滤芯水过滤样品和环境DNA的提取使用
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
在水生环境环境DNA（的eDNA）是指水体中发现的遗传物质。最近的研究表明的eDNA的效用从各种水生环境，包括池塘1-3，4-8江河鱼类检测，溪流9，和海水10-14。这些研究大多集中在检测的单个或几个侵入1,4-6,8,14和珍稀或濒危物种3,9，而最近的一些研究试图在当地鱼类群落7,9多个物种的同时检测， 12,13,15和生物群落11,12。
后一种方法被称为“metabarcoding”和的eDNA metabarcoding使用的PCR引物的一个或多个集合横跨分类学上不同的样品coamplify的基因区域。其次是与索引和适配器此外文库制备和索引库由一个高通量平行测序分析平台。最近宫等人 12开发的通用PCR引物用于从鱼类（称为“MiFish”）metabarcoding的eDNA。所述MiFish引物靶向线粒体12S rRNA基因（163-185碱基），它包含足够的信息，以确定鱼生物分类科，属和除了一些密切相关的同源物种的高变区。随着使用中的eDNA metabarcoding的引物，美哉等 12检测到水族箱230余属亚热带海洋物种，水族馆附近的已知物种组成和珊瑚礁。
同时优化metabarcoding协议容纳天然海水以改变来自鱼类的eDNA浓度的水平，我们已经注意到，MiFish引物偶尔未能扩增后续文库制备目标区域。一个为这个不成功的PCR扩增更可能的原因是缺乏足够数量的TE的中所含的水的小体积的过滤mplate的DNA（ 即 1-2升）。虽然从特定分类群的eDNA浓度放大之前是不可知的大型水量（> 1-2升），过滤是一个简单而有效的手段，从稀缺的鱼类数量和生物量，比如水环境收集更多的eDNA公海和深海生态系统。
相对于磁盘纤维过滤器在许多鱼的eDNA研究16的常规使用的，滤芯具有堵塞17之前容纳较大水量的优点。事实上，最近的一项研究表明，大体积（> 20 L）采用滤芯18沿海海水样品的过滤。此外，它们被单独包装和无菌，实验工作流的若干步骤可以在过滤器外壳中进行，从而降低了从实验室19污染的可能性。后者特征为的eDNA metabarcoding关键，其中污染的风险仍然之间的最大实验挑战20,21。尽管滤芯这些技术优势，它尚未在鱼类的eDNA研究中使用的有两个例外8,15。
在这里，我们提供了从它的过滤器的eDNA的滤筒和提取水样的过滤的协议，而不必切开壳体。我们还提供了根据不同的水量（≤4L或> 4升）两种可供选择的水过滤系统。为了比较新开发协议的性能，在我们的研究小组使用12,14,22,23玻璃纤维过滤先前使用的协议，我们执行的eDNA metabarcoding海水分析从一个巨大的鱼缸（7 500 立方米 ）与已知的物种组成，并示出了从两个协议作为代表性结果得出检测物种的数目。该协议ħ作为已经开发了用于从鱼类metabarcoding的eDNA，但也适用于的eDNA从其他生物体。
Protocol
Representative Results
Discussion
在使用环境样品，例如水和土壤许多metabarcoding研究中，过滤器滤芯的过滤后处理通常如下24,25：1）切开或开裂用手动工具（管切割器或钳子）的外壳; 2）清除墨盒上的过滤器;和3）切割所述滤波器小块用刀片用于DNA提取。为了避免过滤器滤芯的外壳内的这种麻烦的和费时的，它们很容易在实验室污染的程序，我们尝试几种DNA提取方法使用一种广泛使用的，价格低廉市售的试剂盒，并成功?…
Divulgations
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
- 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).
- Takahara, T., Minamoto, T., Yamanaka, H., Doi, H., Kawabata, Z. Estimation of fish biomass using environmental DNA. PLoS ONE. 7, e35868 (2012).
- 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).
- 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).
- 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).
- 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).
- Minamoto, T., Yamanaka, H., Takahara, T., Honjo, M. N., Kawabata, Z. Surveillance of fish species composition using environmental DNA. Limnology. 13, 193-197 (2012).
- Keskin, E. Detection of invasive freshwater fish species using environmental DNA survey. Biochem. Syst. Ecol. 56, 68-74 (2014).
- Wilcox, T. M., et al. Robust detection of rare species using environmental DNA: the importance of primer specificity. PLoS ONE. 8, e59520 (2013).
- Thomsen, P. F., et al. Detection of a diverse marine fish fauna using environmental DNA from seawater samples. PLoS ONE. 7, e41732 (2012).
- Kelly, R. P., et al. Harnessing DNA to improve environmental management. Science. 344, 1455-1456 (2014).
- 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).
- Port, J. A., et al. Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA. Mol. Ecol. 25, 527-541 (2015).
- 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).
- Valentini, A., et al. Next generation monitoring of aquatic biodiversity using environmental DNA metabarcoding. Mol. Ecol. 25, 929-942 (2016).
- 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).
- Stewart, F. J., DeLong, E. E. . Microbial metagenomics, Metatranscriptomics, and metaprotenomics Vol. 531 Methods in Enzymology. 10, 187-218 (2013).
- Walsh, D. A., Zaikova, E., Hallam, S. J. Large volume (20L+) filtration of coastal seawater samples. J Vis Exp. (28), e1161 (2009).
- Smalla, K., Akkermans, D. L., Elsas, J. D., Bruijn, F. J. . Molecular Microbial Ecology Manual. , 13-22 (1995).
- Thomsen, P. F., Willerslev, E. Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv. 183, 4-18 (2014).
- Pedersen, M. W., et al. Ancient and modern environmental DNA. Phil. Trans. R. Soc. B. 370, 20130383 (2015).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- Turner, C. R., et al. Particle size distribution and optimal capture of aqueous macrobial eDNA. Methods Ecol. Evol. 5, 676-684 (2014).
- Barnes, M. A., Turner, C. R. The ecology of environmental DNA and implications for conservation genetics. Conserv. Genet. 17, 1-17 (2016).
- Sorokulova, I., Olsen, E., Vodyanoy, V. Biopolymers for sample collection, protection, and preservation. Appl. Microbiol. Biotechnol. 99, 5397-5406 (2015).
- 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).
