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. 2016 May 31:6:26775.
doi: 10.1038/srep26775.

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

Satyabrata Bag  1 , Bipasa Saha  1 , Ojasvi Mehta  1 , D Anbumani  1 , Naveen Kumar  1 , Mayanka Dayal  1 , Archana Pant  1 , Pawan Kumar  1 , Shruti Saxena  1 , Kristine H Allin  2 , Torben Hansen  2 , Manimozhiyan Arumugam  2 , Henrik Vestergaard  2 , Oluf Pedersen  2 , Verima Pereira  3 , Philip Abraham  3 , Reva Tripathi  4 , Nitya Wadhwa  5 , Shinjini Bhatnagar  5 , Visvanathan Gnana Prakash  6 , Venkatesan Radha  6 , R M Anjana  6 , V Mohan  6 , Kiyoshi Takeda  7 , Takashi Kurakawa  7 , G Balakrish Nair  1 , Bhabatosh Das  1
Affiliations

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

Satyabrata Bag et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Diverse microbial species living in different ecosystems have different cell membranes and different types of cell wall encasing their cytoplasm.
Outermembrane cover and cell wall can be removed by treating the microbial community with specific enzymes that use polymer or transpeptide bridge of cell wall as their substrate.
Figure 2
Figure 2. Different environmental and human samples used in this study to isolate community DNA from culturable and uncultured microbial residents.
Figure 3
Figure 3. Agarose gel electrophoresis of microbial genomic DNA isolated from environmental and human samples.
Genomic DNA was electrophoresed on a 0.8% agarose gel, stained with ethidium bromide and photographed in a gel imaging system. (A) Genomic DNA isolated by THSTI method. Lane 1: Lambda genomic DNA digested with restriction endonuclease HindIII; lane 2: Genomic DNA isolated from Sewage water (SW), lane 3: Genomic DNA isolated from soil sample, lane 4: Genomic DNA isolated from stool, lane 5: Genomic DNA isolated from vaginal swab (VS), lane 6: Genomic DNA isolated from gastric tissue biopsy (GTB) sample. (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.
Figure 4
Figure 4. PCR amplification of 16S rRNA gene from community DNA isolated from environmental and human origin samples.
(A) Organization of conserved and variable regions of 16S rRNA gene. Small arrows indicate different primers used in this study to amplify partial or complete 16S rRNA gene. C denotes conserved while V indicates variable. (B) PCR amplification of complete or partial 16S rRNA gene using primers tagged with or without different barcode and adaptor sequences for 454 GS FLX+ pyrosequencer. Genomic DNA isolated both from environmental (SW, Soil) or human samples (Stool, VS, GTB) were used as template. Lane 1: 1-kb DNA ladder; Lane 2–6: complete 16S rRNA gene amplicons from SW, Soil, Stool, VS, GTB, respectively; Lane 7–11: V1-V5 region amplicons of 16S rRNA gene of SW, Soil, Stool, VS, GTB, respectively; Lane 12–16: V1-V3 region amplicons of 16S rRNA gene of SW, Soil, Stool, VS, GTB, respectively.
Figure 5
Figure 5. Restriction endonuclease (EcoRI) digestion of genomic DNA isolated from environmental and human origin samples using kit, ALHS and THSTI methods.
Lane 1, Lambda genomic DNA digested with restriction endonuclease HindIII; Lane 2, undigested genomic DNA isolated from stool sample; Lane 3–5, EcoRI digested stool genomic DNA sample isolated by kit, ALHS and THSTI methods, respectively; Lane 6–8: 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.
Figure 6
Figure 6. PCR amplification and cloning of complete 16S rRNA gene of sewage water samples.
Lane 1: 1-kb DNA ladder. Lane 2: 16S rRNA gene PCR amplicon. Lane 3: Cloning vector pCR2.1. Lane 4–11: Cloning vector containing complete 16S rRNA gene isolated from eight randomly selected clones.
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