplitude"
 MeanAmplitudeTotal: "Total Mean Amplitude" ExperimentComments:
 "Experiment Notes" MergedWells: "Merged Wells Info"
 TotalConfidenceMax68: "68% Confidence Max" TotalConfidenceMin68:
 "68% Confidence Min" PoissonConfidenceMax68: "68% Poisson
 Max" PoissonConfidenceMin68: "68% Poisson Min"
 TotalCNVMax68: "Total CNV 68% Max" PoissonCNVMax68: "Max
 Poisson CNV 68%" PoissonCNVMin68: "Min Poisson CNV 68%"
 TotalCNVMin68: "Total CNV 68% Min" TotalRatioMax68: "Total
 Ratio 68% Max" TotalRatioMin68: "Total Ratio 68% Min"
 PoissonRatioMax68: "Max Poisson 68% Ratio" PoissonRatioMin68:
 "Min Poisson 68% Ratio" TotalFractionalAbundanceMax68:
 "Fractional Abundance 68% Max" TotalFractionalAbundanceMin68:
 "Fractional Abundance 68% Min" PoissonFractionalAbundanceMax68:
 "Poisson Fractional Abundance 68% Max"
 PoissonFractionalAbundanceMin68: "Poisson Fractional Abundance 68%
 Min" TiltCorrected: "Tilt Correction Status"
 ***Semi-processed data*** **kit_compare_orig.csv** Sample: "Sample
 number" ID: "Sample lab ID" Conc: "Target gene
 concentration (copies)" Type: "Material type" Spiked:
 "Spiked sample Y/N" Core: "Core material" Species:
 "Tree species code" Label: "Full sample label"
 **Extraction_Masses.csv** Sample ID: "Full sample ID" Rep Lab
 ID: "Replicate number" Lab No: "Lab ID number" Well
 No: "Plate well number" Well: "Plate well identifier"
 Mass Weighed: "Sample mass (mg)" Base_Type: "Source
 locale" Spike Type: "Spiking input material" Spike Before:
 "Spiking temporal stage" Species: "Tree species code"
 Core_Type: "Input material form" Dilution: "Dilution
 correction factor" Dilution_mcraP: "Dilution correction factor
 for mcA gene reading" Orig Mass: "Original sample mass (g)"
 Spike Reps: "Spike replicate" Perc_Orig: "Percent original
 mass" Perc_Spike: "Percent spike" Super Attempted :
 "Supernatant transfer attempt (ul)" Tube Super:
 "Supernatant processed (ul)" Correction Factor:
 "Supernatant correction factor" ## Code/Software All analysis
 was performed using RStudio (tree_methods_analyses.R). Microbiome
 processing was conducted using the standard dada2 pipeline using
 NIH's Nephele computing cluster.