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 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 &#62;4 L.
<ol>
	<li>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.</li>
	<li>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&#39;s instructions).</li>
	<li>Insert the male luer-lock connector into the hole of the screw cap. Wait until complete adhesive bonding of the two parts (usually &#62;24 hr). Sterilize the screw cap with the male luer-lock connector with 10% commercial bleach (ca. 0.6% sodium hypochlorite) before use.</li>
	<li>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.</li>
</ol>
2. Assembly of the Filtration System
<ol>
	<li>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 &#34;off position&#34; (i.e., horizontal).</li>
	<li>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.</li>
	<li>Carefully attach the female luer fitting to an outlet port of the filter cartridge and attach the vacuum connector to a silicone stopper.</li>
</ol>
3. Filtration of Water Samples (&#8804;4 L) using the Filter Cartridge
NOTE: Skip this step if the filtration volume is &#62;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.
<ol>
	<li>Pour the 1 L sampled water into the plastic bag and close the screw cap with the male luer-lock connector.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Carefully remove the filter cartridge from the plastic bag and the vacuum connector.</li>
	<li>Cap both ends of the cartridge with the inlet and outlet luer caps.</li>
	<li>Label the cartridge and inlet luer cap appropriately using a solvent-proof pen for fast-drying and non-smearing labeling.</li>
	<li>Store the filter cartridge at -20 &#730;C before DNA extraction.</li>
</ol>
4. Filtration of Water (&#62;4 L) Samples using the Filter Cartridge
Note: Skip this step if the water filtration volume is &#8804;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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C before DNA extraction.</li>
</ol>
5. Extraction of eDNA from the Filter
NOTE: In steps 4 and 5, we use a commercial kit, largely following a protocol for &#34;nucleated blood&#34; 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.
<ol>
	<li>Preheat an incubator to 56 &#176;C.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Remove the collection tube from the filter cartridge and discard the tube. Re-cap the inlet port of the cartridge.</li>
	<li>Prepare a mixture of 20 &#181;l proteinase-K solution, 220 &#181;l PBS (phosphate buffered saline; not provided by the kit) and 200 &#181;l buffer AL. 
	NOTE: The filter cartridge accommodates up to four times the volumes of the mixture (ca. 1.8 ml).</li>
	<li>Remove the inlet cap and add the mixture (440 &#181;l) 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.</li>
	<li>Re-cap the inlet port and place the filter cartridge on a rotary shaker.</li>
	<li>Place the rotary shaker in the preheated incubator at 56 &#730;C and turn on the shaker at a speed of 20 rpm for 20 min.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Purification of Extracted DNA
NOTE: We elute eDNA with 100 &#181;l buffer AE instead of 200 &#181;l specified in the manual of the commercial kit.
<ol>
	<li>Add 200 &#181;l ethanol (96-100%) to the extracted DNA in the 2.0-ml tube from the above step (ca. 440 &#181;l), and mix thoroughly by vortexing.</li>
	<li>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.</li>
	<li>Place the spin column in a new 2 ml collection tube, add 500 &#181;l buffer AW1, and centrifuge for 1 min at 5,000 x g. Discard the flow-through and the collection tube.</li>
	<li>Place the spin column in a new 2 ml collection tube, add 500-&#181;l buffer AW2, and centrifuge for 3 min at 20,000 x g. Discard the flow-through and the collection tube.</li>
	<li>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.</li>
	<li>Elute the DNA by adding 100 &#181;l 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.</li>
	<li>Discard the spin column and cap the tube. Store the purified DNA at -20 &#176;C.</li>
</ol>

<ol><li>Takahara, T., Minamoto, T., Doi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+environmental+DNA+to+estimate+the+distribution+of+an+invasive+fish+species+in+ponds%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Using environmental DNA to estimate the distribution of an invasive fish species in ponds.</a> PLoS ONE. 8, e56584(2013).</li>
<li>Takahara, T., Minamoto, T., Yamanaka, H., Doi, H., Kawabata, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimation+of+fish+biomass+using+environmental+DNA%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Estimation of fish biomass using environmental DNA.</a> PLoS ONE. 7, e35868(2012).</li>
<li>Sigsgaard, E. E., Carl, H., Møller, P. R., Thomsen, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+the+near-extinct+European+weather+loach+in+Denmark+based+on+environmental+DNA+from+water+samples%5BTitle%5D)+AND+%22Biol.+Conserv%22%5BJournal%5D)">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/pubmed?term=((Detection+of+Asian+carp+DNA+as+part+of+a+Great+Lakes+basin-wide+surveillance+program%5BTitle%5D)+AND+%22Can.+J.+Fish.+Aquat.+Sci%22%5BJournal%5D)">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/pubmed?term=((%26%2334%3BSight-unseen%26%2334%3B+detection+of+rare+aquatic+species+using+environmental+DNA%5BTitle%5D)+AND+%22Conserv.+Lett%22%5BJournal%5D)">&#34;Sight-unseen&#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/pubmed?term=((Validation+of+eDNA+surveillance+sensitivity+for+detection+of+Asian+carps+in+controlled+and+field+experiments%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Surveillance+of+fish+species+composition+using+environmental+DNA%5BTitle%5D)+AND+%22Limnology%22%5BJournal%5D)">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/pubmed?term=((Detection+of+invasive+freshwater+fish+species+using+environmental+DNA+survey%5BTitle%5D)+AND+%22Biochem.+Syst.+Ecol%22%5BJournal%5D)">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/pubmed?term=((Robust+detection+of+rare+species+using+environmental+DNA%3A+the+importance+of+primer+specificity%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Detection+of+a+diverse+marine+fish+fauna+using+environmental+DNA+from+seawater+samples%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Harnessing+DNA+to+improve+environmental+management%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Mifish%2C+a+set+of+universal+PCR+primers+for+metabarcoding+environmental+DNA+from+fishes%3A+detection+of+more+than+230+subtropical+marine+species%5BTitle%5D)+AND+%22Roy.+Soc.+Open+Sci%22%5BJournal%5D)">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. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+vertebrate+biodiversity+in+a+kelp+forest+ecosystem+using+environmental+DNA%5BTitle%5D)+AND+%22Mol.+Ecol%22%5BJournal%5D)">Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA.</a> Mol. Ecol. 25, 527-541 (2015).</li>
<li>Yamamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+DNA+provides+a+%27snapshot%27+of+fish+distribution%3A+a+case+study+of+Japanese+jack+mackerel+in+Maizuru+Bay%2C+Sea+of+Japan%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Next+generation+monitoring+of+aquatic+biodiversity+using+environmental+DNA+metabarcoding%5BTitle%5D)+AND+%22Mol.+Ecol%22%5BJournal%5D)">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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review%3A+The+detection+of+aquatic+animal+species+using+environmental+DNA+-+a+review+of+eDNA+as+a+survey+tool+in+ecology%5BTitle%5D)+AND+%22J.+Appl.+Ecol%22%5BJournal%5D)">Review: The detection of aquatic animal species using environmental DNA - a review of eDNA as a survey tool in ecology.</a> J. Appl. Ecol. 51, 1450-1459 (2014).</li>
<li>Stewart, F. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aStewart%2C+FJ+%22Microbial+metagenomics%2C+Metatranscriptomics%2C+and+metaprotenomics+Vol.+531+Methods+in+Enzymology%22">Microbial metagenomics, Metatranscriptomics, and metaprotenomics Vol. 531 Methods in Enzymology</a>. DeLong, E. E. 10, Elsevier. Ch. 10 187-218 (2013).</li>
<li>Walsh, D. A., Zaikova, E., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large+volume+%2820L%2B%29+filtration+of+coastal+seawater+samples%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Large volume (20L+) filtration of coastal seawater samples.</a> J Vis Exp. (28), e1161(2009).</li>
<li>Smalla, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSmalla%2C+K+%22Molecular+Microbial+Ecology+Manual%22">Molecular Microbial Ecology Manual</a>. Akkermans, D. L., Elsas, J. D., Bruijn, F. J. , Springer. 13-22 (1995).</li>
<li>Thomsen, P. F., Willerslev, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+DNA+-+An+emerging+tool+in+conservation+for+monitoring+past+and+present+biodiversity%5BTitle%5D)+AND+%22Biol.+Conserv%22%5BJournal%5D)">Environmental DNA - An emerging tool in conservation for monitoring past and present biodiversity.</a> Biol. Conserv. 183, 4-18 (2014).</li>
<li>Pedersen, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ancient+and+modern+environmental+DNA%5BTitle%5D)+AND+%22Phil.+Trans.+R.+Soc.+B%22%5BJournal%5D)">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/pubmed?term=((A+basin+scale+application+of+environmental+DNA+assessment+for+rare+endemic+species+and+closely+related+exotic+species+in+rivers%3A+a+case+study+of+giant+salamanders+in+Japan%5BTitle%5D)+AND+%22J.+Appl.+Ecol%22%5BJournal%5D)">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. Ecol. 52, 358-365 (2015).</li>
<li>Yamanaka, H., Minamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+environmental+DNA+of+fishes+as+an+efficient+method+of+determining+habitat+connectivity%5BTitle%5D)+AND+%22Ecol.+Indicators%22%5BJournal%5D)">The use of environmental DNA of fishes as an efficient method of determining habitat connectivity.</a> Ecol. Indicators. 62, 147-153 (2016).</li>
<li>Moss, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ciliated+protists+from+the+nepheloid+layer+and+water+column+of+sites+affected+by+the+Deepwater+Horizon+oil+spill+in+the+Northeastern+Gulf+of+Mexico%5BTitle%5D)+AND+%22Deep+Sea+Res.+Pt+I%22%5BJournal%5D)">Ciliated protists from the nepheloid layer and water column of sites affected by the Deepwater Horizon oil spill in the Northeastern Gulf of Mexico.</a> Deep Sea Res. Pt I. 106, 85-96 (2015).</li>
<li>Hilton, J. A., Satinsky, B. M., Doherty, M., Zielinski, B., Zehr, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metatranscriptomics+of+N2-fixing+cyanobacteria+in+the+Amazon+River+plume%5BTitle%5D)+AND+%22The+ISME+journal%22%5BJournal%5D)">Metatranscriptomics of N2-fixing cyanobacteria in the Amazon River plume.</a> The ISME journal. 9, 1557-1569 (2015).</li>
<li>Deiner, K., Walser, J. -C., Mächler, E., Altermatt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Choice+of+capture+and+extraction+methods+affect+detection+of+freshwater+biodiversity+from+environmental+DNA%5BTitle%5D)+AND+%22Biol.+Conserv%22%5BJournal%5D)">Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA.</a> Biol. Conserv. 183, 53-63 (2015).</li>
<li>Eichmiller, J. J., Miller, L. M., Sorensen, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+techniques+to+capture+and+extract+environmental+DNA+for+detection+and+quantification+of+fish%5BTitle%5D)+AND+%22Mol.+Ecol.+Res%22%5BJournal%5D)">Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.</a> Mol. Ecol. Res. 16, 56-68 (2016).</li>
<li>Lemarchand, K., Pollet, T., Lessard, V., Badri, M. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLemarchand%2C+K+%22Sample+Preparation+Techniques+for+Soil%2C+Plant%2C+and+Animal+Samples%27Springer+Protocols+Handbooks%22">Sample Preparation Techniques for Soil, Plant, and Animal Samples'Springer Protocols Handbooks</a>. Micic, M. , Springer. 325-339 (2016).</li>
<li>Turner, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particle+size+distribution+and+optimal+capture+of+aqueous+macrobial+eDNA%5BTitle%5D)+AND+%22Methods+Ecol.+Evol%22%5BJournal%5D)">Particle size distribution and optimal capture of aqueous macrobial eDNA.</a> Methods Ecol. Evol. 5, 676-684 (2014).</li>
<li>Barnes, M. A., Turner, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ecology+of+environmental+DNA+and+implications+for+conservation+genetics%5BTitle%5D)+AND+%22Conserv.+Genet%22%5BJournal%5D)">The ecology of environmental DNA and implications for conservation genetics.</a> Conserv. Genet. 17, 1-17 (2016).</li>
<li>Sorokulova, I., Olsen, E., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biopolymers+for+sample+collection%2C+protection%2C+and+preservation%5BTitle%5D)+AND+%22Appl.+Microbiol.+Biotechnol%22%5BJournal%5D)">Biopolymers for sample collection, protection, and preservation.</a> Appl. Microbiol. Biotechnol. 99, 5397-5406 (2015).</li>
<li>Renshaw, M. A., Olds, B. P., Jerde, C. L., McVeigh, M. M., Lodge, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+room+temperature+preservation+of+filtered+environmental+DNA+samples+and+assimilation+into+a+Phenol-Chloroform-Isoamyl+alcohol+DNA+extraction%5BTitle%5D)+AND+%22Mol.+Ecol.+Res%22%5BJournal%5D)">The room temperature preservation of filtered environmental DNA samples and assimilation into a Phenol-Chloroform-Isoamyl alcohol DNA extraction.</a> Mol. Ecol. Res. 2014, (2014).</li>
</ol>

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

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...

Watch this Scientific Journal Video about Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA at JoVE.com

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 ...

use-filter-cartridge-for-filtration-water-samples-extraction

Research

JoVE Journal

Environment

25.0K Views.  Natural History Museum and Institute, Chiba. 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.

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

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Genetics

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

Biology

Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

Capture of Environmental DNA eDNA from Water Samples by Flocculation

Environmental DNA (eDNA) is typically captured from water samples using alcohol precipitation, filtration, or flocculation. Presented here is an alternative protocol that uses lanthanum chloride to enable collection of dissolved and particulate-bound nucleic acids from water samples containing eukaryotic cells, prokaryotic cells, and virus.

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species ...

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking&#39;s impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.

Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil ...

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...