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

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

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 ponds^1-3, rivers^4-8, streams⁹, and seawater^10-14. Most of these studies focused on detection of a single or a few invasive^1,4-6,8,14 and rare or threatened species^3,9, while some recent studies attempted simultaneous detection of multiple species in local fish communities^7,9,12,13,15 and mesocosms^11,12.

The latter approach is called "met....

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 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-fi....

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 follows^24,25: 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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"