ろ過水のサンプルおよび環境DNAの抽出のためのフィルターカートリッジの使用

要約

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.

要約

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.

概要

水生環境における環境DNA（エドナ）は水柱で見つかった遺伝物質を指します。最近の研究では、池^1-3、河川^4-8を含む様々な水生環境から魚を検出するためのエドナの有用性を実証した^{9ストリーム} 、および海水^10-14。いくつかの最近の研究では、地元の魚のコミュニティ^7,9で複数種の同時検出を試みながら、これらの研究のほとんどは^、単一または侵襲^1,4-6,8,14と希少または絶滅危惧種^3,9数の検出に焦点を当て^12,13,15とメソコスム^11,12。

後者のアプローチは、「metabarcoding」と呼ばれ、エドナのmetabarcodingは分類学的に多様なサンプル全体で遺伝子領域をcoamplifyするためのPCRプライマーの一つまたは複数のセットを使用しています。これは、インデックス作成とアダプタを添加したライブラリの準備が続いている、してインデックスを作成ライブラリーは、ハイスループット並列配列決定により分析されていますプラットフォーム。最近宮ら ^{12は、（「MiFish」}と呼ばれる）魚類からエドナをmetabarcodingためのユニバーサルPCRプライマーを開発しました。 MiFishプライマーは、いくつかの密接に関連した同族体を除き分類学上の科、属および種に魚を識別するのに十分な情報が含まれているミトコンドリア12S rRNA遺伝子（163から185塩基対）の超可変領域を標的とします。エドナのmetabarcodingにおけるそれらのプライマーを用いることで、宮ら ^12は、水槽の近くに既知の種組成やサンゴ礁と水槽タンクから230以上の亜熱帯海洋生物種を検出しました。

魚類からエドナ濃度のさまざまなレベルでの天然海水を収容するためにmetabarcodingプロトコルを最適化しながら、我々はMiFishプライマーは、時折、その後のライブラリーの調製のための標的領域を増幅するために失敗したことに気づきました。この失敗したPCR増幅のための可能性が高い理由の一つは、TEの十分な量の欠如でありますろ過された水の小容積に含まれるmplate DNA（ すなわち、1-2 L）。特定の分類群からエドナ濃度が増幅前に不可知であるが、大規模な水の体積（> 1-2 L）のろ過のような、希少魚の豊富さとバイオマスと水環境からより多くのエドナを収集するためのシンプルで効果的な手段であろうオープン海や深海の生態系。

従来魚エドナ研究¹⁶の数で使用されるディスクファイバーフィルターに対して、フィルタカートリッジ^17を詰まらせる前に、より大きな水容積を収容するという利点を有します。実際、最近の研究は、大容量（> 20 L）は、フィルタカートリッジ^18を使用して^、沿岸海水サンプルのろ過を示しました。さらに、それらは、個別に包装され、滅菌、および実験ワークフローのいくつかのステップは、このように研究室¹⁹からの汚染の可能性を低減する、フィルターハウジング内で行うことが可能とされています。後者特徴は、最大の実験は^20,21に挑戦間で汚染の危険性が残っているエドナのmetabarcoding、のために重要です。フィルタカートリッジのこれらの技術的な利点にもかかわらず、それは2つの例外^8,15と魚類のエドナ研究で使用されていません。

ここでは、筐体を開くカットすることなく、そのフィルタからエドナのフィルターカートリッジと抽出と水試料の濾過のためのプロトコルを提供します。また、水の体積（≤4Lまたは> 4 L）に応じて、2つの代替水のろ過システムを提供します。私たちの研究グループ^12,14,22,23に新たに開発されたプロトコルの性能とガラス繊維フィルターを使用して、以前に使用されるプロトコルを比較するために、我々は7500メートル^3（巨大な水槽から海水のエドナmetabarcoding分析を行います）既知の種組成とし、代表的な結果として、2つのプロトコル由来検出種の数を示しています。このプロトコルの時間魚類からエドナをmetabarcodingために開発されたが、他の生物からもエドナにも適用可能です。

プロトコル

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.

結果

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...

ディスカッション

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...

資料

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"