TITLE: Sakata et al. Fish SedDNA Extraction Protocol
AUTHORS: Masayuki K. Sakata

[DESCRIPTION]
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

[STEPS]
SECTION: Alkaline extraction
2. VORTEX samples to mix

INCUBATE samples at 94 °C for 50 min 
SECTION: Alkaline extraction
3. ALLOW samples to cool to Room temperature 

CENTRIFUGE at 5000 x g for 30 s 
SECTION: Alkaline extraction
1. 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
 

SECTION: Alkaline extraction
4. TRANSFER 7.5 mL of supernatant to a new 50 mL tube

NEUTRALIZE by adding 7.5 mL Tris-HCL (1M)
SECTION: Ethanol precipitation
5. ADD 1.5 mL  sodium acetate

VORTEX well to mix
SECTION: Ethanol precipitation
7. INCUBATE at -20 °C for at least 60 min  
 

SECTION: Ethanol precipitation
8. WIPE off condensation from around the 50 mL tube and centrifuge at 5,350xG for 20 min

CENTRIFUGE at 5350 x g for 20 min 

SECTION: Ethanol precipitation
9. 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
SECTION: DNeasy PowerSoil extraction
10. ADD 60 µL of Solution C1

VORTEX at max speed for 10 min 

CENTRIFUGE at 10000 x g for 30 s

 

SECTION: DNeasy PowerSoil extraction
11. TRANSFER supernatant to a new 2 mL tube
 

SECTION: DNeasy PowerSoil extraction
12. ADD 250 µL of Solution C2

VORTEX briefly to mix

INCUBATE at 4 °C for 5 min 

CENTRIFUGE at 10000 x g 1 min 
SECTION: DNeasy PowerSoil extraction
13. 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 5 min 

CENTRIFUGE at 10000 x g  for 1 min 
SECTION: DNeasy PowerSoil extraction
14. TRANSFER 750 µL of supernatant to a new 2 mL tube

ADD 1.2 mL of Solution C4

VORTEX well to mix
SECTION: DNeasy PowerSoil extraction
15. TRANSFER 675 µL to a Spin Filter

CENTRIFUGE at 10000 x g for 1 min 

DISCARD the liquid filtrate
SECTION: DNeasy PowerSoil extraction
16. REPEAT the above step until all liquid has passed through the Spin Filter
SECTION: DNeasy PowerSoil extraction
17. ADD 500 µL of Solution C5 to the Spin Filter

CENTRIFUGE at 10000 x g for 30 s

DISCARD the liquid filtrate
SECTION: DNeasy PowerSoil extraction
18. TRANSFER the Spin Filter to a new 1.5 mL tube

ADD 100 µL of Solution C6 to the Spin Filter

LET stand for 1 min 

CENTRIFUGE at 10000 x g for 30 s 
SECTION: DNeasy PowerSoil extraction
19. DISCARD the Spin Filter

DNA is now ready for downstream applications
SECTION: Ethanol precipitation
6. ADD 30 mL of ethanol

VORTEX well to mix