Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

1. Collection of sediment samples for nucleic acid extraction

1. Submerge a sterile 50 mL conical tube into the stream water. Wear gloves during sample collection to avoid introducing unwanted human contamination. Perform this step either from the shore or facing upstream if in the water.
2. While the conical tube is submerged, remove the cap, and use it to scoop approximately 3 mL of sediment from a depth of 1 to 3 cm into the conical tube.
3. Remove the conical tube from the water and dump out all water, except for a thin layer covering the sediment sample (approximately 1 mL).
4. Using a 1000 µL pipette and appropriate pipette tips, add 3 mL of DNA/RNA preservative (see Table of Materials for the preservative specifications) to the collected sample. Keep the pipette tips in a sterile pipette tip box and only attach them immediately before use and discarded after use. Invert the capped conical tube 10 times to ensure the preservative and sample are thoroughly mixed.
   NOTE Step 1.4 is not necessary, but it is strongly recommended if RNA is to be extracted from the sediments later.
5. Place the samples on ice for the rest of sample collection. Upon returning from collection, store in a freezer at -20 °C if the samples are to be used for 16S analysis (DNA), or -70 °C, if they are to be used for metatranscriptomics analysis (RNA).

2. Filter collection for nucleic acid extraction

1. Remove the cap of a sterile 1 L bottle. While facing upstream or from the shore, fill the bottle with stream water to the top and then dump it out. Repeat this process two more times to condition the bottle. Fill the entire bottle a fourth time and cap it.
   NOTE: If reusing a 1 L bottle, it can be sterilized by rinsing with 10% bleach for 2 min, followed by rinsing three times with deionized water and then once with 70% ethanol, and finally autoclaving with settings: 30 min exposure time at 121.1 °C and 15 min drying time. During autoclaving, the cap on the bottle should be very loose to avoid the bottle being compressed in the process.
2. Once on a stable surface, use a sterile Luer lock syringe and draw up a full volume. Then connect the syringe to a sterile and DNA/RNA-free 1.7 cm diameter polyethersulfone filter with a pore size of 0.22 µm and push the entire volume through the filter by pressing the plunger all the way down. Repeat this process until the total volume collected in the bottle (1 L) is pushed through the filter.
   NOTE: The volume of the syringe can be variable, if, the total amount of water pushed through the filter is tracked. However, generally, 60 mL is preferred. While 1 L is ideal, anecdotally, a volume of at least 200 mL would likely still collect enough biomass (assuming ~20,000 cells per mL) for the extraction of DNA and RNA.
3. Remove excess water from the filter by drawing up roughly 20 mL worth of air into the syringe and pushing it through the filter.
   NOTE: This will help prevent loss of the preservative if step 2.4 is performed.
4. Using a P1000 micropipette, add 2 mL of a DNA/RNA preservative by discharging it through the filter's larger opening (where it was attached to the syringe) while holding the filter horizontally. The tip of the pipette should be within the barrel of the filter when the pipette is depressed to ensure the preservative enters the filter. Change the tip after each use.
   NOTE: As with the sediment collection, this step is not necessary, but it is strongly recommended for increased nucleic acid yield later, especially for RNA.
5. Peel off one square of paraffin film and wrap it tightly around each opening/end of the filter to seal. Place the paraffin film wrapped filter into a sterile sample bag and then place the entire bag on ice during collection.
   NOTE: Ensure that the side used to wrap the filter is sterile, i.e., not previously exposed to the environment.
6. Upon return from sampling, store filters at -20 °C for 16S or -70 °C for meta-transcriptomics.

3. Nucleic acid extraction and quantification

1. Clean the work area with 10% Bleach and 70% Ethanol before beginning sample transfer.
2. For sediment (from step 1.5), generally, use ~0.25 g of sample. Flame sterilize a metal tool by dipping it in a beaker of 70% ethanol and burning the ethanol off between samples.
3. For filters (from step 2.6), move the filter paper into a sterile tube for extraction. To do so follow the steps below.
   1. Create a sterile, DNA and RNA free-surface by folding aluminum foil so that the inner part of the fold is not exposed to the outside environment and autoclaving the folded piece with the settings: 121.1 °C and 5 min drying time.
   2. Sterilize a vise-grip with 70% ethanol and an open flame. Then use the vise-grip to break open the filter casing on the sterile surface and remove the core from the casing.
   3. Use a sterile scalpel to cut the filter paper away from the core by slicing at the top and bottom and then along the seam. Fold the filter paper using sterile tweezers and then cut the filter into small pieces using the scalpel.
   4. Place the filter pieces in a microcentrifuge tube for extraction. Make sure that the filter paper does not come into contact with any surfaces which are not sterilized or that could have nucleic acid present, as this would lead to unwanted contamination of the sample.
4. Perform DNA isolation as described previously¹³ or by using a commercially available column-based kit (see Table of Materials). The steps for the commercial kit listed are briefly described below.
   1. Lyse the cells within the sample by transferring it to a bead tube and subjecting it to a cell disruptor at high speed for at least 5 min. Centrifuge and transfer the supernatant to a sterile microcentrifuge tube.
   2. Add lysis buffer to the supernatant (1:1 volume) and transfer to the provided filter (yellow). Centrifuge the filter.
   3. Transfer the filter to a new sterile microcentrifuge tube. Add the preparation buffer (400 µL), centrifuge, and discard the flow through.
   4. Add wash buffer (700 µL), centrifuge, and discard the flow through. Then add wash buffer (400 µL), centrifuge, and discard the flow through again.
   5. Transfer the filter to a new sterile microcentrifuge tube. Elute with 50 µL of DNase/RNase free water and let sit for 5 min at room temperature before centrifuging.
   6. During that in cubation period, prepare the III-HRC filter by placing it in a collection tube and adding the HRC prep solution (600 µL) to it, followed by a centrifugation step of 3 min at 8,000 x g.
   7. Move the prepared filter onto a sterile microcentrifuge tube. Transfer the eluted DNA from step 3.4.5 to this filter and centrifuge at 16,000 x g for 3 min. The flow through contains the extracted DNA.
5. Store DNA extracts for both sediments and filters at -20 °C.
   NOTE: DNA extracts can be stored for around 8 years at -20 °C assuming stable temperature, limited light exposure, and no harmful contaminants¹⁴.
6. Perform RNA isolation as per the manufacturer's protocol. Store RNA extracts at -80 °C.
   1. Lyse the cells within the sample by transferring it to a bead tube and subjecting it to a cell disruptor at high speed for at least five minutes. Centrifuge and transfer the supernatant to a sterile microcentrifuge tube.
   2. Add lysis buffer to the supernatant (1:1 volume) and transfer to the provided column (yellow). Centrifuge the column.
   3. Add an equal volume of 95-100% ethanol to the flow through and mix by pipetting up and down five times.
   4. Place the IICG Column (green) on a sterile microcentrifuge tube. Transfer the mixed solution to the column and centrifuge.
   5. Add wash buffer (400 µL), centrifuge, and discard the flow through.
   6. Add 5 µL of DNase I and 75 µL of DNA digestion buffer to the column and incubate at room temperature for 15 minutes.
   7. Add prep buffer (400 µL), centrifuge, and discard the flow through.
   8. Add wash buffer (700 µL), centrifuge, and discard the flow through. Then add wash buffer (400 µL), centrifuge, and discard the flow through again.
   9. Transfer the column to a new sterile microcentrifuge tube. Elute with 50 µL of DNase/RNase free water and let sit for 5 min before centrifuging.
   10. During that incubation period, prepare the III-HRC filter by placing it in a collection tube and adding the HRC prep solution (600 µL) to it, followed by a centrifugation step of 3 min at 8,000 x g.
   11. Move the prepared filter onto a sterile microcentrifuge tube. Transfer the eluted RNA from step 3.6.9 to this filter and centrifuge at 16,000 x g for 3 min. The flow through contains the extracted RNA.
       NOTE: RNA extracts can only be stored for one year before they start to degrade¹⁵. Both DNA and RNA extracts are degraded by repeated freeze-thawing. Some protocols allow for the extraction of both DNA and RNA from the same sample^16,17.
7. Quantify the extracted DNA and RNA samples using a fluorometer or a spectrophotometer. See Table 1 for example fluorometer DNA concentration values. For an example spectrophotometer quantification protocol, see reference¹⁸. Sediment DNA concentration values with the kit listed in Table of Materials generally range from 1 to 40 ng/µL, while filter DNA concentration values tend to range from 0.5 to 10 ng/µL. Sediment RNA concentration values with the kit listed in Table of Materials generally range from around 1 to 20 ng/µL, while filter RNA concentration values tend to be lower, typically ranging from 0.5 to 5 ng/µL.

4. 16S rRNA library creation

1. Clean the work area with 10% Bleach and 70% Ethanol. The work area should be an enclosed space capable of producing laminar flow conditions (laminar flow hood).
2. Use the DNA extracts (from step 3.5) and prepare samples for 16S rRNA amplicon sequencing with a standard PCR protocol, such as the one described on the Earth Microbiome's website that amplifies the V4 hypervariable region of 16S rRNA¹⁹ under laminar flow conditions.
3. Prepare a 2% agarose gel as described previously and let it solidify¹⁷. Mix 7 µL of PCR product and 13 µL of DNase free water. Add a gel loading dye to a final concentration of 1x. Once agarose is solidified, load this PCR products mix on a 2% agarose gel.
   NOTE: Alternatively, a pre-cast gel can be used instead, as these gels run faster and come pre-made.
4. Run the gel at 90 V for 60-90 min to check for the band size of 386 as successful amplification for 16S rRNA V4 amplicons, using the Earth Microbiome's protocol.

5. DNA 16S rRNA library purification

1. Pool 10 µL of PCR products for the samples that yielded bright bands and 13 µL for the samples that yielded faint bands in an appropriately sized sterile microcentrifuge tube.
2. Check the concentration of the resulting pool using a fluorometer or spectrophotometer and prepare a 2% agarose gel as before. Ideally, the pool should have a concentration of at least 10 ng/µL, and most samples should have had a concentration of around 25 ng/µL.
3. Concentration and volume permitting, load around 150-200 ng in a well of 2% agarose gel.
4. Run the gel for 60-90 min at 90 volts.
5. Purify the pooled library by running a 2% agarose gel.
   1. Excise the 386 bp DNA band from the gel and purify the pooled library using a commercially available kit as described previously²⁰. Elute the purified DNA with 30 µL of 10 mM Tris-Cl (pH 8.5). Perform this step in a different area than DNA or RNA extraction to prevent future contamination, as cutting the gel will spread PCR amplicons onto both the experimenter and the surrounding area.
6. Check the concentration of the purified pool using a fluorometer or spectrophotometer. If purification went well, its concentration should be at least half of the unpurified pool's. Generally, the final concentration should range from 5 to 20 ng/µL.
7. Send the purified libraries for next generation sequencing. Ensure that they are kept cold during transport by including dry ice in the shipping container.

6. RNA library creation and purification

1. Several commercial kits can be utilized to create RNA libraries. For whichever one is used, follow the manufacturer's protocol as written while working in a sterile laminar flow environment. A very summarized version of the protocol for kit in the Table of Materials is presented below²¹.
   1. Make the first strand cDNA synthesis master mix (8 µL of nuclease-free water and 2 µL of First Strand Synthesis Enyzme Mix) and add it to the sample. Place the sample in the thermocycler with the conditions specified in the protocol.
   2. Make the second strand cDNA synthesis master mix (8 µL of Second Strand Synthesis Reaction Buffer, 4 µL Second Strand Synthesis Enzyme Mix, and 48 µL of nuclease-free water) on ice and add it to the sample. Place in a thermocycler set to 16 °C for one hour.
   3. Purify the reaction by adding the provided beads (144 µL) and performing two 80% ethanol washes (200 µL).
   4. Elute with the provided TE buffer (53 µL) and transfer 50 µL of the supernatant to a clean PCR tube. Place the PCR tube on ice.
   5. Make the end prep master mix (7 µL of End Prep Reaction Buffer and 3 µL of End Prep Enzyme Mix) on ice and add it to the PCR tube. Place the PCR tube in a thermocycler with the conditions specified in the protocol.
   6. Mix the Diluted Adaptor (2.5 µL), Ligation Master Mix (30 µL) and Ligation Enhancer (1 µL) solutions on ice. Add the mixed solutions to the sample and place in a thermocycler for 15 min at 20 °C.
   7. Purify the reaction by adding the provided beads (87 µL) and performing ethanol washes (200 µL) and elution as before, except only add 17 µL of TE.
   8. Add indices (10 µL) and the Q5 Master Mix (25 µL) solution and place in a thermocycler with the conditions described in the protocol.
   9. Purify the reaction by adding the provided beads (45 µL) and performing an addition two ethanol washes (200 µL) and elute with 23 µL of TE. Transfer 20 µL to a clean PCR tube.
2. Check the libraries for detectable concentrations of RNA using a Bioanalyzer, fluorometer, or spectrophotometer.
3. Pool the metatranscriptomic libraries in a roughly equimolar ratio.
4. Purify the library following the same protocol for the 16S library purification, except excise fragments between 250 and 400 bp. Whereas the 16S library had a distinct band representing the amplified region, the result here is a smear.
5. Check the concentration of the purified library as before.
6. Ship the purified library with dry ice to a sequencing facility.
   ​NOTE: Alternatively, RNA extracts can be sent to a university or private company for library preparation and sequencing.

7. Microbial community analysis

1. Once sequencing is complete, access the sample data. Download it to a usable computer.
   NOTE: Ideally, the device should have at least 16 gigabytes of RAM. For a discussion of computing requirements (for Qiime2), see https://forum.qiime2.org/t/recommended-specifications-to-run-qiime2/9808.
2. Use software, such as mothur, QIIME2, and R, to analyze 16S rRNA data. See here https://docs.qiime2.org/2020.11/tutorials/moving-pictures/ for an example QIIME2 16S analysis tutorial.
3. For metatranscriptomics (RNA) data, use HUMAnN2 and ATLAS to determine which genes and pathways are present in the samples.
   NOTE: An example metatranscriptomics pipeline culminating in diversity and random forest analysis is presented in the Supplemental Information file. All commands are run through command line, e.g., Terminal for Mac users.