An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples

To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

rare representatives of each taxonomic groups possessing different thickness of cell wall and different layer of cell membranes with different embedded components casing their genomic contents.
Lyses of microbial cells expose their genomic DNA to different cellular and extracellular molecules including different type of nucleases. Despite its inert nature, double stranded DNA is physically fragile and highly susceptible to exo-and endonucleases, active forms of which are widely present in the matrix of most of the environmental and human samples analyzed in this study. Therefore, it is important to inactivate all the nucleases in lysis solution by incorporating strong denaturing agents or chemicals that chelate residual metallic ions from the suspension. Although, several commercial kits are now available to extract DNA from human and environmental samples, most of which uses silica-based column where DNA adsorb selectively to a stationary solid phase at high pH and high salt concentration. The major disadvantage for most of the commercial kits is insufficient recovery of genomic DNA from marginal amount of clinical or environmental samples. Furthermore, different DNA extraction kits have different biases, which can produce dramatically different results for the same sample 1 . Several laboratories working on metagenomics reported different methods of community DNA extraction depending on the type of samples they used for analysis [2][3][4][5][6][7][8] . Recently, International Human Microbiome Standards (IHMS) launched a guideline for standard operating procedures to optimize community DNA extraction methods from human fecal samples (http://www.microbiome-standards.org). So far, no attempt has been taken to develop a gold standard for community DNA extraction from both human and environmental origin samples.
In this study, we developed a highly sensitive method, by combining physical, mechanical and chemical lysis approaches, to isolate community bacterial DNA from different human and environmental samples (Fig. 2). All the selected samples harbor culturable and uncultured bacteria belonging to closely or distantly related taxonomic groups and having different thickness of cell wall and different layer of cell membranes (Fig. 1). We compared both the quality and quantity of isolated community DNA with existing methodologies and observed that this approach worked best compared to currently available approaches. The isolated DNAs are suitable for all types of high-resolution downstream applications including shotgun metagenomics sequencing where HMW genomic DNA is preferable.

Results and Discussion
Spheroplast formation and DNA isolation. Both, environmental and human samples contain large numbers of microbial cells belonging to different phyla and they are reasonably heterogeneous in terms of their genomic contents, morphology and architecture of their cell wall (Fig. 1). To obtain sufficient amount of quality community DNA from Gram-positive and Gram-negative bacterial cells, it is important to preprocess the samples before adding lysis reagents. In this study, we used three different enzymes lysozyme, lysostaphin and mutanolysin that target either 1,4-beta glycoside-linkages or transpeptide bond in Gram-positive and Gramnegative bacterial cell wall and help in spheroplast formation. Spheroplast is highly susceptible to lysis reagents and labile to mechanical and physical forces.
For lysis, first we treated the spheroplast with Guanidinium thiocyanate (GITC) to disrupt the bacterial cell membrane and inactivate nucleases and other enzymes. Combining mechanical (bead beating) and thermal (heat) forces enabled final lysis. The recovery and quality of the isolated DNAs were confirmed by running the samples on agarose gel (Fig. 3). We used both environmental and human samples (Fig. 2), containing diverse range of bacterial species including Gram-positive and Gram-negative bacteria possessing different types of cell wall, to confirm the suitability of the same method in wide range of samples. We successfully isolated reasonably good amount of quality DNA from all the tested samples ( Fig. 3 and Table 1). DNA yield was typically ~1-109 μ g, depending on the initial sample size and the way the sample was stored (Table 1). Total yield of DNA irrespective of the sample types was always higher in THSTI method compare to Kit and ALHS methods (Table 1). Average size of the DNA fragments recovered by THSTI method was ~20 kb (Fig. 3).  (B) Genomic DNA isolated from equal amount of samples using commercial kits or automated liquid handling system. Lane 1: Lambda genomic DNA digested with restriction endonuclease HindIII; Lane 2-3: Genomic DNA isolated from stool samples using commercial kit. Lane 4-5: Genomic DNA isolated from GTB samples using commercial kit. Lane 6-7: Genomic DNA isolated from stool DNA samples using automated liquid handling system. Lane 8-9: Genomic DNA isolated from VS samples using automated liquid handling system. Assessment of the quality of isolated DNA. Both the quality and quantity of isolated DNA were assessed by measuring the absorbance at 260 and 280 nm wavelengths (Table 1) and by visualizing extracted community DNA on agarose gel (Fig. 3). Most of the isolated DNA samples had OD 260 /OD 280 ratio in between ~1.6 and ~1.9 except the genomic DNA isolated from soil sample (Table 1). We further confirmed the quality of isolated DNA by visualizing all the samples on 0.8% agarose gel containing DNA-intercalating agent ethidium bromide. Although, the gel electrophoresis is not very sensitive to measure the quantity of DNA but this is useful to analyze the stable RNA contamination, short fragment DNA contamination, and also shown the average size of isolated DNA. It is important to note that in THSTI and kit methods nucleic acids were treated with RNase to remove stable RNA while in automated liquid handling system the RNAse treatment step was absent. Thus, in terms of quality of DNA, the present method is free of from other nucleic acid impurities.
Comparison of current method with available DNA isolation kits and automated nucleic acid extraction system. Several methods have been described for community microbial DNA extraction from human and environmental origin samples [7][8][9][10][11][12][13] . We compared the quality and quantity of DNA obtained from equal amount of same samples for all, except gastric tissue biopsy, using DNA isolation kit (Qiagen, Germany), and automated nucleic acid extraction system (MagNA pure, Roche Diagnostics, Swizerland). We observed that, when the tested samples, like stool specimen, contained large numbers of bacterial species, both automated nucleic acid extraction system and kit method could recover adequate amount of quality DNA for downstream applications. However, both the methods are not efficient to recover sufficient amount of DNA from low amount of microbial cells including vaginal swabs, where bacterial number was limited ( Fig. 3 and Table 1). In contrast, the method developed in this study efficiently recoverd sufficient amount of genomic DNA even in samples with limited amount of bacterial cells ( Fig. 3 and Table 2).

Suitability of isolated DNA in different downstream applications.
To assure the quality of isolated nucleic acid, the samples were used for different downstream applications including PCR amplification (Fig. 4), restriction digestion (Fig. 5), cloning and sequencing of PCR products (Fig. 6). The PCR amplification of complete and partial 16S rRNA gene of bacterial DNA was done by using set of primer tagging with or without NGS   specific adaptor and barcode sequences. The adaptor was selected based on the recommendation of 454 GS FLX+ pyrosequencing platform (Table 3). We used different NGS primers specific for C1, C3 and C5 and C9 regions of  EcoRI digested HVS sample isolated by kit, ALHS and THSTI methods, respectively; Lane 9-11: EcoRI digested genomic DNA of soil sample isolated by kit, ALHS and THSTI methods, respectively; Lane 12-14: EcoRI digested genomic DNA of sewage water sample isolated by kit, ALHS and THSTI methods, respectively. 16S rRNA gene ( Fig. 4 and Table 3). Sufficient amount of desired amplicon from each set of amplification reaction confirmed the suitability of isolated DNA for NGS application (Fig. 4). The complete 16S rRNA genes were amplified from the sewage water, soil, stool, GTB and vaginal swabs genomic DNA and subset of them were used for cloning and sequencing reactions. Among thousands of clones obtained during cloning of 16S rRNA gene, few of them were randomly picked up for plasmid isolation. Eight representative recombinant clones of 16S rRNA gene amplified from sewage water DNA are shown (Fig. 6). Insert of subset of plasmids were sequenced in a capillary sequencer using universal M13F and/or M13R primers. Identity of 16S rRNA genes amplified from DNA sample of sewage water, soil, stool, GTB and vaginal swabs were examined by using NCBI BLASTN program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE= BlastSearch) database. Although the sample size was small (n = 36), still we have identified multiple Gram-positive and Gram-negative bacterial species in different samples belonging to different bacterial classes (Table 4). Restriction digestions of subset of DNA samples were done using type II restriction endonuclease EcoRI (Fig. 5). Complete digestion of genomic DNA indicates absence of inhibitory compounds, possibly, in the isolated DNA samples.

Conclusion
The method reported in this study is very efficient and economic to isolate community bacterial DNA from minimal amount of human and environmental samples. The quality and quantity of extracted DNA are suitable for various downstream applications including restriction enzyme digestion, PCR amplification using sequencing adaptor and barcode tagged primers used for NGS reactions. Compared to testified two methods, kit and automated nucleic acid extraction system, the recovery of community DNA in THSTI method is substantially higher. A limitation of the present method is the duration for extraction of DNA from the sample. This can be afforded, considering the quality, quantity and suitability of the isolated DNA for subsequent downstream applications.

Methods and Materials
Samples. Sewage  The human origin samples were collected after receiving approval from THSTI ethics committee and informed consent from the study subjects. Recombinant DNA works were carried out in "accordance" with the approved guidelines of THSTI biosafety committee. All other experimental protocols used in this study were carried out in "accordance" with the relevant guidelines and standard operating procedure (SOP) of Centre for human microbial ecology (CHME).
Enzymes. Lysozyme (10 mg/ml), mutanolysin (25 KU/ml) and lysostaphin (4 KU/ml) were used for removal of cell wall from Gram-positive and Gram-negative bacterial cells. All three enzymes were purchased from Sigma-Aldrich, USA. Both mRNA and stable RNA species were removed from the pool of nucleic acids by treating the samples with RNase (10 mg/ml). Glass Beads processing. The glass beads are very useful to detach microbes from the matrix of collected samples. 2.5 mm glass beads are suitable for bacterial cells. First, the glass beads (Biospec USA) were kept in 1.0% Triton-X solution for 30 minutes at room temperature and then washed 6-7 times with water. The washed beads were kept in an incubator over night at 55 °C. Beads were autoclaved before use.  USA). Around 300 mg of zirconia beads was added to the suspension and cell lysis was done by mechanical disruption using SpeedMill PLUS bead beater (Analytical Jena, Germany). Beating was done in two cycles (30 seconds each). Total program time for bacteria was 2 minutes. After completion of bead beating, 15 mg Polyvinylpolypyrrolidone (PVPP) was added to the suspension and mixed well by gentle vortexing of the sample.

Buffers. Tris
To remove the added beads, PVPP and all other cell debris, the suspension was spun down at 14000 rcf for 5 minutes in a microcentrifuge (5427R, Eppendorf, Germany).

Organic extraction and precipitation of nucleic acids. The supernatant was transferred into a fresh
MCT. The pellet was washed with 500 μ l Tris (50 mM)-EDTA(20 mM)-NaCl(100 mM)-PVPP(1%) and the supernatants were pooled. The genomic DNA was precipitated from the supernatant by adding two volumes of 96% ethanol. The organic solvent was mixed gently for one minute and kept five minutes at room temperature and the nucleic acids were recovered by centrifugation at maximum speed, 14000 rcf, for 10 minutes at 4 °C in a microcentrifuge. The supernatant was removed by mild aspiration and keeping the tube in an inverted position on adsorbent paper to let the fluid drain away. The pelleted nucleic acids were dried for 10-15 minutes at room temperature.

Removal of RNA and purification of genomic DNA.
To remove all the RNA species that are present in the nucleic acid preparation, the pellet was dissolved in 450 μ l phosphate buffer supplemented with 50 μ l 3 M-potassium acetate. The pellet was dissolved by pipetting and incubated on ice for 90 minutes. The tube was removed from ice and 2 μ l RNase (10 mg/ml) was added and placed in a heating block (37 °C) for 30 minutes.
The suspension was supplemented with 50 μ l sodium-acetate (3 M) and 1 ml of ice-cold 96% ethanol. The DNA was precipitated by centrifugation at 14000 rcf for 10 minutes at 4 °C. To remove the excess salts, the pellet was washed with 70% ice-cold ethanol. The pellet was dried at room temperature and re-suspended in 200 μ l Tris (10 mM)-EDTA (1 mM) buffer (pH 8.0) and dissolved DNA was stored at 4 °C.
PCR amplification and cloning of community 16S rRNA gene. PCR

Highlights
• Sensitive method to isolate community bacterial DNA from different human origin and environmental samples. • Efficient recovery and high purity of isolated DNA made this method attractive for high-resolution molecular applications. • Would be gold standard for wide range of studies including environmental and clinical samples.
• Very economic compared to kits and automated DNA extraction methods.

Box 1
Lysozyme, well known antimicrobial peptide, is a lytic enzyme that disrupts bacterial cell walls by catalyzing hydrolysis of 1,4-beta glycoside-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues present in the peptidoglycan layer.
Lysostaphin, a 27 KDa glycylglycine endopeptidase, used as antimicrobial agent against Gram-positive bacteria 14 . The endopeptidase works on the transpeptide bond of bacterial cell wall and removes the crosslinking peptide bridges.
Mutanolysin is a an N-acetylmuramidase that catalyzes the cleavage of β -N-acetylmuramyl-(1 → 4)-Nacetylglucosamine linkage of the Gram-positive bacterial cell wall 15 . Its N-terminal end carries enzymatic domain where the C-terminal moieties are involved in substrate recognition and binding to the unique cell wall polymers. The enzyme is preferably used in the formation of spheroplasts and isolation of DNA from bacterial culture.
Guanidinium thiocyanate (GITC) is a chaotropic agent, used as strong denaturant to isolate nucleic acids from viral particles and bacterial cells16. GITC is used to lyse cells and inactivate RNase and DNase, the enzymes that is present in all bacterial cells and degrade RNA and DNA, respectively.
Sodium lauroyl sarcosinate, an amphiphilic amino acid anionic surfactant comprising hydrophobic 12-carbon aliphatic chain and the hydrophilic carboxylate, most often used in nucleic acid isolation from bacterial cells17. It helps in lysis of host cells and removing protein and broken cell walls from the suspension.
Polyvinylpolypyrrolidone (PVPP) is an insoluble, cross-linked form of polyvinylpyrrolidone. PVPP helps to remove the host secondary metabolites and other phenolic impurities from aqueous solution.
Isopropanol and Ethanol. Since, isopropanol is less volatile than ethanol and it co-precipitates simple sugars and salts with nucleic acids, precipitation of DNA with ice cold, 96% ethanol is preferable. DNA is a highly polar molecule, because of its negatively charged phosphate residues in the nucleotide backbone. The repulsive forces that arise because of the exposed phosphate group between the polynucleotide chains need to be neutralized for effective precipitation of DNA. In the presence of 70% ethanol and 300 mM Na + ions, the negative charges of the polynucleotide chains are reduced to the point where the DNA precipitates. It is important to note that ethanol precipitation of DNA can only be done if the cations are available in sufficient amount.