Whole soil DNA extraction followed by Next-Generation Sequencing (NGS) is the best available technology for the analysis of the soil microbiome. Nevertheless, the characterization of the soil complex microbial assemblages requires careful experimental design and a priori selection of the most suitable DNA extraction and purification method. The DNA extraction from soil is challenging due to its reaction with the soil inorganic and organic solid phases after cell lysis, whereas the amplification step is potentially hampered by low quality of template DNA and presence of PCR inhibitors. Owing to the broad differences in soil properties, after more than 25 y of studies, there is not an optimal DNA extraction protocol for all soils and all purposes. These peculiar features often limit the potentials of the microbiome analysis either by PCR-metagenetic or metagenomic studies. The objective of this study was to compare different soil DNA extraction methods with the purpose of optimizing NGS, in particular of 16S and Internal Transcribed Spacers (ITS) amplicons were sequenced for evaluating soil bacterial and fungal communities, respectively. In particular, we evaluated mechanical, chemical and enzymatic pre-treatments to enhance DNA yield. Quality and quantity of the extracted DNA were determined by high-sensitivity DNA electrophoresis and fluorimetry, respectively. As case study, we analyzed samples from agricultural soils and polluted and remediated soils covering a broad range of soil physico-chemical properties. The suitability of extracted DNA for downstream molecular analyses was further verified using Ion S5 semiconductor sequencing and real-time PCR targeting the archaeal and bacterial genes coding for enzymes involved in the N cycle.

Improving automated DNA extraction from soil for Next Generation Sequencing applications

Laura Maretto;Claudia Chiodi;Diego Pizzeghello;Serenella Nardi;Giancarlo Renella;Massimo Cagnin;Giuseppe Concheri;Andrea Squartini;Piergiorgio Stevanato
2020

Abstract

Whole soil DNA extraction followed by Next-Generation Sequencing (NGS) is the best available technology for the analysis of the soil microbiome. Nevertheless, the characterization of the soil complex microbial assemblages requires careful experimental design and a priori selection of the most suitable DNA extraction and purification method. The DNA extraction from soil is challenging due to its reaction with the soil inorganic and organic solid phases after cell lysis, whereas the amplification step is potentially hampered by low quality of template DNA and presence of PCR inhibitors. Owing to the broad differences in soil properties, after more than 25 y of studies, there is not an optimal DNA extraction protocol for all soils and all purposes. These peculiar features often limit the potentials of the microbiome analysis either by PCR-metagenetic or metagenomic studies. The objective of this study was to compare different soil DNA extraction methods with the purpose of optimizing NGS, in particular of 16S and Internal Transcribed Spacers (ITS) amplicons were sequenced for evaluating soil bacterial and fungal communities, respectively. In particular, we evaluated mechanical, chemical and enzymatic pre-treatments to enhance DNA yield. Quality and quantity of the extracted DNA were determined by high-sensitivity DNA electrophoresis and fluorimetry, respectively. As case study, we analyzed samples from agricultural soils and polluted and remediated soils covering a broad range of soil physico-chemical properties. The suitability of extracted DNA for downstream molecular analyses was further verified using Ion S5 semiconductor sequencing and real-time PCR targeting the archaeal and bacterial genes coding for enzymes involved in the N cycle.
2020
Book of Abstracts
978-88-98362-09-7
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3456659
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