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Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses
Accepted Manuscript Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses Xiaohu Dai, Sisi Chen, Ning Li, Han Yan PII:
S0003-2697(16)00005-1
DOI:
10.1016/j.ab.2016.01.003
Reference:
YABIO 12280
To appear in:
Analytical Biochemistry
Received Date: 25 October 2015 Revised Date:
2 January 2016
Accepted Date: 5 January 2016
Please cite this article as: X. Dai, S. Chen, N. Li, H. Yan, Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.01.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses
Xiaohu Dai1,2, Sisi Chen1,2, Ning Li1,*, Han Yan
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1. State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University 2. These authors contributed equally in this work
[email protected];[email protected]
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*Corresponding author:
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ACCEPTED MANUSCRIPT Abstract The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called standard DNA extraction method - the commercial kit method. Microbial cells in the digested sludge
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sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid, protein etcs., were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the
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in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the DNA-based method.
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Keywords: DNA extraction, Dilution, Sonication, Fluorescent dye, Pyrosequencing
Introduction
Microorganisms are widely distributed in natural and engineered environments, where they play significant ecological roles in global elemental cycles. Traditionally,
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microbial communities have been studied using cultivation-dependent techniques, whereas it is generally accepted that the bulk of microorganisms in environment are unculturable. During the last several decades, the development of culture independent
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molecular biology technology, basing on Polymerase Chain Reaction (PCR), provided researchers access to investigate the previously hidden information of microbial populations contained within environmental samples. Therefore, DNA probing,
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PCR-based amplification of specific targets, and the construction of clonal libraries have been extensively achieved. And in particular, the recent application of comprehensive investigation of microbial populations using high-throughput sequencing technology (such as 454 or Illumina pyrosequencing) has been reported as well, which allows the scientific community to assess the discriminations between different extraction methods with enough resolution (Dionisi et al., 2003; Terrat et al., 2012; Satoh et al., 2013). The prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extractions. DNA, as the template of PCR, is 2
ACCEPTED MANUSCRIPT embedded in the microbes by other components, and is further surrounded by extracellular polymer substances (EPS) and co-exists with other inhibitory contaminants (Frolund et al., 1996). Numerous protocols have described different approaches for DNA extraction with microorganism lysis, which include alkaline
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extraction (Birnboim and Doly, 1979), enzymatic extraction (Pitcher et al., 1989), microwave heating (Picard et al., 1992), salt extraction (Aljanabi et al., 1997), freeze-thawing extraction (Zhou et al., 1996), thermal shocks (Bourrain et al., 1999), bead lysis (Miller et al., 1999), and sonication (Krsek and Wellington, 1999).
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However, during the extraction processes, the inhibitory substances of PCR including environmental molecules (e.g., humic acid, metals, and proteins) and the carry-over of
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compounds used can still remain. The high-quality and high-fidelity DNA extraction is thus still the hurdle and the technical challenge (Martin-Laurent et al., 2001; Hazen et al. 2013).
Meanwhile, as the extraction of DNA is the first step in molecular analytical techniques, the procedures proper or not have a significant impact on the results
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obtained. And hence, cautions have been raised that the use of different DNA extraction protocols may result in different microbial community structures. The potential for such bias may also have a critical impact on quantitative molecular
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analyses (e.g., measurement of gene quantity or copy number largely relies on stable amplification of the gene, as well as the DNA coverage of the primers or probe used). Commercial kits such as FastSpin Soil DNA kit (MP Biomedicals, USA) and
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PowerSoil DNA Isolation kit (MOBIO, USA) have been widely applied in the preparation of DNA template, and become almost the standard method due to the high quality of extracted DNA (Sagova-Mareckova et al., 2008). However, unbiased genetic material from microorganisms with varying cell wall compositions and differentially accessible DNA has been reported to be difficult to be obtained (Yergeau et al., 2010; Mackelprang et al., 2011), as the extraction condition might not represent the overall picture of the indigenous community which hampered attempts in applying metagenomics approaches to environmental samples such as soils and sediments (Hazen et al., 2013). Sonication based method, on the other hand, is widely 3
ACCEPTED MANUSCRIPT considered one of the most efficient for the lysis of microbes if combined with ethanol purification as the homogeneous decomposition by it needs just several to dozens of seconds, and seems to be a promising, efficient and cost-effective method (Gunawardana et al., 2014).
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In this study, in consideration of a high extraction efficiency, low costs and less labour intensive technique, the sonication-based DNA extraction method was evaluated the feasibility of the replacement of the so-called standard DNA extraction method (the commercial kit). The anaerobically digested samples, representative in
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the highly contaminated samples with inhibitory substances, such as humic acid, metals, proteins, as well as other simple carry-over compounds, was utilized in this
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study. The in-depth understanding of the varied effectiveness and the potential bias triggered by the two different methods for the applications of molecular biology approaches was provided. To address these methodological issues, the amount of target biomolecule extracted from the sample must first be determined. The process of the DNA based method was comprised of sample washing by organic and inorganic
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eluents, DNA fixing in 50% ethanol, dilution, and sonication, which were combined to thoroughly extract DNA and inhibited the co-extracted contaminants. Furthermore, the sequencing depths afforded by the pyrosequencing approach allowed us to obtain
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the detailed information regarding the dominant, subdominant, and rare taxa within the fingerprints of the microbial communities. The experimental findings from this work will expand ideas in choosing the appropriate DNA extraction technology
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(and/or PCR amplification procedure) to accommodate the specific needs when analyzing microbial community diversity of samples from various regions of the world.
Material and methods Sample origin and characteristics Sludge samples were obtained from a laboratory-scale high solid anaerobic digester with SRT of 20 days. The digester was fed daily with dewatered sludge. Total solids (TS) and the ratio of volatile solids to total solids (VS/TS) of digestate were 18.8 ± 4
ACCEPTED MANUSCRIPT 0.1% and 41.5 ± 0.1%, respectively.
Sonication-based DNA extraction One gram of digested sludge was transferred to a 50 mL centrifuge tube using a
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spatula, and was then washed with 20 mL ethanol to reduce alteration of the microbial population and prevent the inhibitory effects of organic substances. Samples were shaken at 250 rpm for 10 min on a shaker (KS-260; IKA; Germany), and centrifuged at 10,000 × g for 5 min; the supernatants were then discarded. The pellets were
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resuspended in 50 mL double-distilled (dd) water to dissolve the salt contained in the samples, and the samples were again shaken and centrifuged as described above. After
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discarding the supernatants, the pellets were resuspended in 50 mL 50% (v/v) ethanol eluent, and sonicated by FS-1200N ultrasonic processor (Ultrasonics, China) under the predetermined optimal conditions with intensity of 30% amplitude (15W/cm2) and duration of 30s. Finally, all samples were stored at −80°C until use. The LIVE/DEAD BacLight™ Kit (Invitrogen; Eugene, OR, USA) was used to
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evaluate the decomposition of bacterial cells using the determined sonication conditions. Microscopic images of the samples were observed under an IX73 inverted fluorescence microscope (Olympus; Japan).
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The optical density of DNA templates were measured by Nanodrop 2000 (Thermo Scientific, USA) with the absorbance values of 260/280 to evaluate the
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quality of the extracted DNA templates.
PCR
The optimal concentration of the DNA template was pre-determined as described below. Firstly, the DNA template was extracted from sludge samples using PowerSoil DNA Isolation Kit (MO BIO, USA), which was the experimental alternative of commercial kits in this study and has been widely used in labs all over the world. The concentration of DNA template was measured by following the instruction for PicoGreen fluorescent dye from the manufacturer (Invitrogen; Eugene, OR, USA) and adjusted to 10 ng/µL afterward. Samples were then serially diluted to 10−7 ng/µL. 5
ACCEPTED MANUSCRIPT Each sample was added to triplicate PCR reactions. The PCR products were quantified using PicoGreen fluorescent dye. The minimum DNA concentration to be used for subsequent PCR reactions was determined by the final concentration of PCR products that were >10 ng/µL.
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The universal primers used to amplify the bacterial DNA template were the 27f forward primer (5′-AGAGTTTGATC(A/C)TGGCTCAG-3′) and 519r reverse primer (5′-G(A/T)ATTACCGCGGC(G/T)GCTG-3′) (Lane,
1991). The PCR cycling
conditions were as the reported by Li et al. (2012): 95°C for 10 min, 30 cycles of
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98°C for 10 s, 55.3°C for 30 s, and 72°C for 1 min, followed by a final extension step of 72°C for 10 min. The reaction mixtures contained 0.125 µL TaKaRa Ex Taq hot
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start (5 U/µL, Japan), 2.5 µL 10× Ex Taq buffer (TaKaRa; Japan), 2 µL dNTP mixture (2.5 mM each, TaKaRa; Japan), 0.5 µL forward primer (10 pmol/µL), 0.5 µL reverse primer (10 pmol/µL), and 2.5 µL template DNA. The volume was adjusted to 25 µL using sterilized ultrapure Milii-Q water (Millipore, Geremany). For the comparisons of the molecular biological results, the DNA templates
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obtained by both the commercial kit and the sonication-based method were amplified.
Real-time quantitative PCR (qPCR)
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The extracted DNA was analyzed by qPCR in triplicate to evaluate the PCR efficiency with the universal primer set (27f/519r). Reactions were performed in optical 96-well reaction plates in an Applied Biosystems 7500 Real-Time PCR system (Applied
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Biosystems; Foster City, CA, USA). The reaction mixtures consisted of 10 µL SYBR Premix DimerEraser (2×), 0.6 µL 27f forward primer (10 pmol/µL), 0.6 µL 519r reverse primer (10 pmol/µL), 0.4 µL ROX Reference Dye II (50×), and 2 µL DNA template. The volume was adjusted to 20 µL using sterile ultrapure water. The real-time cycling conditions were as follows: 50°C for 2 min, 95°C for 30 s, 40 cycles of 95°C for 5 s, 55.3°C for 30 s, and 72°C for 34 s, and a final melting curve of 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. Cycle threshold (Ct) values obtained from the real-time PCR results were used to statistically evaluate whether the template concentration was appropriate. Survival 6
ACCEPTED MANUSCRIPT analyses were performed to determine whether the Ct values differed between samples processed using the sonication-based extraction method and the commercial DNA extraction kits (PowerSoil DNA Isolation Kit, MO BIO, USA). The ∆Rn values, displayed as a function of cycle number, were calculated to evaluate the efficiency of
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PCR reaction, by which the efficiency of pyrosequencing with the same universal primer set (27f/519r) was to some extent evaluated due to the same PCR-based reactions. The magnitude of the normalized fluorescence generated by the reporter at
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each cycle of the PCR amplification was then determined.
High-throughput sequencing
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The microbial communities in the DNA samples extracted using both the sonication-based extraction method and the PowerSoil DNA Isolation Kit were investigated by pyrosequencing. Eight-base barcode sequences (AGTGTATG and ATAGATAG) were attached to the 27f and 519r bacterial primer set, respectively, for the identification of the two different samples. The PCR reaction mixtures contained
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0.125 µL TaKaRa Ex Taq HS (5 U/µL), 2.5 µL 10× Ex Taq Buffer, 2 µL dNTP Mixture (2.5 mM each), 0.5 µL forward primer (10 pmol/µL), 0.5 µL reverse primer (10 pmol/µL), and 2.5 µL template DNA. The volume was adjusted to 25 µL using
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sterilized ultrapure water. The thermal program was the same as presented in section 2.3. The concentration of the PCR products was assessed using a PicoGreen dsDNA assay kit. The PCR products were purified using a QIAquick gel extraction kit
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(Qiagen; USA). The quality of the purified PCR products was verified using gel electrophoresis. And the similarity of the two maps of microbial populations in the amplified samples were analyzed, whose original DNA template were extracted by the two different methods.
Results Removal of extracellular inhibitory substances during pre-washing process The amount of soluble inhibitory substances removed during extraction was assessed by measuring the weight lost during the pre-washing process. Figure 1 shows the solid 7
ACCEPTED MANUSCRIPT weight changes after each washing step. The total solids (TS) of sludge used in extractions was ~18.8%. After pre-purification using ethanol followed by ddH2O, the TS decreased to 17.5% (a ~7% decrease to TS) and 17.0% (a further ~3% decrease to TS), respectively. The ratio of volatile solids to total solids (VS/TS) also decreased
dissolving organic matters and salts.
The minimum DNA template for molecular analysis
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from 41.5% to 38.1% during the pre-washing steps, suggesting the removal of the
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Figure 2 shows the results of PCR reactions performed using a series of 10-fold-diluted DNA templates to determine the minimum DNA concentration that
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could be used. DNA samples containing <10−3 ng/µL were poorly amplified probably due to the insufficient templates provided (Figure S1). Thus, the minimum template concentration required for the PCR reactions was ~1 × 10−3 ng/µL. As long as the amount of template in samples is higher than that concentration, it will be amplified to satisfactory levels within 30 cycles.
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The yields of extracted DNA obtained using the sonication-based method for different treatment time and the commercial kit are shown in Figure 3. The optical density measurement obtained at 260/280 nm revealed that the commercial kit
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extracted purer DNA (not shown). In contrast, the sonication-based method resulted in a superior yield of DNA than that extracted by the commercial kit.
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Feasibility of the sonication-based method The feasibility of the sonication-based method was confirmed by applying the method to the high solid digested sludge sample. Figure 4 shows the PCR products obtained from the serially diluted original templates, as well as DNA extracted using the commercial kit. The DNA template concentration of 10−3 ng/µL resulted in the highest concentration of PCR product with 30 cycles, except for the reaction based on template extracted using the commercial kit (diluted to10 ng/ul). Results based on fluorescence specific in cell quantification might be more reliable on presenting the decomposition of microbial cells because impurities in the 8
ACCEPTED MANUSCRIPT DNA extracts could also exhibit ultraviolet absorbance. One example is the LIVE/DEAD BacLight Kit, which contains two fluorescent dyes: red fluorescence to identify dead cells, and green fluorescence to label live cells. This allows the evaluations of the decomposition degree of the microbial populations. Figure 5 (a)
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shows an image of the debris resulting from 30 s sonication treatment at 30% amplitude, whereas Figure 5 (b) shows an image of live cells after sonication treatment. The obvious green fluorescence confirmed that bacteria to some extent still
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remained intact after sonication.
Bias of the presented microbial populations using different extraction methods
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As described above, the sonication-based and the commercial kit-based extraction methods used in this study might cause bias when profiling microbial populations in samples. Therefore, detailed analyses using pyrosequencing and real-time PCR were performed to further evaluate the effects of the two different extraction methods on PCR reactions.
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Figure 6 shows the different presentations of the microbial structure caused by the difference in the two extraction methods. In total, 21482 and 15572 reads were sequenced from the amplified samples extracted using the sonication-based method
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and the commercial kit method, respectively. The dominance of bacteria differed in samples extracted using these two methods. For example, 46.6% and 54.0% of the detected species in samples belonged to Firmicutes for the sonication-based method
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and the kit, respectively (Figure 6 (a)). Meanwhile, Venn diagram was constructed to show the common and the difference in terms of OTU numbers between the revealed bacterial communities (Figure 6 (b)), by which the bacterial community obtained by the sonication-based method showed totally different to that by the commercial kit. In addition, rarefaction curves were also drawn based on the results of the theoretical calculation, demonstrating that the sonication-based method extracted more representative bacterial community with the values presenting the higher diversity. The PCR efficiency of samples extracted using the sonication-based method and the commercial kit were also compared using qPCR in triplicate. Survival analysis 9
ACCEPTED MANUSCRIPT was conducted to investigate whether Ct values differed statistically between samples with DNA extracted by the different methods. The Ct values were both around 25 cycles with the same original template concentration of 10-3 ng/µl, and the ∆Rn values were relatively low (0.13 and 0.14, respectively), and were comparable between
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samples processed using the sonication-based method and the DNA extraction kits (Figure S2).
Discussion
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D’Abzac and Bordas (2010) compared different physical and chemical extraction methods, and found that ethanol could not only assist in the decomposition of the
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sludge structure, but also remove organic contaminants. On the other hand, if without ethanol fixation, DNA can be altered in two ways: first, the bacterial community could change rapidly during transportation and storage because of the changing environment (Guo and Zhang, 2013). In addition, DNA might leak from cells into the bulking water, and then be washed away. For environmental samples, such as the digested
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sludge samples containing much amount of contaminants in this study, fixation in a final concentration of 50% ethanol was recommended to improve the recovery of DNA templates, as which is the same as that used to fix samples for fluorescence in
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situ hybridization (FISH) (Xia et al., 2007). Meanwhile, unlike other environmental samples, sludge is composed of nearly all microbial cells and their products (Frolund et al., 1996). In general, 1 g of dry mass of
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activated sludge contains more than 1010–1012 bacterial cells (Guo and Zhang, 2013). The amount of DNA is usually not a great concern for PCR-based community analyses because concentrations as low as 10–100 ng DNA, equivalent to ~106–107 cells, are sufficient for amplification followed by sequencing (Nielsen and Nielsen, 2001). The VS/TS value of high solid anaerobically digested sludge is ~30–50%. This abundance ensures that biomass is not a limiting factor, since small amounts (several hundred microliters to several milliliters) of sludge are sufficient for DNA extraction and consequent PCR reactions. However, inhibitory substances may remain in the samples even after prewashing, which affects the efficiency of PCR reactions at 10
ACCEPTED MANUSCRIPT certain concentrations. As EPS are innate protectors of bacterial cells, these complex biopolymers constitute a very large proportion of digested sludge, and the organic or inorganic matters that adsorb to the sludge are difficult to be separated thoroughly from the DNA during extraction (Flemming et al., 2007). These co-extracted
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contaminants should be removed or diluted appropriately to avoid any effect on PCR reactions, and meet the minimum amount requirement of DNA templates in PCR reactions as well. As shown in Figure 4, even the low quantity (10−3 ng/µL) and low quality (because of contamination with co-extracted substances) of the extracted DNA
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used in this study could provide an adequate profile of the microbial community.
Other than yield and fidelity, an additional problem is that DNA extracts from
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both the sonication-based extraction method and the commercial kit are usually small in terms of segment size. In both methods, a high shear force is applied to the samples during extraction. Small pieces of DNA are not suitable for the construction of fosmid and cosmid BAC libraries, which prefer genomic DNA fragments >25 k. Therefore, they are usually extracted using laboratory-developed methods (Robe et al., 2003).
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Moreover, if the extracted DNA is used for full-length 16S rRNA gene (~1.5 kb) amplification, ~10 kb-sized fragments theoretically lose ~15% of the genes (Guo and Zhang, 2013). However, the current high-throughput sequencing method is unlikely to
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be affected because of the short amplified regions (mostly <400 bp). Previous studies reported that microbes belonging to the gram-positive Actinobacteria, Nitrospirae, and Chloroflexi were difficult to lyse because of their
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thick cell wall (Bollet et al., 1991), or the formation of spores (Kuske et al., 1998). In general, if samples contain dominant lysis-resistant species, such as Actinobacteria and Nitrospirae, the lysing intensity should increase. Snaidr et al. (1997) used untreated sludge to perform PCR and cloning, and were unable to detect Actinobacteria, even though FISH revealed that ~13% of the cells belonged to this phylum. In contrast, the cell walls of Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and other rare microbes can be readily broken (Guo and Zhang, 2013). In addition, Actinobacteria and Chloroflexi were the two biomarkers used to 11
ACCEPTED MANUSCRIPT assess cellular decomposition and to confirm that bacterial cells had been disrupted efficiently by both extraction methods. However, more Chloroflexi was detected in samples extracted using the sonication-based method, whereas Actinobacteria accounted for more in the samples extracted using the commercial kit.
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In this study, the more diverse microbial structure presented in the samples with DNA extracted by the sonication-based method which proved the findings that the difference in the microbial fractions might not be due to the bias in the PCR reactions, but from a different recovery of DNA by the different methods which might result
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from variations in cell lysis, the binding of DNA to the matrix, and elution (Dionisi et al., 2003; Guo and Zhang, 2013). As confirmed by Morono et al.(2014), even with
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commonly used kits and protocols employing cell separation and enumeration techniques, ~75% of sedimentary microbial cells remained intact in the residue of DNA extraction, indicating that large fractions of many microbial communities have been missed in previous molecular ecological surveys. On the other hand, other researchers have also pointed out that PCR-based molecular approaches might
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overlook some evolutionarily distinct microbial populations because of bias introduced by PCR reactions (Morono et al., 2014). Thus, the observed significant inconsistencies between cell abundance and molecular quantification using
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PCR-based techniques indicated that the results must be understood to represent only the DNA-extractable and PCR-amplifiable fraction. Additionally, qPCR is a powerful and robust tool, the accurate quantification of
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specific microbial populations by it strongly depends on the quality and yield of the DNA extracted from the environmental samples, which could be thus utilized to present inherent variability associated with the PCR amplification reactions as well (Bonot et al., 2010). Teske and Sorensen (2008) have clearly illustrated the possible bias in the amplification of archaeal sequences introduced by the use of conventional PCR primer sequences, such as the frequently produced mismatches to sequences of predominantly sedimentary archaea in deep sediments. Therefore, although the presence of PCR inhibitors, such as humic acids in organic-rich samples, may also significantly diminish the amplification efficiency and cycle threshold (CT) values in 12
ACCEPTED MANUSCRIPT quantitative real-time PCR analyses, the insufficient PCR reactions for both samples might be mainly caused by the non-specific universal primers used in the current study. In summary, this study provided the quantitative and qualitative validations of a
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sonication-based DNA extraction approach for PCR-based molecular biological analyses, and concluded that the efficient sonication-based DNA extraction method could be widely applied in PCR-based analyses instead of the costly commercial kit methods for the following reasons: first, the sonication-based method enhances the
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adequate lysis of bacterial cells to produce DNA template of sufficient quality and quantity, and represents an alternative to the commercial kit method. Second, the
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minimum concentration of DNA template that could be used for PCR-based analysis of environmental samples (high solid anaerobically digested sludge in this study) was found to be 10 − 3 ng/mL, which is easily achieved. Third, the pyrosequencing technology used in this study analyzed the bias triggered by the different DNA extraction methods, which highlighted the utility of the sonication-based method. The
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sonication method can be further refined and validated in spike and recover studies to define the analytical assay performance parameters - the limits of detection (LOD) and the limits of quantitation (LOQ) by utilizing specific microorganism within
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complex samples.
Conflict of Interest
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The authors declare no competing financial interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No.51308402 and 51538008).
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. 13
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Terrat, S., Christen, R., Dequiedt, S., Lelievre, M., Nowak, V., Regnier, T., Bachar, D., Plassart, P., Wincker, P., Jolivet, C., Bispo, A., Lemanceau, P., Maron P. A., Mougel, C., Ranjard, L. 2012. Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure. Microbial Biotechnology. 5 (1),135-141
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ACCEPTED MANUSCRIPT Teske, A., Sorensen, K. B., 2008. Uncultured archaea in deep marine subsurface sediments: have we caught them all? The ISME Journal, 2(1), 3-18 Xia, Y., Kong, Y., Nielsen, P. H. 2007. In situ detection of protein-hydrolysing microorganisms in activated sludge. FEMS Microbiology Ecology. 60,156-165
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Zhou, J., Bruns, M. A. N. N., Tiedje, J. M. 1996. DNA recovery from soils of diverse
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composition. Applied and Environmental Microbiology. 62 (2), 316-322
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ACCEPTED MANUSCRIPT Figure 1. Changes in solids weight during the pre-treatment processes. The bars show standard deviation (SD), n=3. Figure 2. The concentration of amplified PCR products using a 10-fold serial diluted template DNA (n = 3). The bars show standard deviation (SD), n=3. NC denotes the
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negative control Figure 3. Figure 3. The yield of extracted DNA obtained from the sonication-based method using different sonication time and by the commercial kit (n = 3), the bars show standard deviation (SD). S denotes the samples pretreated by the
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sonication-based method. The figure behind S denotes the duration of sonication time. The lowercase s denotes the unit of sonication time -second.
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Figure 4. Comparisons of the PCR yields obtained from the differently diluted and the kit-extracted templates (n = 3), the bars show standard deviation (SD). The original concentration of the sonicated samples was 5 ng/µl. The concentration of DNA templates extracted by the kit was adjusted to 10 ng/µl.
Figure 5. Cell images after sonication. (a) optical microscopy; (b) live cell imaging
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Figure 6. The structure of bacterial communities with the original DNA extracted using the sonication-based method and the commercial kit. (a) Pyrosequencing results
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presented at phylum level. (b) Venn diagram of OTU distribution. A denotes the sonication-based method, B denotes the commercial kit extraction method. (c) rarefaction curves with Shannon index.
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Figure S1. The gel image of the amplified PCR products using a 10-fold serial diluted template DNA.
Figure S2. The quantitative PCR results of the two DNA samples from the commercial approach and the sonication-based method. (a) Amplification plots. (b) Dissociation curve. The green solid lines denote the DNA samples from the commercial approach, while the grey solid line denote the DNA samples from the sonication-based approach.
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S0003-2697(16)00005-1
DOI:
10.1016/j.ab.2016.01.003
Reference:
YABIO 12280
To appear in:
Analytical Biochemistry
Received Date: 25 October 2015 Revised Date:
2 January 2016
Accepted Date: 5 January 2016
Please cite this article as: X. Dai, S. Chen, N. Li, H. Yan, Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.01.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses
Xiaohu Dai1,2, Sisi Chen1,2, Ning Li1,*, Han Yan
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1. State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University 2. These authors contributed equally in this work
[email protected];[email protected]
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*Corresponding author:
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ACCEPTED MANUSCRIPT Abstract The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called standard DNA extraction method - the commercial kit method. Microbial cells in the digested sludge
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sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid, protein etcs., were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the
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in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the DNA-based method.
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Keywords: DNA extraction, Dilution, Sonication, Fluorescent dye, Pyrosequencing
Introduction
Microorganisms are widely distributed in natural and engineered environments, where they play significant ecological roles in global elemental cycles. Traditionally,
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microbial communities have been studied using cultivation-dependent techniques, whereas it is generally accepted that the bulk of microorganisms in environment are unculturable. During the last several decades, the development of culture independent
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molecular biology technology, basing on Polymerase Chain Reaction (PCR), provided researchers access to investigate the previously hidden information of microbial populations contained within environmental samples. Therefore, DNA probing,
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PCR-based amplification of specific targets, and the construction of clonal libraries have been extensively achieved. And in particular, the recent application of comprehensive investigation of microbial populations using high-throughput sequencing technology (such as 454 or Illumina pyrosequencing) has been reported as well, which allows the scientific community to assess the discriminations between different extraction methods with enough resolution (Dionisi et al., 2003; Terrat et al., 2012; Satoh et al., 2013). The prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extractions. DNA, as the template of PCR, is 2
ACCEPTED MANUSCRIPT embedded in the microbes by other components, and is further surrounded by extracellular polymer substances (EPS) and co-exists with other inhibitory contaminants (Frolund et al., 1996). Numerous protocols have described different approaches for DNA extraction with microorganism lysis, which include alkaline
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extraction (Birnboim and Doly, 1979), enzymatic extraction (Pitcher et al., 1989), microwave heating (Picard et al., 1992), salt extraction (Aljanabi et al., 1997), freeze-thawing extraction (Zhou et al., 1996), thermal shocks (Bourrain et al., 1999), bead lysis (Miller et al., 1999), and sonication (Krsek and Wellington, 1999).
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However, during the extraction processes, the inhibitory substances of PCR including environmental molecules (e.g., humic acid, metals, and proteins) and the carry-over of
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compounds used can still remain. The high-quality and high-fidelity DNA extraction is thus still the hurdle and the technical challenge (Martin-Laurent et al., 2001; Hazen et al. 2013).
Meanwhile, as the extraction of DNA is the first step in molecular analytical techniques, the procedures proper or not have a significant impact on the results
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obtained. And hence, cautions have been raised that the use of different DNA extraction protocols may result in different microbial community structures. The potential for such bias may also have a critical impact on quantitative molecular
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analyses (e.g., measurement of gene quantity or copy number largely relies on stable amplification of the gene, as well as the DNA coverage of the primers or probe used). Commercial kits such as FastSpin Soil DNA kit (MP Biomedicals, USA) and
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PowerSoil DNA Isolation kit (MOBIO, USA) have been widely applied in the preparation of DNA template, and become almost the standard method due to the high quality of extracted DNA (Sagova-Mareckova et al., 2008). However, unbiased genetic material from microorganisms with varying cell wall compositions and differentially accessible DNA has been reported to be difficult to be obtained (Yergeau et al., 2010; Mackelprang et al., 2011), as the extraction condition might not represent the overall picture of the indigenous community which hampered attempts in applying metagenomics approaches to environmental samples such as soils and sediments (Hazen et al., 2013). Sonication based method, on the other hand, is widely 3
ACCEPTED MANUSCRIPT considered one of the most efficient for the lysis of microbes if combined with ethanol purification as the homogeneous decomposition by it needs just several to dozens of seconds, and seems to be a promising, efficient and cost-effective method (Gunawardana et al., 2014).
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In this study, in consideration of a high extraction efficiency, low costs and less labour intensive technique, the sonication-based DNA extraction method was evaluated the feasibility of the replacement of the so-called standard DNA extraction method (the commercial kit). The anaerobically digested samples, representative in
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the highly contaminated samples with inhibitory substances, such as humic acid, metals, proteins, as well as other simple carry-over compounds, was utilized in this
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study. The in-depth understanding of the varied effectiveness and the potential bias triggered by the two different methods for the applications of molecular biology approaches was provided. To address these methodological issues, the amount of target biomolecule extracted from the sample must first be determined. The process of the DNA based method was comprised of sample washing by organic and inorganic
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eluents, DNA fixing in 50% ethanol, dilution, and sonication, which were combined to thoroughly extract DNA and inhibited the co-extracted contaminants. Furthermore, the sequencing depths afforded by the pyrosequencing approach allowed us to obtain
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the detailed information regarding the dominant, subdominant, and rare taxa within the fingerprints of the microbial communities. The experimental findings from this work will expand ideas in choosing the appropriate DNA extraction technology
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(and/or PCR amplification procedure) to accommodate the specific needs when analyzing microbial community diversity of samples from various regions of the world.
Material and methods Sample origin and characteristics Sludge samples were obtained from a laboratory-scale high solid anaerobic digester with SRT of 20 days. The digester was fed daily with dewatered sludge. Total solids (TS) and the ratio of volatile solids to total solids (VS/TS) of digestate were 18.8 ± 4
ACCEPTED MANUSCRIPT 0.1% and 41.5 ± 0.1%, respectively.
Sonication-based DNA extraction One gram of digested sludge was transferred to a 50 mL centrifuge tube using a
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spatula, and was then washed with 20 mL ethanol to reduce alteration of the microbial population and prevent the inhibitory effects of organic substances. Samples were shaken at 250 rpm for 10 min on a shaker (KS-260; IKA; Germany), and centrifuged at 10,000 × g for 5 min; the supernatants were then discarded. The pellets were
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resuspended in 50 mL double-distilled (dd) water to dissolve the salt contained in the samples, and the samples were again shaken and centrifuged as described above. After
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discarding the supernatants, the pellets were resuspended in 50 mL 50% (v/v) ethanol eluent, and sonicated by FS-1200N ultrasonic processor (Ultrasonics, China) under the predetermined optimal conditions with intensity of 30% amplitude (15W/cm2) and duration of 30s. Finally, all samples were stored at −80°C until use. The LIVE/DEAD BacLight™ Kit (Invitrogen; Eugene, OR, USA) was used to
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evaluate the decomposition of bacterial cells using the determined sonication conditions. Microscopic images of the samples were observed under an IX73 inverted fluorescence microscope (Olympus; Japan).
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The optical density of DNA templates were measured by Nanodrop 2000 (Thermo Scientific, USA) with the absorbance values of 260/280 to evaluate the
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quality of the extracted DNA templates.
PCR
The optimal concentration of the DNA template was pre-determined as described below. Firstly, the DNA template was extracted from sludge samples using PowerSoil DNA Isolation Kit (MO BIO, USA), which was the experimental alternative of commercial kits in this study and has been widely used in labs all over the world. The concentration of DNA template was measured by following the instruction for PicoGreen fluorescent dye from the manufacturer (Invitrogen; Eugene, OR, USA) and adjusted to 10 ng/µL afterward. Samples were then serially diluted to 10−7 ng/µL. 5
ACCEPTED MANUSCRIPT Each sample was added to triplicate PCR reactions. The PCR products were quantified using PicoGreen fluorescent dye. The minimum DNA concentration to be used for subsequent PCR reactions was determined by the final concentration of PCR products that were >10 ng/µL.
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The universal primers used to amplify the bacterial DNA template were the 27f forward primer (5′-AGAGTTTGATC(A/C)TGGCTCAG-3′) and 519r reverse primer (5′-G(A/T)ATTACCGCGGC(G/T)GCTG-3′) (Lane,
1991). The PCR cycling
conditions were as the reported by Li et al. (2012): 95°C for 10 min, 30 cycles of
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98°C for 10 s, 55.3°C for 30 s, and 72°C for 1 min, followed by a final extension step of 72°C for 10 min. The reaction mixtures contained 0.125 µL TaKaRa Ex Taq hot
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start (5 U/µL, Japan), 2.5 µL 10× Ex Taq buffer (TaKaRa; Japan), 2 µL dNTP mixture (2.5 mM each, TaKaRa; Japan), 0.5 µL forward primer (10 pmol/µL), 0.5 µL reverse primer (10 pmol/µL), and 2.5 µL template DNA. The volume was adjusted to 25 µL using sterilized ultrapure Milii-Q water (Millipore, Geremany). For the comparisons of the molecular biological results, the DNA templates
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obtained by both the commercial kit and the sonication-based method were amplified.
Real-time quantitative PCR (qPCR)
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The extracted DNA was analyzed by qPCR in triplicate to evaluate the PCR efficiency with the universal primer set (27f/519r). Reactions were performed in optical 96-well reaction plates in an Applied Biosystems 7500 Real-Time PCR system (Applied
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Biosystems; Foster City, CA, USA). The reaction mixtures consisted of 10 µL SYBR Premix DimerEraser (2×), 0.6 µL 27f forward primer (10 pmol/µL), 0.6 µL 519r reverse primer (10 pmol/µL), 0.4 µL ROX Reference Dye II (50×), and 2 µL DNA template. The volume was adjusted to 20 µL using sterile ultrapure water. The real-time cycling conditions were as follows: 50°C for 2 min, 95°C for 30 s, 40 cycles of 95°C for 5 s, 55.3°C for 30 s, and 72°C for 34 s, and a final melting curve of 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. Cycle threshold (Ct) values obtained from the real-time PCR results were used to statistically evaluate whether the template concentration was appropriate. Survival 6
ACCEPTED MANUSCRIPT analyses were performed to determine whether the Ct values differed between samples processed using the sonication-based extraction method and the commercial DNA extraction kits (PowerSoil DNA Isolation Kit, MO BIO, USA). The ∆Rn values, displayed as a function of cycle number, were calculated to evaluate the efficiency of
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PCR reaction, by which the efficiency of pyrosequencing with the same universal primer set (27f/519r) was to some extent evaluated due to the same PCR-based reactions. The magnitude of the normalized fluorescence generated by the reporter at
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each cycle of the PCR amplification was then determined.
High-throughput sequencing
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The microbial communities in the DNA samples extracted using both the sonication-based extraction method and the PowerSoil DNA Isolation Kit were investigated by pyrosequencing. Eight-base barcode sequences (AGTGTATG and ATAGATAG) were attached to the 27f and 519r bacterial primer set, respectively, for the identification of the two different samples. The PCR reaction mixtures contained
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0.125 µL TaKaRa Ex Taq HS (5 U/µL), 2.5 µL 10× Ex Taq Buffer, 2 µL dNTP Mixture (2.5 mM each), 0.5 µL forward primer (10 pmol/µL), 0.5 µL reverse primer (10 pmol/µL), and 2.5 µL template DNA. The volume was adjusted to 25 µL using
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sterilized ultrapure water. The thermal program was the same as presented in section 2.3. The concentration of the PCR products was assessed using a PicoGreen dsDNA assay kit. The PCR products were purified using a QIAquick gel extraction kit
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(Qiagen; USA). The quality of the purified PCR products was verified using gel electrophoresis. And the similarity of the two maps of microbial populations in the amplified samples were analyzed, whose original DNA template were extracted by the two different methods.
Results Removal of extracellular inhibitory substances during pre-washing process The amount of soluble inhibitory substances removed during extraction was assessed by measuring the weight lost during the pre-washing process. Figure 1 shows the solid 7
ACCEPTED MANUSCRIPT weight changes after each washing step. The total solids (TS) of sludge used in extractions was ~18.8%. After pre-purification using ethanol followed by ddH2O, the TS decreased to 17.5% (a ~7% decrease to TS) and 17.0% (a further ~3% decrease to TS), respectively. The ratio of volatile solids to total solids (VS/TS) also decreased
dissolving organic matters and salts.
The minimum DNA template for molecular analysis
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from 41.5% to 38.1% during the pre-washing steps, suggesting the removal of the
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Figure 2 shows the results of PCR reactions performed using a series of 10-fold-diluted DNA templates to determine the minimum DNA concentration that
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could be used. DNA samples containing <10−3 ng/µL were poorly amplified probably due to the insufficient templates provided (Figure S1). Thus, the minimum template concentration required for the PCR reactions was ~1 × 10−3 ng/µL. As long as the amount of template in samples is higher than that concentration, it will be amplified to satisfactory levels within 30 cycles.
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The yields of extracted DNA obtained using the sonication-based method for different treatment time and the commercial kit are shown in Figure 3. The optical density measurement obtained at 260/280 nm revealed that the commercial kit
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extracted purer DNA (not shown). In contrast, the sonication-based method resulted in a superior yield of DNA than that extracted by the commercial kit.
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Feasibility of the sonication-based method The feasibility of the sonication-based method was confirmed by applying the method to the high solid digested sludge sample. Figure 4 shows the PCR products obtained from the serially diluted original templates, as well as DNA extracted using the commercial kit. The DNA template concentration of 10−3 ng/µL resulted in the highest concentration of PCR product with 30 cycles, except for the reaction based on template extracted using the commercial kit (diluted to10 ng/ul). Results based on fluorescence specific in cell quantification might be more reliable on presenting the decomposition of microbial cells because impurities in the 8
ACCEPTED MANUSCRIPT DNA extracts could also exhibit ultraviolet absorbance. One example is the LIVE/DEAD BacLight Kit, which contains two fluorescent dyes: red fluorescence to identify dead cells, and green fluorescence to label live cells. This allows the evaluations of the decomposition degree of the microbial populations. Figure 5 (a)
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shows an image of the debris resulting from 30 s sonication treatment at 30% amplitude, whereas Figure 5 (b) shows an image of live cells after sonication treatment. The obvious green fluorescence confirmed that bacteria to some extent still
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remained intact after sonication.
Bias of the presented microbial populations using different extraction methods
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As described above, the sonication-based and the commercial kit-based extraction methods used in this study might cause bias when profiling microbial populations in samples. Therefore, detailed analyses using pyrosequencing and real-time PCR were performed to further evaluate the effects of the two different extraction methods on PCR reactions.
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Figure 6 shows the different presentations of the microbial structure caused by the difference in the two extraction methods. In total, 21482 and 15572 reads were sequenced from the amplified samples extracted using the sonication-based method
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and the commercial kit method, respectively. The dominance of bacteria differed in samples extracted using these two methods. For example, 46.6% and 54.0% of the detected species in samples belonged to Firmicutes for the sonication-based method
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and the kit, respectively (Figure 6 (a)). Meanwhile, Venn diagram was constructed to show the common and the difference in terms of OTU numbers between the revealed bacterial communities (Figure 6 (b)), by which the bacterial community obtained by the sonication-based method showed totally different to that by the commercial kit. In addition, rarefaction curves were also drawn based on the results of the theoretical calculation, demonstrating that the sonication-based method extracted more representative bacterial community with the values presenting the higher diversity. The PCR efficiency of samples extracted using the sonication-based method and the commercial kit were also compared using qPCR in triplicate. Survival analysis 9
ACCEPTED MANUSCRIPT was conducted to investigate whether Ct values differed statistically between samples with DNA extracted by the different methods. The Ct values were both around 25 cycles with the same original template concentration of 10-3 ng/µl, and the ∆Rn values were relatively low (0.13 and 0.14, respectively), and were comparable between
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samples processed using the sonication-based method and the DNA extraction kits (Figure S2).
Discussion
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D’Abzac and Bordas (2010) compared different physical and chemical extraction methods, and found that ethanol could not only assist in the decomposition of the
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sludge structure, but also remove organic contaminants. On the other hand, if without ethanol fixation, DNA can be altered in two ways: first, the bacterial community could change rapidly during transportation and storage because of the changing environment (Guo and Zhang, 2013). In addition, DNA might leak from cells into the bulking water, and then be washed away. For environmental samples, such as the digested
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sludge samples containing much amount of contaminants in this study, fixation in a final concentration of 50% ethanol was recommended to improve the recovery of DNA templates, as which is the same as that used to fix samples for fluorescence in
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situ hybridization (FISH) (Xia et al., 2007). Meanwhile, unlike other environmental samples, sludge is composed of nearly all microbial cells and their products (Frolund et al., 1996). In general, 1 g of dry mass of
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activated sludge contains more than 1010–1012 bacterial cells (Guo and Zhang, 2013). The amount of DNA is usually not a great concern for PCR-based community analyses because concentrations as low as 10–100 ng DNA, equivalent to ~106–107 cells, are sufficient for amplification followed by sequencing (Nielsen and Nielsen, 2001). The VS/TS value of high solid anaerobically digested sludge is ~30–50%. This abundance ensures that biomass is not a limiting factor, since small amounts (several hundred microliters to several milliliters) of sludge are sufficient for DNA extraction and consequent PCR reactions. However, inhibitory substances may remain in the samples even after prewashing, which affects the efficiency of PCR reactions at 10
ACCEPTED MANUSCRIPT certain concentrations. As EPS are innate protectors of bacterial cells, these complex biopolymers constitute a very large proportion of digested sludge, and the organic or inorganic matters that adsorb to the sludge are difficult to be separated thoroughly from the DNA during extraction (Flemming et al., 2007). These co-extracted
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contaminants should be removed or diluted appropriately to avoid any effect on PCR reactions, and meet the minimum amount requirement of DNA templates in PCR reactions as well. As shown in Figure 4, even the low quantity (10−3 ng/µL) and low quality (because of contamination with co-extracted substances) of the extracted DNA
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used in this study could provide an adequate profile of the microbial community.
Other than yield and fidelity, an additional problem is that DNA extracts from
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both the sonication-based extraction method and the commercial kit are usually small in terms of segment size. In both methods, a high shear force is applied to the samples during extraction. Small pieces of DNA are not suitable for the construction of fosmid and cosmid BAC libraries, which prefer genomic DNA fragments >25 k. Therefore, they are usually extracted using laboratory-developed methods (Robe et al., 2003).
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Moreover, if the extracted DNA is used for full-length 16S rRNA gene (~1.5 kb) amplification, ~10 kb-sized fragments theoretically lose ~15% of the genes (Guo and Zhang, 2013). However, the current high-throughput sequencing method is unlikely to
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be affected because of the short amplified regions (mostly <400 bp). Previous studies reported that microbes belonging to the gram-positive Actinobacteria, Nitrospirae, and Chloroflexi were difficult to lyse because of their
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thick cell wall (Bollet et al., 1991), or the formation of spores (Kuske et al., 1998). In general, if samples contain dominant lysis-resistant species, such as Actinobacteria and Nitrospirae, the lysing intensity should increase. Snaidr et al. (1997) used untreated sludge to perform PCR and cloning, and were unable to detect Actinobacteria, even though FISH revealed that ~13% of the cells belonged to this phylum. In contrast, the cell walls of Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and other rare microbes can be readily broken (Guo and Zhang, 2013). In addition, Actinobacteria and Chloroflexi were the two biomarkers used to 11
ACCEPTED MANUSCRIPT assess cellular decomposition and to confirm that bacterial cells had been disrupted efficiently by both extraction methods. However, more Chloroflexi was detected in samples extracted using the sonication-based method, whereas Actinobacteria accounted for more in the samples extracted using the commercial kit.
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In this study, the more diverse microbial structure presented in the samples with DNA extracted by the sonication-based method which proved the findings that the difference in the microbial fractions might not be due to the bias in the PCR reactions, but from a different recovery of DNA by the different methods which might result
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from variations in cell lysis, the binding of DNA to the matrix, and elution (Dionisi et al., 2003; Guo and Zhang, 2013). As confirmed by Morono et al.(2014), even with
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commonly used kits and protocols employing cell separation and enumeration techniques, ~75% of sedimentary microbial cells remained intact in the residue of DNA extraction, indicating that large fractions of many microbial communities have been missed in previous molecular ecological surveys. On the other hand, other researchers have also pointed out that PCR-based molecular approaches might
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overlook some evolutionarily distinct microbial populations because of bias introduced by PCR reactions (Morono et al., 2014). Thus, the observed significant inconsistencies between cell abundance and molecular quantification using
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PCR-based techniques indicated that the results must be understood to represent only the DNA-extractable and PCR-amplifiable fraction. Additionally, qPCR is a powerful and robust tool, the accurate quantification of
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specific microbial populations by it strongly depends on the quality and yield of the DNA extracted from the environmental samples, which could be thus utilized to present inherent variability associated with the PCR amplification reactions as well (Bonot et al., 2010). Teske and Sorensen (2008) have clearly illustrated the possible bias in the amplification of archaeal sequences introduced by the use of conventional PCR primer sequences, such as the frequently produced mismatches to sequences of predominantly sedimentary archaea in deep sediments. Therefore, although the presence of PCR inhibitors, such as humic acids in organic-rich samples, may also significantly diminish the amplification efficiency and cycle threshold (CT) values in 12
ACCEPTED MANUSCRIPT quantitative real-time PCR analyses, the insufficient PCR reactions for both samples might be mainly caused by the non-specific universal primers used in the current study. In summary, this study provided the quantitative and qualitative validations of a
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sonication-based DNA extraction approach for PCR-based molecular biological analyses, and concluded that the efficient sonication-based DNA extraction method could be widely applied in PCR-based analyses instead of the costly commercial kit methods for the following reasons: first, the sonication-based method enhances the
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adequate lysis of bacterial cells to produce DNA template of sufficient quality and quantity, and represents an alternative to the commercial kit method. Second, the
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minimum concentration of DNA template that could be used for PCR-based analysis of environmental samples (high solid anaerobically digested sludge in this study) was found to be 10 − 3 ng/mL, which is easily achieved. Third, the pyrosequencing technology used in this study analyzed the bias triggered by the different DNA extraction methods, which highlighted the utility of the sonication-based method. The
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sonication method can be further refined and validated in spike and recover studies to define the analytical assay performance parameters - the limits of detection (LOD) and the limits of quantitation (LOQ) by utilizing specific microorganism within
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complex samples.
Conflict of Interest
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The authors declare no competing financial interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No.51308402 and 51538008).
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. 13
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ACCEPTED MANUSCRIPT Teske, A., Sorensen, K. B., 2008. Uncultured archaea in deep marine subsurface sediments: have we caught them all? The ISME Journal, 2(1), 3-18 Xia, Y., Kong, Y., Nielsen, P. H. 2007. In situ detection of protein-hydrolysing microorganisms in activated sludge. FEMS Microbiology Ecology. 60,156-165
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ACCEPTED MANUSCRIPT Figure 1. Changes in solids weight during the pre-treatment processes. The bars show standard deviation (SD), n=3. Figure 2. The concentration of amplified PCR products using a 10-fold serial diluted template DNA (n = 3). The bars show standard deviation (SD), n=3. NC denotes the
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negative control Figure 3. Figure 3. The yield of extracted DNA obtained from the sonication-based method using different sonication time and by the commercial kit (n = 3), the bars show standard deviation (SD). S denotes the samples pretreated by the
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sonication-based method. The figure behind S denotes the duration of sonication time. The lowercase s denotes the unit of sonication time -second.
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Figure 4. Comparisons of the PCR yields obtained from the differently diluted and the kit-extracted templates (n = 3), the bars show standard deviation (SD). The original concentration of the sonicated samples was 5 ng/µl. The concentration of DNA templates extracted by the kit was adjusted to 10 ng/µl.
Figure 5. Cell images after sonication. (a) optical microscopy; (b) live cell imaging
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Figure 6. The structure of bacterial communities with the original DNA extracted using the sonication-based method and the commercial kit. (a) Pyrosequencing results
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presented at phylum level. (b) Venn diagram of OTU distribution. A denotes the sonication-based method, B denotes the commercial kit extraction method. (c) rarefaction curves with Shannon index.
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Figure S1. The gel image of the amplified PCR products using a 10-fold serial diluted template DNA.
Figure S2. The quantitative PCR results of the two DNA samples from the commercial approach and the sonication-based method. (a) Amplification plots. (b) Dissociation curve. The green solid lines denote the DNA samples from the commercial approach, while the grey solid line denote the DNA samples from the sonication-based approach.
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