Dec 10, 2024

Public workspaceAn optimized CTAB based RNA extraction from rhizosphere, followed by universal rRNA depletion for metatranscriptomics

DOI
dx.doi.org/10.17504/protocols.io.6qpvr8weolmk/v1
  • 1Arkansas State University;
  • 2Arkansas Biosciences Institute
Kipa Tamrakar
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr8weolmk/v1
Protocol Citation: Kipa Tamrakar, Maureen Dolan, Asela Wijeratne 2024. An optimized CTAB based RNA extraction from rhizosphere, followed by universal rRNA depletion for metatranscriptomics. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8weolmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 20, 2024
Last Modified: December 10, 2024
Protocol Integer ID: 106010
Keywords: Soybean, Rhizosphere, Soil, Metatranscriptomics, RNA, rRNA-depletion
Funders Acknowledgements:
USDA
Abstract
Metatranscriptomics is an approach to analyze the gene expression dynamics to find the functional microbial population in soil. This protocol describes an optimized CTAB based Phenol Chloroform method to extract good-quality RNA from soybean rhizosphere. Furthermore, it incorporates Zymo-Seq RiboFree Total RNA Library Kit to remove highly abundant ribosomal RNA (rRNA) that impedes the isolation of messenger RNA (mRNA)needed for transcriptomics analysis.
Guidelines
The step duration is calculated based on time required to process 3 rhizosphere samples.
Materials
Reagents & Equipment:
- ReagentRNaseZap®Thermo ScientificCatalog #AM9780
- ReagentDiethyl pyrocarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5758

RNA Extraction:
  1. Reagent0.1 mm Zirconia/Silica BeadsBio Spec Products Inc.Catalog #11079101z
  2. Reagent0.5 mm Zirconia/Silica BeadsBio Spec Products Inc.Catalog #11079105z
  3. ReagentCTAB (Hexadecyltrimethylammonium bromide)Merck MilliporeSigma (Sigma-Aldrich)Catalog #52365-50G
  4. ReagentSodium ChlorideFisher ScientificCatalog #S271
  5. ReagentPolyvinylpyrrolidoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #PVP40
  6. ReagentSodium phosphate monobasic monohydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9638
  7. ReagentSodium Phosphate Dibasic Heptahydrate, ACS Reagent Grade, 500g Poly BottleMerck MilliporeSigma (Sigma-Aldrich)Catalog #7914-04
  8. Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
  9. ReagentUltraPure™ PhenolThermo Fisher ScientificCatalog #15509-037
  10. ReagentOmniPur® ChloroformThermo Fisher ScientificCatalog #31-509-50ML
  11. ReagentIsoamyl Alcohol (Clear, Colorless/Certified ACS), Fisher Chemical™Thermo Fisher ScientificCatalog #A393-4
  12. ReagentSodium acetate, anhydrous, 99%, Thermo Scientific ChemicalsThermo Fisher ScientificCatalog #AAA1318430
  13. ReagentGlacial acetic acidFisher ScientificCatalog #A38-500
  14. ReagentPEG 8000 Powder (Polyethylene Glycol), 500gmPromegaCatalog #V3011
  15. Deionized water - DI water

RNA Clean up:
  1. ReagentRNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
  2. ReagentDNase I Set (RNase-free)Zymo ResearchCatalog #E1010
  3. ReagentEthanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500

RNA Quality Control:
  1. ReagentQubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
  2. ReagentRNA ScreenTapeAgilent TechnologiesCatalog #5067-5576
  3. ReagentRNA ScreenTape Sample BufferAgilent TechnologiesCatalog #5067-5577
  4. ReagentRNA ScreenTape LadderAgilent TechnologiesCatalog #5067-5578
RNAseq Library Preparation:
  1. ReagentZymo-Seq RiboFree Total RNA Library KitZymo ResearchCatalog #R3000

Consumables:
RNA extraction:
  1. 1 L Glass bottles
  2. 100 mL Glass bottles
  3. 50 mL Nuclease free tubes
  4. Weighing boats
  5. 2 mL Screw-capped tubes
  6. 0.1 mm & 0.5 mm zirconium beads
  7. 2 mL Nuclease free microfuge tubes
  8. 1.5 mL Nuclease free microfuge tubes
  9. 50 mL Serological pipettes
  10. 10 mL Serological pipettes
  11. 20 μL pipette and tips
  12. 200 μL pipette and tips
  13. 1000 μL pipette and tips

RNA Clean-up:
  1. 1.5 mL Nuclease free microfuge tubes
  2. 20 μL pipette and tips
  3. 200 μL pipette and tips
  4. 1000 μL pipette and tips

RNA quality Control:
  1. Qubit assay tubes
  2. Strip tubes and caps
  3. Loading tips
  4. 20 μL pipette and tips
  5. 2.5 μL pipette and tips

RNAseq Library Preparation:
  1. 0.2 mL PCR tubes
  2. Magnetic stand
  3. 200 μL pipette and tips
  4. 20 μL pipette and tips
  5. 2.5 μL pipette and tips

Reagent Preparation:
1. DEPC-treated water
  • Add 1 mL of Diethyl Pyrocarbonate (DEPC) to 1 L DI water in a 1 L glass bottle.
  • Mix by stirring and treat the DEPC overnight.
  • Next day, autoclave at 121ºC at 15psi for 30 mins.

2. Cetyltrimethylammonium bromide (CTAB) extraction buffer:
A. 100ml of 5M Sodium Chloride (NaCl) stock
  • Dissolve Amount14.61 g of NaCl in Amount70 mL DI water. Stir to mix
  • Bring the volume to Amount100 mL by adding DI water.
  • Autoclave to sterilize.

B. 100ml stock of 500mM Sodium Phosphate (NaP) Buffer, pH 5.8
  • Prepare Concentration500 millimolar (mM) sodium phosphate monobasic by dissolving Amount6.8995 g of sodium phosphate monobasic (monohydrate, M.W. 138.0) in Amount100 mL of DI water.
  • Prepare Concentration500 millimolar (mM) sodium phosphate dibasic by dissolving Amount13.4035 g of sodium phosphate dibasic (heptahydrate, M.W. 268.0) in Amount100 mL of DI water.
  • Take Amount95 mL of 1M sodium phosphate monobasic in a beaker.
  • Adjust the pH to 5.8 by adding 1M sodium phosphate dibasic (usually around 5ml).
  • Autoclave to sterilize.

C. Mix all reagents as described in the following table to prepare 500mM NaP buffer, pH 5.8:
Preparation of CTAB extraction buffer


3. Water-saturated Phenol:
  • Thaw the bottle in water bath set at Temperature50 °C
  • Add DI water to fill the amber bottle to the base of the neck. (A saturated solution of phenol contains 12.36 mL of deionized water per 100 g of phenol.)
  • Cap securely then mix the organic and aqueous phases until they form a fine emulsion.
  • Store at Temperature4 °C overnight until phases separate.
  • Aspirate the upper aqueous layer.
  • Once saturated, store with some water phase on top of the phenol.
  • Store at 4oC. Reagent is typically stable for a month or two.
NOTE: The phenol phase will eventually turn pink upon oxidation – DO NOT USE when pink!

4. 100 mL of Chloroform: Isoamyl alcohol (49:1) solution:
  • Take Amount98 mL of chloroform in a glass amber bottle.
  • Add Amount2 mL of Isoamyl alcohol to it. Mix properly.

5. 100 mL of 3M Sodium acetate (NaOAc), pH 4.6:
  • Prepare Concentration3 Molarity (M) acetic acid solution by mixing Amount8.62 mL of glacial acetic acid (17.4N) inAmount41.38 mL DI water.
  • Prepare Concentration3 Molarity (M) NaOAc solution by dissolving Amount24.61 g NaOAc (82.03 g/mol) in Amount100 mL DI water)
  • Mix Amount21 mL 3M acetic acid with Amount79 mL 3M NaOAc solution.
  • Autoclave to sterilize.

5. Precipitation Buffer:
A. Oven bake glass bottle atTemperature225 °C DurationOvernight . For caps, perform DEPC treatment DurationOvernight These bottle will be used for preparing 2M NaCl, 50% PEG, and precipitation buffer.

B. Prepare Amount100 mL of Concentration2 Molarity (M) NaCl:
  • Dissolve Amount11.68 g of NaCl in Amount60 mL deionized water in a beaker. Stir to mix
  • Bring the volume toAmount100 mL by adding DI water.
  • Add Amount100 µL DEPC to solution. Treat DurationOvernight .
  • Transfer the solution to an oven baked glass bottle.
  • Autoclave to inactivate DEPC.

C. Prepare Amount100 mL ofConcentration50 % (v/v) PEG solution:
  • Weigh Amount50 g of PEG-8000.
  • Dissolve in Amount50 mL DEPC treated water. Stir to mix.
  • Transfer the solution to an oven baked glass bottle using serological pipette.
  • Add DEPC water to bring volume to 100ml.

D. Mix the solution as per the following table to prepare NaCl-PEG precipitation buffer:
Preparation of PEG-NaCl precipitation buffer

6. Concentration75 % (v/v) Ethanol:
  • DispenseAmount37.5 mL Absolute ethanol in a nuclease free 50ml tubes.
  • Add Amount12.5 mL DEPC treated water to bring the total volume to 50ml.
  • Store in -20⁰C freezer until use.



Safety warnings
DEPC, Phenol, Chloroform, Isoamyl alcohol and 2-Mercaptoethanol are hazardous chemical, which should be handled under fume hood.
Before start

  • Clean the workspace with RNase decontamination solution (i.e. RNaseZap)
  • Treat pipettes, tip boxes and racks used during experiment with RNase decontamination solution.
RNA Extraction
RNA Extraction
4h
4h
Prepare bead beating tubes by adding 950 mg 0.1 mm and 50 mg 0.5 mm silica beads in 2 mL screw-capped tube
5m
Weigh 250 mg of rhizosphere in a clean weigh boat and transfer sample to bead tubes.

10m
Add the following reagents to each sample:
  • 600 µL water-saturated phenol
  • 400 µL 500 mM NaP buffer, pH 5.8
  • 200 µL Chloroform: Isoamyl alcohol (49:1)
Note
Set water bath at 60°C and place tube containing CTAB extraction buffer.

  • 200 µL pre-heated CTAB extraction buffer
  • 10 µL 14.3 M 2-Mercaptoethanol
  • Vortex to mix
10m
Toxic
Incubate samples on ice for 2 mins.

2m
Homogenize using Mini beadbeater (Biospec, Cat# 112011) in two bead-beating steps (3.5 x 1000 strokes per min for 40 secs) with one intermediate cooling step on ice for 1 min.

5m
Completely mix samples using a series of vortex cycles (one cycle with 20 secs vortexing/40 secs cooling on ice; followed by two cycles of 10 secs vortexing/30 secs cooling on ice)
5m
Critical
Centrifuge at 10,000 xg for 10 mins at 4ºC. Transfer the aqueous phase to a new 2 mL microfuge tube.



15m
Add 350 µL 3M Sodium acetate, pH 4.6 to the tube and mix by vortexing. Incubate on ice for 3 mins.

5m
Add 600 µL water-saturated phenol and vortex to mix. Incubate on ice for 3 mins.

Note
STOP POINT: Samples can be stored on ice bath for few hours

5m
Pause
Toxic
Add 300 µL Chloroform: Isoamyl alcohol and pulse vortex to mix till milky solution is obtained. Incubate on ice for 3 mins.
5m
Toxic
Centrifuge at 10,000 xg for 10 mins at 4ºC. Transfer (~800 µL) aqueous phase into a new 2 mL microfuge tube.
10m
Add 800 µL Chloroform: Isoamyl alcohol (49:1) and pulse vortex to mix sample until a milky consistency is attained. Incubate on ice for 3 mins.
5m
Toxic
Centrifuge samples at 10,000 xg for 10 mins at 4ºC.
Note
After centrifugation, carry out the rest of the steps in a clean area. An isolated area properly sterilized with RNase decontamination solution can be considered as a clean area.

15m
Transfer aqueous phase into a new 1.5 mL microfuge tube.

Add one volume of PEG-NaCl precipitation buffer and quick vortex.
3m
Incubate on ice bath in a 4°C refrigerator for 20 mins.
20m
Centrifuge samples at 20,000 xg for 20 mins at 4°C.
20m
Add 1500 µL of 70% ice-cold ethanol. Centrifuge at 20,000 xg at 4 °C for 5mins. Discard ethanol and retain pellet.
10m
Go togo to step #18

10m
Minifuge samples to enable removal of any remining ethanol.
Air dry pellet under laminar hood and resuspend pellet in 50 µL Nuclease free water.

Note
Do not leave the tubes to dry for more than 15 mins

15m
Critical
RNA samples can be stored at -80°C until further use or proceed directly to RNA clean-up step (Go togo to step #23 )

RNA Clean up using ZYMO RNA Clean & Concentrator Kits-5
RNA Clean up using ZYMO RNA Clean & Concentrator Kits-5
45m
45m
Add 100 µL of RNA binding buffer to each 50 µL total RNA sample and mix by pipetting.

3m
Add 150 µL of absolute ethanol and mix by pipetting.

3m
Transfer 300 µL of this mixture to the Zymo-Spin IC Column placed in a collection tube.

5m
Critical
Centrifuge 10,000 xg for 30 secs at room temperature and discard the flow-through.
5m
Add 400 µL RNA Wash Buffer to the column. Centrifuge at 10,000 xg for 30 secs at room temperature and discard the flow-through.

5m
Prepare DNase I Reaction Mix as follows in a Nuclease free tube:
ZYMO DNase Mix
  • Mix by gentle inversion.
  • Add 40 µL of the mixture directly onto the column matrix.
  • Incubate column at room temperature for 15 minutes.

20m
Optional
Add 400 µL RNA Prep Buffer to the column and centrifuge at 10,000 xg for 30 secs at room temperature Discard the flow-through.

3m
Add 700 µL RNA Wash Buffer to the column and centrifuge at 10,000 xg for 30 secs at room temperature. Discard the flow-through.

3m
Add 400 µL RNA Wash Buffer to the column and centrifuge at 10,000 xg for 1 min at room temperature to ensure complete removal of the wash buffer. Carefully, transfer the column into a RNase-free 1.5 mL tube.

5m
Add 15 µL DNase/RNase-Free Water directly to the column matrix. Incubate for 1 min and centrifuge at 10,000 xg for 30 secs at room temperature

3m
Purified RNA samples can be stored at -80°C until further use.
RNA Quality Control
RNA Quality Control
30m
30m
Measure RNA quantity using 1 µL of each purified total RNA sample and a Qubit RNA HS kit assay in Qubit fluorometer (ThermoFisher, Cat# Q33238).

10m
Measure RNA quality with 1.5 µL of sample on a Nanodrop spectrophotometer (ThermoFisher, Cat# ND-ONE-W).

5m
Confirm RNA samples were intact (not degraded) using an Agilent Tapestation (Cat# G2992AA) with 1 µL of each RNA sample.

10m
RNAseq Library Preparation using Zymo-Seq RiboFree® Total RNA Library Kit
RNAseq Library Preparation using Zymo-Seq RiboFree® Total RNA Library Kit
2d
2d
cDNA Synthesis

ZYMOseq cDNA Synthesis Cycle
  • Transfer 250 ng RNA to a clean PCR tube and adjust the total volume to 8 µL with Nuclease free water.
  • Add 2 µL of cDNA Synthesis Reagent 1 to the sample tube for a total of 10 µL. Mix thoroughly by pipetting and spin down.
  • Place the tube in the thermal cycler and run Primer Annealing program (Stage 1-2); see detail settings in Table below.
  • Add 10 µL of cDNA Synthesis Reagent 2 to each sample during the 4°C hold of Stage 2. Mix thoroughly by pipetting and spin down.
  • Resume the Reverse Transcription program on the Thermal Cycler (Stage 3-5); see detail settings in Table below.
Note
Proceed directly to RiboFree Universal Depletion for the depletion of ribosomal RNA.

1h
RiboFree Universal Depletion
ZYMOseq Ribo-depletion Cycle
  • Following cDNA synthesis, transfer sample tubes from Step 37 to ice and add 10 µL of Depletion Reagent 1 for a total of 30 µL. Mix thoroughly by pipetting and spin down.
  • Return sample tubes to the thermal cycler and run PreDepletion Incubation program (Stage 1-3); see detail settings in Table above.
Note
Do not remove the tube from the thermal cycler at the 68ºC hold of Stage 3.
  • Add 10 µL of Depletion Reagent 2 to the tube in the thermocycler. Mix thoroughly by pipetting.
  • Close the thermal cycler lid and continue Depletion Reaction program (Step 4); see detail settings in Table above

Note
DO NOT remove the tube from the thermal cycler at the Stage 5 hold.
  • Add 10 µL of Depletion Reagent 3 to each sample tube in the thermocycler. Mix thoroughly by pipetting.
  • Close the thermal cycler lid and continue through Stop Depletion (Stage 6-7).
  • Add 2 µL of Depletion Reagent 4 to each sample tube. Quickly remove the tube from the thermal cycler and gently flick to mix. Spin down briefly to collect the reaction. Immediately return the tube to the thermal cycler to continue and continue through Depletion Cleanup program (Stage 8-10).
  • Remove the tube from the thermal cycler and spin down. Add 26 µL of 95% ethanol to the sample for a total of 78 µL. Mix thoroughly by pipetting.
  • Add 156 µL of Select-a-Size MagBeads to the tubes and incubate at room temperature for 5mins.
  • Pellet the beads by placing the tubes on a magnetic stand. Wash the beads with 200ul of Zymo-Seq Wash Buffer.
  • Repeat the washing step.
  • Remove the tube from the magnetic stand. Spin down and place back on the magnetic stand. Without disturbing the pellet, aspirate any residual Zymo-Seq Wash Buffer.
  • Air dry all sample pellets while tubes remain on the magnetic stand
  • Resuspend the beads in 11 µL of DNA Elution Buffer and transfer the tube to the thermal cycler to complete DNA Elution program (Stage 11-12).
  • Remove the tube from the thermal cycler, collect sample by centrifugation and place each tube on the magnetic stand to elute.
  • Transfer 10 µL of the eluate Air dry all sample pellets while tubes remain on the magnetic standto a new 0.2 mL PCR tube.

Note
STOP POINT: Purified cDNA can be safely stored at -20°C short term until proceeding to next step in the process.

2h 30m
Adapter Ligation
  • Preheat a thermal cycler to 98°C (lid temperature at 105°C).
  • For each sample from step 28, combine the following components (see Table below) in a 0.2 mL PCR tube, on ice. Mix by pipetting thoroughly and spin down.

ZYMOseq Adapter Ligation Mix 1
  • Incubate the tube on ice for 2 minutes.
  • Heat shock by immediately placing the tube at 98°C (lid temperature at 105°C) for 3 minutes.
  • Immediately return the tube to ice and incubate for at least 2 minutes.
Note
During this incubation, reset the thermal cycler to 37°C (lid temperature at 50°C) for next step in the process. Leave the lid open to allow for faster cooling.
  • Mix the Adapter Ligation Master Mix by vortexing for 30 secs before use. For each sample, add the following components, in the order listed in the Table below. Carry this step out on ice.

ZMOseq Adapter Ligation Mix 2
  • Mix by vortexing for 1 min and spin down.
  • Incubate the sample tubes at 37°C (lid temperature at 45°C) for 1 hour.
  • Remove the tube from the thermal cycler. At room temperature, add 85 μL of DNA Elution Buffer to the sample and mix thoroughly by pipetting.
  • Add 48.6 µL of Select-a-Size MagBeads to the tubes and incubate at room temperature for 5mins.
  • Pellet the beads by placing the tubes on a magnetic stand. Wash the beads with 200 µL of ZymoSeq Wash Buffer.
  • Repeat the washing step.
  • Remove the tube from the magnetic stand. Spin down and place back on the magnetic stand. Without disturbing the pellet, aspirate any residual Zymo-Seq Wash Buffer.
  • Air dry all sample pellets while tubes remain on the magnetic stand.
  • Resuspend the beads in 16 µL of DNA Elution Buffer and spin down. Incubate at room temperature for 1 min.
  • Place the tube on the magnetic stand to elute.
  • Transfer 15 µL of the eluate to a new 0.2 mL PCR tube.

Note
STOP POINT: Purified cDNA can be safely stored at -20°C short term until proceeding to next step in the process.

2h 30m
RNAseq Library Amplification
  • Add 10 µL of the appropriate Zymo-Seq UDI Primers to each sample tube (15 µL eluate from Step 39) for a total volume of 25 µL. Mix thoroughly by pipetting.
  • Add 25 µL of Amplification PreMix to the tube for a total volume of 50 µL. Mix thoroughly by gently pipetting and spin down.
  • Place the tube in the thermal cycler and run the following program:

ZYMOseq Library Amplification Cycle
  • Remove the tube from the thermal cycler and spin down. Add 50 µL of DNA Elution Buffer to the tube.
  • Add 80 µL of Select-a Size MagBeads to the tube. Mix thoroughly by pipetting until homogenous. Incubate for 5 mins at room temperature.
  • Place the sample tube on a magnetic stand until the beads have fully separated from the solution. Without dislodging the bead pellet, aspirate slowly and discard the supernatant.
  • Remove the sample tube from the magnetic stand. Add 100 µL of DNA Elution Buffer to the beads and mix thoroughly by pipetting until homogenous.
  • Add 80 µL of Library Binding Solution to the tube. Mix thoroughly by pipetting. Incubate for 5 mins at room temperature.
  • Pellet the beads by placing the tubes on a magnetic stand. Wash the beads with 200 µL of ZymoSeq Wash Buffer.
  • Repeat the washing step.
  • Remove the tube from the magnetic stand. Spin down and place back on the magnetic stand. Without disturbing the pellet, aspirate any residual Zymo-Seq Wash Buffer.
  • Air dry all sample pellets while tubes remain on the magnetic stand.
  • Resuspend the beads in 21 µL of DNA Elution Buffer and spin down. Incubate at room temperature for 1 min.
  • Place the tube on the magnetic stand to elute.
  • Transfer 20 µL of the eluate to a new 0.2 mL PCR tube.
  • Use 1 µL of each generated cDNA library sample for quantification using a Qubit DNA HS kit assay.
  • The eluate is the final RNA-Seq library. Libraries may be stored at ≤ 4°C overnight or ≤ -20°C for long-term storage.



1h 30m
Protocol references
​Poursalavati, A., Javaran, V. J., Laforest-Lapointe, I., & Fall, M. L. (2023). Soil Metatranscriptomics: An Improved RNA Extraction Method Toward Functional Analysis Using Nanopore Direct RNA Sequencing. Phytobiomes Journal, 7(1), 42–54. https://doi.org/10.1094/pbiomes-12-22-0108-ta

Chomczynski, P., & Sacchi, N. (2006). The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: Twenty-something years on. Nature Protocols, 1(2), 581–585. https://doi.org/10.1038/nprot.2006.83

Holmes, A., Birse, L., Jackson, R. W., & Holden, N. J. (2014). An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7. Frontiers in Microbiology, 5. https://doi.org/10.3389/fmicb.2014.00286

Hopkins, T. R. (1991). Physical and Chemical Cell Disruption for the Recovery of Intracellular Proteins. In Purification and Analysis of Recombinant Proteins (pp. 57–83). Marcel Dekker, Inc.

Mettel, C., Kim, Y., Shrestha, P. M., & Liesack, W. (2010). Extraction of mRNA from Soil. Applied and Environmental Microbiology, 76(17), 5995–6000. https://doi.org/10.1128/AEM.03047-09

Thorn, C. E., Bergesch, C., Joyce, A., Sambrano, G., McDonnell, K., Brennan, F., Heyer, R., Benndorf, D., & Abram, F. (2019). A robust, cost‐effective method for DNA, RNA and protein co‐extraction from soil, other complex microbiomes and pure cultures. Molecular Ecology Resources, 19(2), 439–455. https://doi.org/10.1111/1755-0998.12979