Environmental DNA
extraction method for a high and stable DNA yield
Short running title: High and stable eDNA extraction method
Takashi Fukuzawa1*, Hiromi
Shirakura1, Naofumi Nishizawa1,
Hisao Nagata1, Yuichi Kameda1,2,
Hideyuki Doi3*
1 GO!FOTON INC., Tsukuba, Japan
2 Department of Anthropology, National Museum of Nature and Science,
Tsukuba, Japan
3 Graduate School of Information Science, University of Hyogo, Kobe,
Japan
*Co-corresponding authors:
Takashi Fukuzawa, Email:takashi.fukuzawa@gofoton.com
Hideyuki Doi, Email:hideyuki.doi@icloud.com
Abstract
Environmental DNA measurement has been widely applied in organism
biomonitoring. Different DNA extraction methods may cause changes in
yield and stability, resulting in an inaccurate estimation of eDNA,
especially when quantitative measurements are performed. This study
focused on the DNA extraction method and compared its yield and
stability for stream fish and spiked DNA samples. Samples were collected
periodically over a year from river and lake water systems and eDNA was
spiked into them. The samples were extracted and compared using three
methods: using Buffer-AL for initial lysis with the DNeasy Blood and
Tissue Kit (Qiagen); using Buffer-ATL for initial lysis and the
microfluidic-channel method (BC method). The method using Buffer-ATL in
the DNeasy Blood and Tissue Kit showed better stability and a higher
yield than the Buffer-AL method. In addition, the BC method, despite
being comparatively simple, performed the extraction stably and with
relatively high yields. We showed that differences in DNA extraction
methods based on the long-term evaluation of eDNA measurements with
various methods may cause alterations in DNA yield and stability.
Keywords: eDNA, extraction, filtering, DNeasy extraction kit, and
Biryu-chip
Introduction
Environmental DNA (eDNA) in aquatic environments has been used to detect
species distributions (Buxton et
al., 2018; Doi et al., 2017; Ficetola et al., 2008; Katano et al., 2017;
Takahara et al., 2012; Wang et al. 2021). eDNA has been detected in
water from various ecosystems, including streams, lakes, ponds,
reservoirs, canals, and oceans
(Doi et al., 2017; Fornillos et
al., 2019; Katano et al., 2017; Suter et al., 2021; Takahara et al.,
2012), and its measurement has been mainly performed using quantitative
real-time PCR (qPCR) (Doi et al., 2017; Fornillos et al., 2019; Katano
et al. 2017; Preece et al., 2021; Takahara, et al., 2012).
The measurement of eDNA is conducted by water sampling, followed by
filtration and extraction, and finally DNA detection by qPCR. To obtain
accurate eDNA measurements, it is necessary to select a reliable method
for each process. Water sampling and filtration processes can provide
relatively precise results if the process conditions are set; however,
few studies have reported on the stability of eDNA extraction. eDNA has
been extracted from filter and sediment samples using the DNeasy Blood
and Tissue Kit (Qiagen, Hilden, Germany; hereafter, DNeasy method)
(Tsuji et al., 2019), PowerWater (Coster et al., 2021; Hinlo et al.,
2017), and PowerSoil (Díaz et al., 2020; Eichmiller et al., 2016; Sakata
et al., 2020). Among these, DNeasy is the most commonly employed (Tsuji
et al., 2019), however there are two groups of methods. One method is
based on blood or cells without the use of ATL (Miya et al., 2016;
Yamamoto et al., 2016), while the other involves tissues that employ ATL
(Coster et al., 2021; Djurhuus et al., 2017). These methods are
described in the manual of the DNeasy Blood and Tissue Kit. Recently,
many studies have referred to the procedure given by Miya et al. (2016),
which does not use ATL, for the specific procedures of this method,
since this is the method on which the manual from the eDNA Society was
based (Minamoto et al., 2021; Wong et al., 2020). However, few studies
have reported the stability of both methods.
The aforementioned DNA extraction methods are difficult for
non-researchers to perform because they require many experimental steps,
and this challenge, along with the questionable reliability, creates a
hurdle for the social implementation of eDNA techniques. In fact, a
stable and simple filtration and extraction method for the social
implementation of eDNA has been suggested (e.g.,, Fukuzawa et al.,
2022).
In evaluating the reliability of several extraction methods, we found
that the DNeasy without ATL described in the manual could cause large
fluctuations in the measured values due to foreign substances in the
water sample, whereas highly stable results can be obtained using ATL.
The stability of the BC method, which we developed based on a simple
method, was simultaneously evaluated and stable measurements were
obtained.
Materials and Methods
The extraction method with Buffer AL (hereafter, the “AL method”)
A method using the DNeasy Blood and Tissue DNA extraction kit was used
according to the procedure described by Miya et al. (2016), and
characterized by the use of Buffer AL as the lysate.
For the AL method using Sterivex:
- The premixes were prepared using proteinase-K, buffer AL, and PBS at
20 µL, 200 µL, and 220 µL, respectively.
- The premixes (440 µL) were added to a Sterivex and incubated at 56 °C
for 30 min.
- The liquid was transferred from the Sterivex to a 1.5 mL tube.
- Ethanol (200 μL) was added to the tube, which was then stirred.
- The solution was transferred to a spin column, centrifuged at 6000 × g
for 1 min, and the solution in the collection tube was discarded.
- The collection tube was replaced, and 500 µL of Buffer AW1 was added
to the spin column, which was then centrifuged at 6000 × g for 1 min,
and the solution in the collection tube was discarded.
- The collection tube was replaced, and 500 µL of Buffer AW2 was added
to the spin column, which was centrifuged at 20000 × g for 3 min, and
the solution in the collection tube was discarded.
- The collection tube was replaced with a new 1.5 mL tube and 200 µL of
Buffer AE was added to the spin column; after for 1 min, the solution
was centrifuged at 6000 × g for 1 min.
The extracted sample was now contained in the 1.5 mL tube.
For the AL method using the GF/F glass filter (GE Healthcare UK)
- A glass filter was placed on the top of a Salivette tube (Zalstat),
and a mixture of 40 µL of proteinase K and 400 µL of Buffer AL was
poured onto the filter paper evenly, and incubated at 56 °C for 30 min
in a thermostatic chamber.
- The tube was placed in a centrifuge at 3000 × g for 3 min.
- TE (220 μL) was poured onto the filter paper, and
after waiting for 1 min, it was
centrifuged at 3000 × g for 3 min.
- The upper part of the Salivette tube containing the filter was removed
and discarded, 400 µL of ethanol (99.5%) was added, and the solution
was stirred.
- Approximately 650 µL (approximately half) of the sample was
transferred to the spin column and centrifuged at 6000 × g for 1 min.
- The solution in the bottom of the collection tube was discarded, and
the remaining solution was transferred to the spin column again, and
centrifuged at 6000 × g for 1 min.
- The collection tube was replaced, and 500 µL of Buffer AW1 was added
to the spin column, which was centrifuged at 6000 × g for 1 min, and
the solution in the collection tube was discarded.
Following this, the Sterivex filtration method was performed, as
described previously.
The extraction method with Buffer ATL (hereafter the “ATL method”)
We modified the method for eDNA following the extraction procedure from
tissue in the DNeasy manual, with the use of Buffer ATL as the lysate.
The detailed procedures for using the Sterivex and GF/F glass filters
are shown below.
For the ATL method using Sterivex:
- Buffer ATL (380 μL) mixed with 20 µL of proteinase K was placed in the
Sterivex and incubated at 56 °C for 30 min.
- The liquid was collected from the Sterivex in a 1.5 mL tube.
- Buffer AL (400 µL) was added to the tube, stirred, and incubated at 56
°C for 10 min.
- Ethanol (400 µL) was added to the tube, which was then stirred.
- Approximately 650 µL (approximately half) of the solution was
transferred to the spin column and centrifuged at 6000 × g for 1 min.
- The solution from the bottom of the collection tube was discarded, and
the remainder was transferred to the spin column again, and
centrifuged at 6000 × g for 1 min.
Following this, the same procedure was performed as described for the AL
method.
For the ATL method using a GF/F glass filter:
- A glass filter was placed on the top of a Salivette tube, a mixture of
40 µL of proteinase K and 400 µL of Buffer ATL was poured onto the
filter paper evenly, and incubated at 56 °C for 30 min in a
thermostatic chamber.
- The tube was placed in a centrifuge at 3000 × g for 3 min.
- Buffer ATL (220 μL) was poured onto the filter paper and then
centrifuged at 3000 × g for 3 min.
- The upper part of the Salivette tube containing the filter was removed
and discarded. The 650 µL of Buffer AL was added to the top of the
tube, which was vortexed for 10 s, and incubated in a thermostatic
chamber at 56 °C for 10 min.
- Ethanol (650 µL; 99.5%) was added and the solution was stirred.
The same procedure was then performed as for the AL method.
The Biryu-Chip (BC) method (Fukuzawa et al., 2022):
- The water sample was injected using a 10 mL syringe into the BC inlet
port and filtered. Using the same syringe, the excess water in the BC
was then drained.
- The tape attached to the BC extract port was removed, and 20 µL of the
extraction solvent was injected into the BC. Solution A from the
Kaneka Easy DNA Extraction Kit version 2 (Kaneka, Tokyo, Japan) was
used as the extraction solvent.
- Two minutes later, the extraction solvent was drawn from the extract
port using a pipette and transferred to a microcentrifuge tube to
obtain the extracted DNA sample.
Experiment 1
As a preliminary experiment, a comparison of the three extraction
methods was conducted over four months from September to December 2020.
First, rainbow trout (Oncorhynchus mykiss ) aquarium water was
mixed well and divided into 40 mL aliquots in centrifuge tubes, which
were frozen and stored as reference samples of eDNA
(rainbow-trout-eDNA). Each week, we sampled 400 mL of surface water from
the shores of the Sagami River (35.575099° N, 139.308802° E) and
Kasumigaura Lake (36.035112° N, 140.257929° E) using a 1 L plastic
bottle. The 40 mL rainbow-trout-eDNA was then spiked into the water
sample and mixed thoroughly. The mixed sample (100 mL) was filtered
using two Sterivex units with a 50 mL syringe and frozen at -20 °C. A
second mixed sample (50 mL) was poured into a centrifuge tube and frozen
to be used in the BC method. The time from water sampling to freezing
was approximately 3 h. Subsequently, all accumulated Sterivex were
extracted monthly using the AL and ATL methods. For the BC method, water
sampling began in October in the Sagami River. Concurrent with water
sampling, another 400 mL of surface water was collected using a 1 L
plastic bottle to confirm the presence of rainbow-trout eDNA in the
water samples. This non-spiked water sample was filtered using a
Sterivex and extracted by the ATL method.
Experiment 2
We continued the sample water collection for approximately one year,
starting in January 2021. Water from a second rainbow trout aquarium was
dispensed and frozen, as described in Experiment 1. We collected 900 mL
of surface water from the same site as that of Experiment 1 and then
spiked 40 mL of rainbow-trout-eDNA to the sample. A GF/F glass filter
was used for filtration for the AL and ATL methods. For the BC method,
filtration and extraction were performed after sampling was completed.
As in Experiment 1, the non-spiked water sample was filtered using a
GF/F glass filter and extracted using the ATL method.
To confirm the stability of rainbow-trout-eDNA, we spiked 40 mL to the
900 mL of DNA-free pure water every month and extracted it using the ATL
method.
DNA measurement with qPCR
Quantification of eDNA from all samples was performed using the
StepOnePlus Real-Time PCR System (Thermo Fisher Scientific,
Massachusetts, USA). Rainbow trout measurements were performed for all
samples. In the summer season (June to September), we also conducted
measurements of the inhabited species, sweetfish (Plecoglossus
altivelis ) in the Sagami River and silver carp
(Hypophthalmichthys molitrix ) in Kasumigaura.
The DNA yield from the AL method decreased considerably during the
experiment. Therefore, we used a specific AL extraction sample to
determine whether the decrease in yield was caused by PCR inhibition or
extraction inhibition. Specifically, we prepared two samples, one with
and one without the extraction sample added to the PCR reagent at a
concentration of 10%, and added an equivalent concentration of horse
mackerel (Trachurus japonicus ) template to each sample for qPCR.
The primer probe sets used are listed in Table 1. Each TaqMan reaction
contained 900 nM of each primer (forward and reverse), 125 nM
TaqMan-Probe, 0.025 U µL-1 qPCR master mix (KAPA3G
Plant PCR Kit, Kapa Biosystems), and 1.5 μL of the eDNA solution. The
final volume for a single PCR assay was increased to 15 μL using
distilled water (DW). The volume of eDNA solution added was 10% of the
volume of the PCR mixture. The qPCR conditions were as follows: 95 °C
for 20 s, followed by 55 cycles of 95 °C for 4 s and 60 °C for 20 s.
Three replicates were performed for each sample and no-template control
(NTC). Standard curves of qPCR measurements showed R2= 0.992–1.000 and PCR efficiency = 94.3%–106.3%.
Statistical analysis
The data were plotted in a time series graph to confirm the variability
of each extraction method and compare the mean value and coefficient of
variation (CV; [standard deviation/mean value] × 100). We compared
the differences in DNA concentrations among the methods using a paired
t-test, multiple comparisons using ”t.test”, and controlled p-value for
multiple comparisons using the Bonferroni method to avoid a Type I
error. We performed Spearman’s rank correlation for time-series data
with the ”cor. test” function. We set the significance criterion at a
p-value of 0.05. All statistical analyses were performed using R ver.
4.2.0 (R Core Team 2022).
Results
Measurements of rainbow trout during two experiments
Weekly changes in the qPCR results of rainbow trout obtained using the
three extraction methods are shown in Fig. 1. Although there was no
continuity between Experiment 1 and Experiment 2 because of the
different experimental conditions, an overall trend of variation can be
observed. The results of the AL method fluctuated greatly even though
the samples contained the same amount of eDNA, whereas the ATL and BC
methods were stable.
The concentrations of rainbow trout DNA were significantly different
among the three methods (paired t-test, t < -4.565, p
< 0.0009). The ATL method yielded more than twice that of the
AL method (Table 2), while the CV was approximately half that of the AL
method. Despite its simplicity, the BC method was more stable and
efficient than the AL method, although it was slightly inferior to the
ATL method, which had more procedures. The results of rainbow trout
measurements on non-spiked water samples extracted by the ATL method
were less than 10% of those of spiked samples in all cases.
Spiked rainbow-trout-eDNA
The spiked rainbow-trout-eDNA showed no significant degradation in
concentration during the one-year experiment (Fig. 2, Spearman’s rank
correlation, rho = -0.132, p = 0.683).
eDNA measurements of the inhabiting species
Aquarium water was spiked as a reference sample (rainbow-trout-eDNA). As
shown in Fig. 1, the results of the reference sample by the AL method
varied greatly; therefore, this behavior was compared to that of the
inhabitant species. The results of the AL and ATL methods for sweetfish
in the Sagami River and silver carp in Kasumigaura are shown in Fig. 3.
A decreased yield obtained by the AL method, comparing to the ATL
method, was observed for both fish species, with a similar trend between
the reference sample and the inhabitant species. In addition, the ATL
method resulted in significantly higher concentrations for both species
than those of the AL method (t < -6.56, p <
0.00000898 for sweetfish; t < -2.27, p < 0.0381 for
silver carp).
The amount of eDNA of the inhabitant species varied, thus the ratio of
the copy numbers obtained by AL and ATL methods is shown in Fig. 4,
where the denominator is the number of copies obtained by the ATL
method, which is stable, and the numerator is that obtained by the AL
method. The ratio was not significantly different between rainbow trout
and the inhabiting fish DNA (t = 0.571, p = 0.577 for sweetfish; t =
-1.48, p= 0.171 for silver carp). Thus, the ratio of both inhabiting
fish showed the same tendency for spiked rainbow-trout-eDNA.
Distinguishing between PCR inhibition and extraction inhibition in the
AL method
The Ct value of the AL method varied significantly. As indicated in
Experiment 2, the DNA yield of the AL method at the Sagami River in the
second week of July 2021 was approximately 10% that of the ATL method.
This indicates the presence of a substance that prevents PCR or DNA
extraction. The Ct values of PCR for horse mackerel with and without the
DNA extraction samples added to the PCR reagent were 31.1 and 30.8,
respectively. Since the addition of the extraction sample resulted in a
slightly higher Ct value, we assumed that the lower yield was not a
result of PCR contamination with foreign substances.
Discussion
From the results of the stability check of the reference sample
(rainbow-trout-eDNA) and the comparison between the reference sample and
eDNA of the inhabitant species, it was suggested that the results of
Experiments 1 and 2 represent changes in the environmental DNA for three
methods. Using this assumption, we evaluated the yields of the AL, ATL,
and BC methods based on the results of Experiments 1 and 2.
The AL method showed a strong seasonal variation that greatly exceeded
the variability of the reference sample (CV<50%), and in some
cases, the yield was less than 10% that of the ATL method. Both the
Sagami River and Kasumigaura results showed a tendency for the measured
values to decrease substantially during the summer, probably because of
the increase in foreign substances generated in this season. In the
Sagami River, the water level rose considerably on a day in the first
week of July, and a similar increase occurred in Kasumigaura in the
second week of August (e.g.,
https://www.ktr.mlit.go.jp/sagami/en/sagami00668.html, Web site of
Sagami River System Management Office), suggesting that the source of
foreign substances is likely the
discharge. In contrast, the ATL and BC methods had stable measurement
results throughout the year.
The ATL method provided close to twice the DNA concentration than that
of the AL method, and the CV value was approximately half that of the AL
method. This indicates that the ATL method was more stable and reliable.
Regarding the yield fluctuation of the AL method, the Sagami River water
flows, while the Kasumigaura (the second largest lake in Japan) water is
still, and the two water bodies are distant from each other. However,
the yield decreased significantly during the summer. Thus, it can be
inferred that this phenomenon occurs at many locations.
Djurhuus et al. (2017) reported that there can be differences in
extraction yields depending on the filter material used, even with the
same extraction method. The Sterivex (filter material PVDF) was used in
Experiment 1 and a glass filter was used in Experiment 2 for the AL
method, although our results showed similar fluctuations in the yield.
Regarding the newly developed BC method, the results show that the
variation is within the same range as that of the reference sample,
indicating the possibility of stable measurement using this simpler
procedure. The yield was slightly lower than that of the ATL method, but
higher than that of the AL method. Therefore, this method may be useful
for future social implementation of eDNA technology.
The yield and stability were compared between two extraction methods
(protocols) using the DNeasy Blood and Tissue DNA extraction kit, which
is commonly used by researchers (Tsuji et al., 2019). However, from
these results, the extraction yield and stability varied greatly
depending on which reagents (i.e., Buffer AL or ATL) were used, and the
results of this experiment suggest that the method using ATL was
superior. The BC method has the potential to be an important extraction
method for the development of eDNA technology.
In conclusion, we showed that differences in DNA extraction methods may
cause differences in yield and stability using long-term evaluation of
eDNA measurements with various methods. The AL method caused significant
fluctuations in yield, whereas the ATL method could allow for extraction
at a high yield. In addition, the BC method could provide a reliable
extraction method for the development of eDNA technology. In this study,
we tested one river and one lake, so further testing of the methods is
needed to confirm their performance in other environments.
Data availability
All the data (Supplementary Table S1) are available at Zenodo
(doi:10.5281/zenodo.6618648).
Acknowledgments
This study was supported by the Environment Research and Technology
Development Fund (JPMEERF20204004).
Conflict of interest
The commercial affiliations of the authors [TF, HS, NN, HN, YK, and
HS] did not alter their adherence to journal policies on sharing data
and materials. TF, NN, HN, and HS were employed by the manufacturer of
the equipment. However, none of the authors would directly benefit from
the publication of this paper.
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Table 1. The qPCR primer-probe sets used in this study