Apr 18, 2023

Sakata et al. Fish SedDNA Extraction Protocol

DOI
https://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
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DOI: https://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 19, 2023
Protocol Integer ID: 80234
Keywords: fish species composition data from modern surface sediment, fish seddna extraction protocol variation, target fish species in lake sediment, environmental dna in lake sediment, fish biodiversity monitoring, lake sediment, fish species composition data, case study for fish biodiversity monitoring, past fauna in lake ecosystem, fish edna, historic japanese lake, fish species composition, river sediment, lake ecosystem, sedimentary edna, target fish species, modern surface sediment, sediment, extraction method, effective sampling method for edna metabarcoding, environmental dna, extraction, lake, reconstructing past fauna, aqueous edna, natural river
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
50m
PLACE 9.0 g of sediment sample into a 50 mL tube

ADD 6 mL NaOH

ADD 3 mL TE buffer

ADD 500 µL G2 enhancer
Note
Sediment volume can be adjusted but reagent volumes should remain the same

VORTEX samples to mix

INCUBATE samples at 94 °C for 00:50:00
50m
ALLOW samples to cool to Room temperature

CENTRIFUGE at 5000 x g for 00:00:30
30s
TRANSFER 7.5 mL of supernatant to a new 50 mL tube

NEUTRALIZE by adding 7.5 mL Tris-HCL (1M)
Ethanol precipitation
1h 20m
ADD 1.5 mL sodium acetate

VORTEX well to mix
ADD 30 mL of ethanol

VORTEX well to mix
INCUBATE at -20 °C for at least 01:00:00
Note
Incubation at -20 °C Overnight is recommended


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

CENTRIFUGE at 5350 x g for 00:20:00

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 100 µL of DW then transfer to the PowerBead Tube
DNeasy PowerSoil extraction
22m
ADD 60 µL of Solution C1

VORTEX at max speed for 00:10:00

CENTRIFUGE at 10000 x g for 00:00:30

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 250 µL of Solution C2

VORTEX briefly to mix

INCUBATE at 4 °C for 00:05:00

CENTRIFUGE at 10000 x g 00:01:00
6m
TRANSFER 600 µL of supernatant to a new 2 mL tube

ADD 200 µL of Solution C3

VORTEX briefly to mix

INCUBATE at 4 °C for 00:05:00

CENTRIFUGE at 10000 x g for 00:01:00
6m
TRANSFER 750 µL of supernatant to a new 2 mL tube

ADD 1.2 mL of Solution C4

VORTEX well to mix
6m
TRANSFER 675 µL to a Spin Filter

CENTRIFUGE at 10000 x g for 00:01:00

DISCARD the liquid filtrate
1m
REPEAT the above step until all liquid has passed through the Spin Filter
ADD 500 µL of Solution C5 to the Spin Filter

CENTRIFUGE at 10000 x g for 00:00:30

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

ADD 100 µL of Solution C6 to the Spin Filter

LET stand for 00:01:00

CENTRIFUGE at 10000 x g for 00:00:30
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