Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Podsumowanie

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Streszczenie

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes ≤4 L or >4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m³) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Wprowadzenie

Environmental DNA (eDNA) in aquatic environments refers to genetic material found in the water column. Recent studies demonstrated the utility of eDNA for detecting fishes from various aquatic environments, including ponds^1-3, rivers^4-8, streams⁹, and seawater^10-14. Most of these studies focused on detection of a single or a few invasive^1,4-6,8,14 and rare or threatened species^3,9, while some recent studies attempted simultaneous detection of multiple species in local fish communities^7,9,12,13,15 and mesocosms^11,12.

The latter approach is called "metabarcoding" and eDNA metabarcoding uses one or multiple sets of PCR primers to coamplify a gene region across taxonomically diverse samples. This is followed by library preparation with indexing and adapter addition, and the indexed libraries are analyzed by a high-throughput parallel sequencing platform. Recently Miya et al.¹² developed universal PCR primers for metabarcoding eDNA from fishes (called "MiFish"). The MiFish primers target a hypervariable region of the mitochondrial 12S rRNA gene (163-185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. With the use of those primers in eDNA metabarcoding, Miya et al.¹² detected more than 230 subtropical marine species from aquarium tanks with known species composition and coral reefs near the aquarium.

While optimizing the metabarcoding protocol to accommodate natural seawater with varying levels of eDNA concentration from fishes, we have noticed that the MiFish primers occasionally failed to amplify the target region for subsequent library preparation. One of the more likely reasons for this unsuccessful PCR amplification is lack of adequate amounts of the template DNA contained in small volumes of water filtered (i.e. 1-2 L). Although eDNA concentration from a specific taxonomic group is unknowable before the amplification, filtration of large water volumes (>1-2 L) would be a simple and effective means to collect more eDNA from the aquatic environments with scarce fish abundance and biomass, such as open-ocean and deep-sea ecosystems.

Relative to disk fiber filters conventionally used in a number of fish eDNA research¹⁶, filter cartridges have the advantage of accommodating larger water volumes before clogging¹⁷. Actually, a recent study showed large volume (>20 L) filtration of coastal seawater samples using filter cartridges¹⁸. In addition, they are individually packaged and sterile, and several steps of the experimental workflow can be performed in the filter housing, thus reducing the probability of contamination from the laboratory¹⁹. The latter feature is critical for eDNA metabarcoding, in which the risk of contamination remains among the greatest experimental challenges^20,21. Despite these technical advantages of filter cartridges, it has not been used in eDNA studies of fishes with two exceptions^8,15.

Here we provide a protocol for filtration of water samples with the filter cartridge and extraction of eDNA from its filter without having to cut open the housing. We also provide two alternative water filtration systems depending on the water volumes (≤4 L or >4 L). To compare the performance of the newly-developed protocol and a previously-used protocol using a glass-fiber filter in our research group^12,14,22,23, we perform eDNA metabarcoding analysis of seawater from a huge aquarium tank (7,500 m³) with known species composition, and show the number of detected species derived from the two protocols as representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Protokół

NOTE: This protocol does not deal with water sampling and metabarcoding methods. Water may be sampled in different manners depending on study purposes¹⁶ and see Miya et al.¹² for details of the metabarcoding methods using MiFish primers. Note that the sampled water should be kept very cold and filtered within a few hours to avoid degradation of eDNA. Also note that this protocol involves the use of a rotary shaker and an incubator, and the latter must be large enough to accommodate the former. In addition, a centrifuge that can accommodate both 15 ml and 50 ml conical tubes is indispensable to remove the remaining liquid from the post-filtration filter and to collect extracted DNA within the cartridge, respectively.

1. Processing a Screw Cap and a 1 L Plastic Bag

NOTE: Skip this step if the filtration volume is >4 L.

1. Drill a hole through the center of a screw cap (attached to a disposable 1 L plastic bag) with the same diameter (4.8 mm) as the tube projecting from a male luer-lock connector. Using diagonal pliers, shorten the tube of the male luer-lock connector to an appropriate length (ca. 3 mm) to avoid clogging of a small amount of water inside the cap after filtration.
2. Apply an adhesive glue specialized for polyethylene (PE) and polypropylene (PP) to both the bottom and surface of the male luer-lock connector and screw cap, respectively. Wait a few minutes for good adhesive bonding (refer to the manufacturer's instructions).
3. Insert the male luer-lock connector into the hole of the screw cap. Wait until complete adhesive bonding of the two parts (usually >24 hr). Sterilize the screw cap with the male luer-lock connector with 10% commercial bleach (ca. 0.6% sodium hypochlorite) before use.
4. Punch two holes at the two bottom corners of the 1 L plastic bag in order to hang it from a mesh panel (step 3). Ensure that the diameter of the holes is larger than that of the prongs on the mesh panel.

2. Assembly of the Filtration System

1. Attach high vacuum tubing to the input connector of an aspirator pump and attach the other end of tubing to a manifold. Be sure that the three red t-valves of the manifold are in the "off position" (i.e., horizontal).
2. Wearing a clean set of gloves, insert a female luer fitting and a vacuum connector at the top and bottom ends of the vacuum rubber tubing for filtration, respectively.
3. Carefully attach the female luer fitting to an outlet port of the filter cartridge and attach the vacuum connector to a silicone stopper.

3. Filtration of Water Samples (≤4 L) using the Filter Cartridge

NOTE: Skip this step if the filtration volume is >4 L. This filtration system requires a self-standing panel for hanging the plastic bag filled with 1 L of water. A mesh panel, multiple prongs, and a stand for the panel, all available from online stores, would be useful for assembling this unit. Autoclave the inlet and outlet luer caps for the filter cartridge before use.

1. Pour the 1 L sampled water into the plastic bag and close the screw cap with the male luer-lock connector.
2. Carefully connect an inlet port of the assembled filter cartridge with the male luer-lock connector of the plastic bag. Do not over-tighten the luer-lock connector; otherwise the unit will leak once the pumping starts. Be sure that all the connections are secure before hanging the plastic bag from a mesh panel.
3. Carefully hang the plastic bag with the filter cartridge from two prongs on the mesh panel and insert a silicone stopper into an inlet port of the manifold.
4. Turn on the aspirator. Open the red t-valves for filtration. Run the manifold until the filter cartridge is dry, then turn the red t-valve to the off position.
5. Repeat 3.1-3.4 steps until the desired amount of water is filtered.
   NOTE: For 1 L of water taken from the subtropical coral reefs, it takes about three min for the filtration.
6. Carefully remove the filter cartridge from the plastic bag and the vacuum connector.
7. Cap both ends of the cartridge with the inlet and outlet luer caps.
8. Label the cartridge and inlet luer cap appropriately using a solvent-proof pen for fast-drying and non-smearing labeling.
9. Store the filter cartridge at -20 ˚C before DNA extraction.

4. Filtration of Water (>4 L) Samples using the Filter Cartridge

Note: Skip this step if the water filtration volume is ≤4 L. This filtration system requires a 10 L book bottle equipped with a valve and a disposable 10-ml pipette tip. An inner diameter of the 10 ml pipette tip (15.0 mm) and a taper of the tip end should fit to an outer diameter of the the valve (15.0 mm) and inlet port of the filter cartridge, respectively. Both connections are retained securely during filtration in a friction fit. Sterilize the pipette tip with 10% commercial bleach (ca. 0.6% sodium hypochlorite) before use.

1. Prepare an appropriate amount of sampled water in the book bottle. Tightly insert the 10 ml pipette tip into the valve of 10 L book bottle.
2. Tightly insert the outlet port of the filter cartridge into the pipette tip end and insert the silicone stopper into the inlet port of the manifold. Turn on the aspirator. Open the red t-valves for filtration. Run the manifold until the filter cartridge is dry, then turn the red t-valve to the off position.
   NOTE: For 10 L of seawater taken from the subtropical outer coral reefs, it takes about 30-40 min for the filtration.
3. Carefully remove the filter cartridge from the plastic bag and the vacuum connector. Cap both ends of the cartridge with the inlet and outlet luer caps. Label the cartridge and inlet luer cap appropriately using a solvent-proof pen for fast-drying and non-smearing labeling. Store the filter cartridge at -20 °C before DNA extraction.

5. Extraction of eDNA from the Filter

NOTE: In steps 4 and 5, we use a commercial kit, largely following a protocol for "nucleated blood" provided by the kit. For simplicity, we describe the procedure for processing an individual cartridge. In practice, we recommend processing 8 (or the maximum number of the centrifuge) or fewer filters at a time.

1. Preheat an incubator to 56 °C.
2. Prepare a 2.0 ml tube by cutting the hinged cap from the tube. Discard the cap.
   NOTE: This tube is used as a collection tube for the remaining liquid in the filter.
3. Remove the inlet (NOT outlet) luer cap from the filter cartridge and insert the inlet port into the collection tube. Tightly seal a connection between the cartridge and collection tube using a self-sealing film.
4. Insert the combined unit into a centrifuge adaptor for a 15-ml conical tube. Centrifuge the cartridge at 5,000 x g for 1 min to remove the remaining liquid in the filter.
5. Remove the collection tube from the filter cartridge and discard the tube. Re-cap the inlet port of the cartridge.
6. Prepare a mixture of 20 µl proteinase-K solution, 220 µl PBS (phosphate buffered saline; not provided by the kit) and 200 µl buffer AL.
   NOTE: The filter cartridge accommodates up to four times the volumes of the mixture (ca. 1.8 ml).
7. Remove the inlet cap and add the mixture (440 µl) into the filter cartridge using a pipette tip. Insert the pipette completely into the inlet port so that the pipette tip is visible inside the cartridge just above the membrane.
8. Re-cap the inlet port and place the filter cartridge on a rotary shaker.
9. Place the rotary shaker in the preheated incubator at 56 ˚C and turn on the shaker at a speed of 20 rpm for 20 min.
10. During the incubation, prepare a new 2.0 ml tube, label the tube appropriately using a solvent-proof pen and place it in a 50-ml conical tube.
    NOTE: The former (2.0 ml) and latter tubes (50 ml) are used for collection of the extracted DNA and for holding the 2.0 ml tube, respectively.
11. After the incubation, remove the inlet cap and insert the inlet (NOT outlet) port of the cartridge into the 2.0 ml tube within the 50 ml conical tube. Close the 50 ml conical tube with a screw cap.
12. Centrifuge the 50-ml conical tube at 5,000 x g for 1 min to collect the extracted DNA from the cartridge. Remove the filter cartridge and the 2.0 ml tube using sterilized forceps and cap the tube. Discard the filter cartridge.

6. Purification of Extracted DNA

NOTE: We elute eDNA with 100 µl buffer AE instead of 200 µl specified in the manual of the commercial kit.

1. Add 200 µl ethanol (96-100%) to the extracted DNA in the 2.0-ml tube from the above step (ca. 440 µl), and mix thoroughly by vortexing.
2. Pipet the mixture into a spin column placed in a 2 ml collection tube. Centrifuge at 5,000 x g for 1 min. Discard the flow-through and the collection tube.
3. Place the spin column in a new 2 ml collection tube, add 500 µl buffer AW1, and centrifuge for 1 min at 5,000 x g. Discard the flow-through and the collection tube.
4. Place the spin column in a new 2 ml collection tube, add 500-µl buffer AW2, and centrifuge for 3 min at 20,000 x g. Discard the flow-through and the collection tube.
5. Prepare a new 1.5 ml tube (not provided by the kit) and label the tube appropriately using a waterproof pen for fast-drying and non-smearing labeling. Transfer the spin column to the 1.5 ml tube.
6. Elute the DNA by adding 100 µl buffer AE to the center of the spin column membrane. Incubate for 1 min at room temperature, and then centrifuge at 6,000 x g for 1 min.
7. Discard the spin column and cap the tube. Store the purified DNA at -20 °C.

Wyniki

It is technically difficult to isolate and quantify only fish eDNA from the extracted bulk eDNA, because the MiFish primers coamplify the target region from some non-fish vertebrates, such as birds and mammals, with PCR products of the same size (ca. 170 bp)¹². Instead of quantifying fish eDNA, we perform MiFish metabarcoding analysis of eDNA from an aquarium tank with known species composition using the two different methods of filtration and DNA extraction, and compa...

Dyskusje

In many metabarcoding studies using environmental samples such as water and soil, post-filtration treatment of the filter cartridge is generally as follows^24,25: 1) cutting open or cracking the housing with hand tools (tubing cutter or pliers); 2) removal of the filter from the cartridge; and 3) cutting the filter into small pieces with a razor blade for DNA extraction. To avoid such cumbersome and time-consuming procedures that are prone to contamination in the laboratory, we have attempted several DNA extrac...

Materiały

Name	Company	Catalog Number	Comments
Mesh panel	Iris Ohyama	MPP-3060-BE
Metal prong	Iris Ohyama	MR12F
Stand for the mesh panel	No brand	4184-9507	available from Amazon Japan
1 L plastic bag with screw cap	Yanagi	DP16-TN1000
Male luer-lock connector	ISIS	11620
10 ml pipette tip	Eppendorf	0030 000.765
10 L book bottle with valve	As One	1-2169-01
Sterivex-HV filter	Millipore	SVHVL10RC	denoted as "filter cartridge" throughout the ms and used in the protocol
Male luer fitting	As One	1-7379-04
Female luer fitting	As One	5-1043-14
Inlet luer cap	ISIS	VRMP6
Outlet luer cap	ISIS	VRFP6
High vacuum tubing	As One	6-590-01
Vacuum connector	As One	6-663-02
Silicone stopper	As One	1-7650-07
Manifold	As One	2-258-01
Aspirator-GAS-1	As One	1-7483-21
DNeasy Blood & Tissue Kit (250)	Qiagen	69506
PowerWater Sterivex DNA Isolation Kit	MO BIO	14600-50-NF	denoted as "optional kit" in the ms
Tabletop Centrifuge	Kubota	Model 4000	Maximum speed 6,000 rpm
Fixed-angle rotor	Kubota	AT-508C
Adaptor for a 15 ml conical tube	Kubota	055-1280
RNAlater Stabilization Solution	Thermo Fisher Scientific	AM7020
	Parafilm	PM992	denoted as "self-sealing film"