Anvendelse af en filterpatron til filtrering af vandprøver og Udvinding af 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
Miljømæssige DNA (eDNA) i vandigt miljø refererer til genetisk materiale, der findes i vandsøjlen. Nylige undersøgelser viste nytten af eDNA til påvisning fisk fra forskellige vandmiljøer, herunder damme 1-3, floder 4-8, vandløb 9, og havvand 10-14. De fleste af disse undersøgelser fokuseret på påvisning af en enkelt eller nogle få invasiv 1,4-6,8,14 og sjældne eller truede arter 3,9, mens nogle nylige undersøgelser forsøgt samtidig påvisning af flere arter i lokale fisk samfund 7,9, 12,13,15 og mesocosmos 11,12.
Sidstnævnte fremgangsmåde kaldes "metabarcoding" og eDNA metabarcoding anvender en eller flere sæt af PCR-primere til coamplify en genregion tværs taksonomisk forskellige prøver. Dette efterfølges af biblioteket forberedelse med indeksering og adapter Desuden, og de indekserede biblioteker analyseres ved en high-throughput parallel sekventeringperron. For nylig Miya et al. 12 udviklede universelle PCR primere til metabarcoding eDNA fra fisk (kaldet "MiFish"). De MiFish primere målretter en hypervariabel region af mitokondrie 12S rRNA-genet (163 til 185 bp), der indeholder tilstrækkelige oplysninger til at identificere fisk at taksonomiske familie, slægt og art undtagen nogle nært beslægtede congenere. Med brugen af disse primere i eDNA metabarcoding, Miya et al. 12 fundet mere end 230 subtropiske marine arter fra akvarium tanke med kendte arter sammensætning og koralrev nær akvariet.
Mens optimere metabarcoding protokollen til at rumme naturlig havvand med varierende niveauer af eDNA koncentration fra fisk, har vi bemærket, at MiFish primere lejlighedsvis undladt at forstærke målområdet til efterfølgende bibliotek forberedelse. En af de mere sandsynlige årsager til denne mislykket PCR-amplifikation er manglen på tilstrækkelige mængder af template DNA indeholdt i små mængder vand filtreret (dvs. 1-2 L). Selvom eDNA koncentration fra en bestemt taksonomiske gruppe er ukendte før forstærkning, filtrering af store vandmængder (> 1-2 L) ville være en enkel og effektiv metode til at indsamle mere eDNA fra vandmiljøer med knappe fisk overflod og biomasse, såsom open-havet og dybhavsøkosystemer.
I forhold til disk fiberfiltre konventionelt anvendes i en række fisk eDNA forskning 16, filterpatroner har den fordel at rumme større vandmængder før tilstopning 17. Faktisk viste en nylig undersøgelse stort volumen (> 20 L) filtrering af havvand prøver kystnære bruger filterpatroner 18. Endvidere er de individuelt pakket og sterilt, og kan udføres flere trin af den eksperimentelle arbejdsgang i filterhuset, hvilket således reducerer sandsynligheden for forurening fra laboratoriet 19. Det sidstefunktion er afgørende for eDNA metabarcoding, hvor risikoen for forurening er fortsat blandt de største eksperimentelle udfordringer 20,21. Trods disse tekniske fordele ved filterpatroner, har det ikke været anvendt i eDNA studier af fisk med to undtagelser 8,15.
Her giver vi en protokol til filtrering af vandprøver med filterpatronen og udvinding af eDNA fra dens filter uden at skulle skære åbne kabinettet. Vi tilbyder også to alternative vand filtrering systemer afhængigt af vandmængder (≤4 L eller> 4 L). For at sammenligne effektiviteten af den nyligt udviklede protokol og en tidligere brugt protokol ved hjælp af et glas-fiber filter i vores forskningsgruppe 12,14,22,23, vi udfører eDNA metabarcoding analyse af havvand fra en enorm akvarium tank (7500 m 3 ) med sammensætning kendt art, og viser antallet af fundne arter stammer fra de to protokoller som repræsentative resultater. Denne protokol hsom er udviklet til metabarcoding eDNA fra fisk, men kan også anvendes til eDNA fra andre organismer.
Protocol
Representative Results
Discussion
I mange metabarcoding undersøgelser med anvendelse af miljøprøver såsom vand og jord, post-filtrering behandling af filterpatronen er generelt som følger 24,25: 1) at skære åben eller revnet huset med håndværktøj (slange cutter eller tang); 2) fjernelse af filteret fra patronen; og 3) skæring af filteret i små stykker med et barberblad til DNA-ekstraktion. For at undgå sådanne besværlige og tidskrævende procedurer, der er tilbøjelige til forurening i laboratoriet, har vi forsøgt flere DNA ek…
Declarações
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" |
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