Summary

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

Published: November 25, 2016
doi:

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 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 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

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 form…

Representative Results

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…

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"

References

  1. Takahara, T., Minamoto, T., Doi, H. Using environmental DNA to estimate the distribution of an invasive fish species in ponds. PLoS ONE. 8, e56584 (2013).
  2. Takahara, T., Minamoto, T., Yamanaka, H., Doi, H., Kawabata, Z. Estimation of fish biomass using environmental DNA. PLoS ONE. 7, e35868 (2012).
  3. Sigsgaard, E. E., Carl, H., Møller, P. R., Thomsen, P. F. Monitoring the near-extinct European weather loach in Denmark based on environmental DNA from water samples. Biol. Conserv. 183, 48-52 (2015).
  4. Jerde, C. L., et al. Detection of Asian carp DNA as part of a Great Lakes basin-wide surveillance program. Can. J. Fish. Aquat. Sci. 70, 522-526 (2013).
  5. Jerde, C. L., Mahon, A. R., Chadderton, W. L., Lodge, D. M. "Sight-unseen" detection of rare aquatic species using environmental DNA. Conserv. Lett. 4, 150-157 (2011).
  6. Mahon, A. R., et al. Validation of eDNA surveillance sensitivity for detection of Asian carps in controlled and field experiments. PLoS ONE. 8, e58316 (2013).
  7. Minamoto, T., Yamanaka, H., Takahara, T., Honjo, M. N., Kawabata, Z. Surveillance of fish species composition using environmental DNA. Limnology. 13, 193-197 (2012).
  8. Keskin, E. Detection of invasive freshwater fish species using environmental DNA survey. Biochem. Syst. Ecol. 56, 68-74 (2014).
  9. Wilcox, T. M., et al. Robust detection of rare species using environmental DNA: the importance of primer specificity. PLoS ONE. 8, e59520 (2013).
  10. Thomsen, P. F., et al. Detection of a diverse marine fish fauna using environmental DNA from seawater samples. PLoS ONE. 7, e41732 (2012).
  11. Kelly, R. P., et al. Harnessing DNA to improve environmental management. Science. 344, 1455-1456 (2014).
  12. Miya, M., et al. Mifish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. Roy. Soc. Open Sci. 2, 150088 (2015).
  13. Port, J. A., et al. Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA. Mol. Ecol. 25, 527-541 (2015).
  14. Yamamoto, S., et al. Environmental DNA provides a ‘snapshot’ of fish distribution: a case study of Japanese jack mackerel in Maizuru Bay, Sea of Japan. PLoS ONE. 11, e0149786 (2016).
  15. Valentini, A., et al. Next generation monitoring of aquatic biodiversity using environmental DNA metabarcoding. Mol. Ecol. 25, 929-942 (2016).
  16. Rees, H. C., Maddison, B. C., Middleditch, D. J., Patmore, J. R., Gough, K. C. Review: The detection of aquatic animal species using environmental DNA – a review of eDNA as a survey tool in ecology. J. Appl. Ecol. 51, 1450-1459 (2014).
  17. Stewart, F. J., DeLong, E. E. . Microbial metagenomics, Metatranscriptomics, and metaprotenomics Vol. 531 Methods in Enzymology. 10, 187-218 (2013).
  18. Walsh, D. A., Zaikova, E., Hallam, S. J. Large volume (20L+) filtration of coastal seawater samples. J Vis Exp. (28), e1161 (2009).
  19. Smalla, K., Akkermans, D. L., Elsas, J. D., Bruijn, F. J. . Molecular Microbial Ecology Manual. , 13-22 (1995).
  20. Thomsen, P. F., Willerslev, E. Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv. 183, 4-18 (2014).
  21. Pedersen, M. W., et al. Ancient and modern environmental DNA. Phil. Trans. R. Soc. B. 370, 20130383 (2015).
  22. Fukumoto, S., Ushimaru, A., Minamoto, T. A basin scale application of environmental DNA assessment for rare endemic species and closely related exotic species in rivers: a case study of giant salamanders in Japan. J. Appl. Ecol. 52, 358-365 (2015).
  23. Yamanaka, H., Minamoto, T. The use of environmental DNA of fishes as an efficient method of determining habitat connectivity. Ecol. Indicators. 62, 147-153 (2016).
  24. Moss, J. A., et al. Ciliated protists from the nepheloid layer and water column of sites affected by the Deepwater Horizon oil spill in the Northeastern Gulf of Mexico. Deep Sea Res. Pt I. 106, 85-96 (2015).
  25. Hilton, J. A., Satinsky, B. M., Doherty, M., Zielinski, B., Zehr, J. P. Metatranscriptomics of N2-fixing cyanobacteria in the Amazon River plume. The ISME journal. 9, 1557-1569 (2015).
  26. Deiner, K., Walser, J. -. C., Mächler, E., Altermatt, F. Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA. Biol. Conserv. 183, 53-63 (2015).
  27. Eichmiller, J. J., Miller, L. M., Sorensen, P. W. Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish. Mol. Ecol. Res. 16, 56-68 (2016).
  28. Lemarchand, K., Pollet, T., Lessard, V., Badri, M. A., Micic, M. . Sample Preparation Techniques for Soil, Plant, and Animal Samples’Springer Protocols Handbooks. , 325-339 (2016).
  29. Turner, C. R., et al. Particle size distribution and optimal capture of aqueous macrobial eDNA. Methods Ecol. Evol. 5, 676-684 (2014).
  30. Barnes, M. A., Turner, C. R. The ecology of environmental DNA and implications for conservation genetics. Conserv. Genet. 17, 1-17 (2016).
  31. Sorokulova, I., Olsen, E., Vodyanoy, V. Biopolymers for sample collection, protection, and preservation. Appl. Microbiol. Biotechnol. 99, 5397-5406 (2015).
  32. Renshaw, M. A., Olds, B. P., Jerde, C. L., McVeigh, M. M., Lodge, D. M. The room temperature preservation of filtered environmental DNA samples and assimilation into a Phenol-Chloroform-Isoamyl alcohol DNA extraction. Mol. Ecol. Res. 2014, (2014).

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Cite This Article
Miya, M., Minamoto, T., Yamanaka, H., Oka, S., Sato, K., Yamamoto, S., Sado, T., Doi, H. Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA. J. Vis. Exp. (117), e54741, doi:10.3791/54741 (2016).

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