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

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.

SUBMITTER: Bag S

PROVIDER: S-EPMC4886217 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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

Bag Satyabrata S Saha Bipasa B Mehta Ojasvi O Anbumani D D Kumar Naveen N Dayal Mayanka M Pant Archana A Kumar Pawan P Saxena Shruti S Allin Kristine H KH Hansen Torben T Arumugam Manimozhiyan M Vestergaard Henrik H Pedersen Oluf O Pereira Verima V Abraham Philip P Tripathi Reva R Wadhwa Nitya N Bhatnagar Shinjini S Prakash Visvanathan Gnana VG Radha Venkatesan V Anjana R M RM Mohan V V Takeda Kiyoshi K Kurakawa Takashi T Nair G Balakrish GB Das Bhabatosh B

Scientific reports 20160531

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 st ...[more]

PMID: 27240745

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Project description:BackgroundPacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing.ResultsWe developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively.ConclusionsDNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.

Project description:BackgroundExtracting high-quality total RNA is pivotal for advanced RNA molecular studies, such as Next-generation sequencing and expression microarrays where RNA is hybridized. Despite the development of numerous extraction methods in recent decades, like the cetyl-trimethyl ammonium bromide (CTAB) and the traditional TRIzol reagent methods, their complexity and high costs often impede their application in small-scale laboratories. Therefore, a practical and economical method for RNA extraction that maintains high standards of efficiency and quality needs to be provided to optimize RNA extraction from human and mice tissues.MethodThis study proposes enhancements to the TRIzol method by incorporating guanidine isothiocyanate (GITC-T method) and sodium dodecyl sulfate (SDS-T method). We evaluated the effectiveness of these modified methods compared to the TRIzol method using a micro-volume UV-visible spectrophotometer, electrophoresis, q-PCR, RNA-Seq, and whole transcriptome sequencing.ResultThe micro-volume UV-visible spectrophotometer, electrophoresis, and RNA-Seq demonstrated that the GITC-T method yielded RNA with higher yields, integrity, and purity, while the consistency in RNA quality between the two methods was confirmed. Taking mouse cerebral cortex tissue as a sample, the yield of total RNA extracted by the GITC-T method was 1,959.06 ± 49.68 ng/mg, while the yield of total RNA extracted by the TRIzol method was 1,673.08 ± 86.39 ng/mg. At the same time, the OD260/280 of the total RNA samples extracted by the GITC-T method was 2.03 ± 0.012, and the OD260/230 was 2.17 ± 0.031, while the OD260/280 of the total RNA samples extracted by the TRIzol method was 2.013 ± 0.041 and the OD260/230 was 2.11 ± 0.062. Furthermore, q-PCR indicated that the GITC-T method achieved higher yields, purity, and greater transcript abundance of total RNA from the same types of animal samples than the TRIzol method.ConclusionThe GITC-T method not only yields higher purity and quantity of RNA but also reduces reagent consumption and overall costs, thereby presenting a more feasible option for small-scale laboratory settings.

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Dataset Information

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

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

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