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
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
In many metabarcoding studies using environmental samples such as water and soil, post-filtration treatment of the filter cartridge is generally as follows24,25: 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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was supported as basic research by CREST from the Japan Science and Technology Agency (JST) and by grants from JSPS/MEXT KAKENHI (Number 26291083) and the Canon Foundation to M.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Materials
| 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" |
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