Data from: A method for sampling the living wood microbiome
Data files
Feb 26, 2024 version files 48.80 MB
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Extraction_Masses.csv
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kit_compare_orig.csv
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mcrA_probe_Stds_curve_20230305_223923_356.csv
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Methods_plate_mcrA_FAM_redos_20230225_233035_079.csv
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Methods_Plate_mcrA_Probe_20230223_182334_914.csv
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Methods_Tree_Serial_Dil_mcrA_P_20230302_214357_917.csv
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README.md
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RM502Inner_16S_S672_R1_001.fastq.gz
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RM502Inner_16S_S672_R2_001.fastq.gz
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RM503Inner_16S_S177_R1_001.fastq.gz
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RM503Inner_16S_S177_R2_001.fastq.gz
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RM5Inner_16S_S684_R1_001.fastq.gz
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RM5Inner_16S_S684_R2_001.fastq.gz
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Tree_Methods_mcrA_EvaGreen_20230228_173602_437.csv
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Tree_Methods_mmoX_EvaGreen_20230301_194341_834.csv
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Tree_Methods_pmoA_Eva_20230226_210141_169.csv
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zymo_1_24_test_core_pmoA_20220616_205334_819.csv
Abstract
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.
README: 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.