Apr 17, 2023

Public workspaceSakata et al. Fish SedDNA Extraction Protocol

DOI
dx.doi.org/10.17504/protocols.io.6qpvr4n8ogmk/v1
  • Masayuki K. Sakata1
  • 1Graduate School of Human Development and Environment, Kobe University, Kobe, Japan
Mark Louie Lopez
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.6qpvr4n8ogmk/v1
Protocol Citation: Masayuki K. Sakata 2023. Sakata et al. Fish SedDNA Extraction Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4n8ogmk/v1
Manuscript citation:
  • Sakata, M. K., Yamamoto, S., Gotoh, R. O., Miya, M., Yamanaka, H., & Minamoto, T. (2020). Sedimentary eDNA provides
different information on timescale and fish species composition compared with aqueous eDNA. Environmental DNA, 2(4),
505– 518. https://doi.org/10.1002/edn3.75
  • Sakata, M. K., Watanabe, T., Maki, N., Ikeda, K., Kosuge, T., Okada, H., … Minamoto, T. (2021). Determining an effective
sampling method for eDNA metabarcoding: a case study for fish biodiversity monitoring in a small, natural river.
Limnology, 22(2), 221–235. https://doi.org/10.1007/s10201-020-00645-9
  • Sakata, M.K., Tsugeki, N., Kuwae, M., Ochi, N., Hayami, K., Osawa, R., Morimoto, T., Yasashimoto, T., Takeshita, D.,
Doi, H., & Minamoto, T. (2022). Fish environmental DNA in lake sediment overcomes the gap of reconstructing past fauna
in lake ecosystems. bioRxiv, https://doi.org/10.1101/2022.06.16.496507
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 10, 2023
Last Modified: April 18, 2023
Protocol Integer ID: 80234
Abstract
Variations of this standardised protocol have been used by Masayuki K. Sakata and colleagues to successfully extract fish eDNA from modern and historic Japanese lake and river sediments.

The method has been used to recover fish species composition data from modern surface sediments from a lake (Sakata et al., 2020) and a small, natural river (Sakata et al., 2021). It has also been applied to detect target fish species in lake sediments up to 100 years old (Sakata et al., 2022).

This extraction method represents a consolidation of the methods applied in the following publications:

  • Sakata, M. K., Yamamoto, S., Gotoh, R. O., Miya, M., Yamanaka, H., & Minamoto, T. (2020). Sedimentary eDNA provides
different information on timescale and fish species composition compared with aqueous eDNA. Environmental DNA, 2(4),
505– 518. https://doi.org/10.1002/edn3.75
  • Sakata, M. K., Watanabe, T., Maki, N., Ikeda, K., Kosuge, T., Okada, H., … Minamoto, T. (2021). Determining an effective
sampling method for eDNA metabarcoding: a case study for fish biodiversity monitoring in a small, natural river.
Limnology, 22(2), 221–235. https://doi.org/10.1007/s10201-020-00645-9
  • Sakata, M.K., Tsugeki, N., Kuwae, M., Ochi, N., Hayami, K., Osawa, R., Morimoto, T., Yasashimoto, T., Takeshita, D.,
Doi, H., & Minamoto, T. (2022). Fish environmental DNA in lake sediment overcomes the gap of reconstructing past fauna
in lake ecosystems. bioRxiv, https://doi.org/10.1101/2022.06.16.496507
Materials
Reagents:
  • NaOH (0.33M)
  • TE buffer (pH 6.7) (Prepared with Tris-HCl buffer: 10 mM, EDTA: 1 mM)
  • Tris-HCl (1M, pH 6.7)
  • NaAc (3M, pH 5.2),
  • Ethanol (99.5%),
  • G2 enhancer (AMPLIQON, liquid type)

Kit: DNeasy PowerSoil Kit (QIAGEN)

Required equipment:
  • Thermostatic bath
  • Centrifuge (capable of centrifuging 50mL, 15mL, 1.5mL tubes)
  • Vortex
  • Vortex adapter (adapter to fix tube)
  • Lo-Bind tubes for storage (recommended)
Alkaline extraction
Alkaline extraction
50m
50m
PLACE Amount9.0 g of sediment sample into a 50 mL tube

ADD Amount6 mL NaOH

ADD Amount3 mL TE buffer

ADD Amount500 µL G2 enhancer
Note
Sediment volume can be adjusted but reagent volumes should remain the same

VORTEX samples to mix

INCUBATE samples at Temperature94 °C for Duration00:50:00
50m
ALLOW samples to cool to TemperatureRoom temperature

CENTRIFUGE at Amount5000 x g for Duration00:00:30
30s
TRANSFER Amount7.5 mL of supernatant to a new 50 mL tube

NEUTRALIZE by adding Amount7.5 mL Tris-HCL (1M)
Ethanol precipitation
Ethanol precipitation
1h 20m
1h 20m
ADD Amount1.5 mL sodium acetate

VORTEX well to mix
ADD Amount30 mL of ethanol

VORTEX well to mix
INCUBATE at Temperature-20 °C for at least Duration01:00:00
Note
Incubation at Temperature-20 °C DurationOvernight is recommended


1h
WIPE off condensation from around the 50 mL tube and centrifuge at 5,350xG for 20 min

CENTRIFUGE at Amount5350 x g for Duration00:20:00

20m
DISCARD supernatant and leave the tube mouth down for a few minutes

TRANSFER the precipitate to a PowerBead Tube (Qiagen Kit)

DISSOLVE the remaining precipitate with Amount100 µL of DW then transfer to the PowerBead Tube
DNeasy PowerSoil extraction
DNeasy PowerSoil extraction
22m
22m
ADD Amount60 µL of Solution C1

VORTEX at max speed for Duration00:10:00

CENTRIFUGE at Amount10000 x g for Duration00:00:30

Note
Vortex for 15-20 minutes if you have more than 12 samples

10m 30s
TRANSFER supernatant to a new 2 mL tube
Note
Expect ~700ul of supernatant, but the more supernatant transferred, the better

ADD Amount250 µL of Solution C2

VORTEX briefly to mix

INCUBATE at Temperature4 °C for Duration00:05:00

CENTRIFUGE at Amount10000 x g Duration00:01:00
6m
TRANSFER Amount600 µL of supernatant to a new 2 mL tube

ADD Amount200 µL of Solution C3

VORTEX briefly to mix

INCUBATE at Temperature4 °C for Duration00:05:00

CENTRIFUGE at Amount10000 x g for Duration00:01:00
6m
TRANSFER Amount750 µL of supernatant to a new 2 mL tube

ADD Amount1.2 mL of Solution C4

VORTEX well to mix
6m
TRANSFER Amount675 µL to a Spin Filter

CENTRIFUGE at Amount10000 x g for Duration00:01:00

DISCARD the liquid filtrate
1m
REPEAT the above step until all liquid has passed through the Spin Filter
ADD Amount500 µL of Solution C5 to the Spin Filter

CENTRIFUGE at Amount10000 x g for Duration00:00:30

DISCARD the liquid filtrate
30s
TRANSFER the Spin Filter to a new 1.5 mL tube

ADD Amount100 µL of Solution C6 to the Spin Filter

LET stand for Duration00:01:00

CENTRIFUGE at Amount10000 x g for Duration00:00:30
1m 30s
DISCARD the Spin Filter

DNA is now ready for downstream applications
Protocol references
  • Sakata, M. K., Yamamoto, S., Gotoh, R. O., Miya, M., Yamanaka, H., & Minamoto, T. (2020). Sedimentary eDNA provides
different information on timescale and fish species composition compared with aqueous eDNA. Environmental DNA, 2(4),
505– 518. https://doi.org/10.1002/edn3.75
  • Sakata, M. K., Watanabe, T., Maki, N., Ikeda, K., Kosuge, T., Okada, H., … Minamoto, T. (2021). Determining an effective
sampling method for eDNA metabarcoding: a case study for fish biodiversity monitoring in a small, natural river.
Limnology, 22(2), 221–235. https://doi.org/10.1007/s10201-020-00645-9
  • Sakata, M.K., Tsugeki, N., Kuwae, M., Ochi, N., Hayami, K., Osawa, R., Morimoto, T., Yasashimoto, T., Takeshita, D.,
Doi, H., & Minamoto, T. (2022). Fish environmental DNA in lake sediment overcomes the gap of reconstructing past fauna
in lake ecosystems. bioRxiv, https://doi.org/10.1101/2022.06.16.496507