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.

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...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+near-extinct+European+weather+loach+in+Denmark+based+on+environmental+DNA+from+water+samples.">Monitoring the near-extinct European weather loach in Denmark based on environmental DNA from water samples.</a> Biol. Conserv. 183, 48-52 (2015).</li><li>Jerde, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Asian+carp+DNA+as+part+of+a+Great+Lakes+basin-wide+surveillance+program.">Detection of Asian carp DNA as part of a Great Lakes basin-wide surveillance program.</a> Can. J. Fish. Aquat. Sci. 70, 522-526 (2013).</li><li>Jerde, C. L., Mahon, A. R., Chadderton, W. L., Lodge, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&amp;#34;Sight-unseen&amp;#34;+detection+of+rare+aquatic+species+using+environmental+DNA.">&amp;#34;Sight-unseen&amp;#34; detection of rare aquatic species using environmental DNA.</a> Conserv. Lett. 4, 150-157 (2011).</li><li>Mahon, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+eDNA+surveillance+sensitivity+for+detection+of+Asian+carps+in+controlled+and+field+experiments.">Validation of eDNA surveillance sensitivity for detection of Asian carps in controlled and field experiments.</a> PLoS ONE. 8, e58316 (2013).</li><li>Minamoto, T., Yamanaka, H., Takahara, T., Honjo, M. N., Kawabata, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+of+fish+species+composition+using+environmental+DNA.">Surveillance of fish species composition using environmental DNA.</a> Limnology. 13, 193-197 (2012).</li><li>Keskin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+invasive+freshwater+fish+species+using+environmental+DNA+survey.">Detection of invasive freshwater fish species using environmental DNA survey.</a> Biochem. Syst. Ecol. 56, 68-74 (2014).</li><li>Wilcox, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+detection+of+rare+species+using+environmental+DNA:+the+importance+of+primer+specificity.">Robust detection of rare species using environmental DNA: the importance of primer specificity.</a> PLoS ONE. 8, e59520 (2013).</li><li>Thomsen, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+a+diverse+marine+fish+fauna+using+environmental+DNA+from+seawater+samples.">Detection of a diverse marine fish fauna using environmental DNA from seawater samples.</a> PLoS ONE. 7, e41732 (2012).</li><li>Kelly, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+DNA+to+improve+environmental+management.">Harnessing DNA to improve environmental management.</a> Science. 344, 1455-1456 (2014).</li><li>Miya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mifish,+a+set+of+universal+PCR+primers+for+metabarcoding+environmental+DNA+from+fishes:+detection+of+more+than+230+subtropical+marine+species.">Mifish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species.</a> Roy. Soc. Open Sci. 2, 150088 (2015).</li><li>Port, J. 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Ecol. 25, 527-541 (2015).</li><li>Yamamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+DNA+provides+a+'snapshot'+of+fish+distribution:+a+case+study+of+Japanese+jack+mackerel+in+Maizuru+Bay,+Sea+of+Japan.">Environmental DNA provides a 'snapshot' of fish distribution: a case study of Japanese jack mackerel in Maizuru Bay, Sea of Japan.</a> PLoS ONE. 11, e0149786 (2016).</li><li>Valentini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+monitoring+of+aquatic+biodiversity+using+environmental+DNA+metabarcoding.">Next generation monitoring of aquatic biodiversity using environmental DNA metabarcoding.</a> Mol. Ecol. 25, 929-942 (2016).</li><li>Rees, H. C., Maddison, B. C., Middleditch, D. J., Patmore, J. R., Gough, K. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+and+modern+environmental+DNA.">Ancient and modern environmental DNA.</a> Phil. Trans. R. Soc. B. 370, 20130383 (2015).</li><li>Fukumoto, S., Ushimaru, A., Minamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> J. Appl. 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The authors have nothing to disclose.

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...

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 ponds1-3, rivers4-8, streams9, and seawater10-14. Most of these studies focused on detection of a single or a few invasive1,4-6,8,14 and rare or threatened species3,9, while some recent studies attempted simultaneous detection of multiple species in local fish communities7,9,12,13,15 and mesocosms11,12.
The latter approach is called &#34;metabarcoding&#34; 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 &#34;MiFish&#34;). 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 (&#62;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 (&#62;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 challenges20,21. Despite these technical advantages of filter cartridges, it has not been used in eDNA studies of fishes with two exceptions8,15.
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 (&#8804;4 L or &#62;4 L). To compare the performance of the newly-developed protocol and a previously-used protocol using a glass-fiber filter in our research group12,14,22,23, 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.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Mesh panel</td>
			<td style="width:167px;">Iris Ohyama</td>
			<td style="width:119px;">MPP-3060-BE</td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Metal prong</td>
			<td style="width:167px;">Iris Ohyama</td>
			<td style="width:119px;">MR12F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stand for the mesh panel</td>
			<td style="width:167px;">No brand</td>
			<td>4184-9507</td>
			<td>available from Amazon Japan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1-L plastic bag with screw cap</td>
			<td style="width:167px;">Yanagi</td>
			<td style="width:119px;">DP16-TN1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Male luer-lock connector</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">11620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">10-mL pipette tip</td>
			<td style="width:167px;">Eppendorf</td>
			<td style="width:119px;">0030 000.765</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">10-L book bottle with valve</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-2169-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sterivex-HV filter</td>
			<td style="width:167px;">Millipore</td>
			<td style="width:119px;">SVHVL10RC</td>
			<td>denoted as &#34;filter cartridge&#34; throughout the ms and used in the protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Male luer fitting</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7379-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Female luer fitting</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">5-1043-14&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Inlet luer cap</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">VRMP6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Outlet luer cap</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">VRFP6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">High vacuum tubing</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">6-590-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Vacuum connector</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">6-663-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone stopper</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7650-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Manifold</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">2-258-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Aspirator-GAS-1</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7483-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DNeasy Blood &#38; Tissue Kit (250)</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">69506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">PowerWater Sterivex DNA Isolation Kit</td>
			<td style="width:167px;">MO BIO</td>
			<td style="width:119px;">14600-50-NF</td>
			<td>denoted as &#34;optional kit&#34; in the ms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tabletop Centrifuge</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">Model 4000</td>
			<td>Maximum speed 6,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fixed-angle rotor</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">AT-508C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adaptor for a 15 mL conical tube</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">055-1280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">RNAlater Stabilization Solution</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;"></td>
			<td style="width:167px;">Parafilm</td>
			<td style="width:119px;">PM992</td>
			<td>denoted as &#34;self-sealing film&#34;</td>
		</tr>
	</tbody>
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use of a filter cartridge for filtration of water samples and extraction of environmental dna

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 &#8804;4 L or &gt;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.

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...

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

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.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

The overall goal of this protocol is to filter water samples with a filter cartridge and extract environmental DNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms. This method can help answer key questions in the ecological field, such as biodiversity monitoring and community ecology. The main advantage of this technique is that the filter cartridges can accommodate larger water volumes and the filtration and DNA extraction can be performed in a sterile manner.To begin this procedure, attach high vacuum tubing to the input connector of an aspirator pump. Attach the other end of the tubing to a manifold. Be sure that the three red T-valves at the manifold are in the off position.Next, put on a clean set of gloves. Insert a female luer fitting at the top end of the vacuum rubber tubing. Then, insert a vacuum connector at the bottom end.Carefully attach the female luer fitting to an outlet port of the filter cartridge. Then, attach the vacuum connector to a silicone stopper. Pour one liter of sampled water into the prepared plastic bag.Enclose the screw cap with the male luer-lock connector. Then, carefully connect an inlet port of the assembled filter cartridge with the male luer-lock connector. Ensure that all connections are secure.Then, hang the plastic bag from two prongs on the mesh panel. Insert a silicone stopper into the inlet port of the manifold. Then, turn on the aspirator.Open the red T-valves for filtration. Run the manifold until the filter cartridge is dry. Then, close the red T-valves.Repeat this entire process to filter the desired amount of water. Once all of the sampled water has been filtered, carefully remove the filter cartridge from the plastic bag and vacuum container. Cap both ends of the cartridge with the appropriate luer caps.Then, using a solvent proof pen, label the cartridge and inlet luer cap. Store at 20 degrees Celsius until ready to perform DNA extraction. For samples larger than four liters, prepare a ten liter book bottle as detailed in the text protocol.Add the water sample to be filtered. Then, tightly insert a ten milliliter pipette tip into the valve of the book bottle. Tightly insert the inlet port of the filter cartridge into the pipette tip.Then, insert a silicone stopper into the inlet port of the manifold. Turn on the aspirator. Then, open the red T-valves for filtration.Run the manifold until the filter cartridge is dry. After the filtration is complete, close the red T-valves. Carefully remove the cartridge from the plastic bag and vacuum connector.Then cap both ends of the cartridge with the appropriate luer caps. Use a solvent proof pen to label the cartridge and the inlet luer cap. Store at 20 degrees Celsius until ready to perform DNA extraction.Preheat an incubator to 56 degrees Celsius. Then, cut the hinged cap from a 2.0 milliliter tube and discard the cap. Remove the inlet luer cap from the filter cartridge.Then, insert the inlet port into the 2.0 milliliter tube. Using a self-sealing film, tightly seal the connection between the cartridge and the collection tube. Next, insert the combined unit into a centrifuge adapter for a 15 milliliter conical tube.Centrifuge the cartridge for one minute at 5000 times g to remove the remaining liquid in the filter. Once centrifugation is complete, remove the collection tube from the filter cartridge and discard the tube. Then, recap the inlet port.Next, mix 20 microliters of protein A-Case solution, 220 microliters of phosphate-buffered saline, and 200 microliters of buffer AL.Remove the inlet cap and add the mixture into the cartridge using a pipette, inserting the pipette completely into the inlet port. Recap the inlet port. Then, place the filter cartridge on a rotary shaker.Place the rotary shaker in the preheated incubator, and set the shaker to 20 rpm for 20 minutes. During the incubation, label a new 2.0 milliliter tube with a solvent proof pen. Then, place it into a 50 milliliter conical tube.After the incubation is complete, remove the inlet cap. Then, insert the inlet port into the new 2.0 milliliter tube. Cap the conical tube with a screw cap.Centrifuge for one minute at 5000 times g to collect the extracted DNA. Then, remove the cartridge in the 2.0 milliliter tube using sterilized forceps. Cap the tube and purify the extracted DNA as outlined in the text protocol.In this procedure, four different volumes of sea water are filtered on the same manifold using two unique filters, and eDNA is subsequently extracted. The filter cartridge detected, on average, 50%more species than the glass fiber filters. Once mastered, this technique can be done in two hours if it is performed properly.After watching this video, you should have a good understanding of how to filter water samples with a filter cartridge, and how to extract eDNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms.

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Research

JoVE Journal

Environment

24.6K vues.  Natural History Museum and Institute, Chiba. The overall goal of this protocol is to filter water samples with a filter cartridge and extract environmental DNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms. This method can help answer key questions in the ecological field, such as biodiversity monitoring and community ecology. The main advantage of this technique is that the filter cartridges can accommodate larger water volumes and the filtration and DNA extraction ca...