Quantitative and qualitative evaluation of the impact of the G2 enhancer, bead sizes and lysing tubes on the bacterial community composition during DNA extraction from recalcitrant soil core samples based on community sequencing and qPCR
| Autor: | Lars H. Hansen, Elisa Filippi, Rui G. Santini, Alex Gobbi, Lea Ellegaard-Jensen, Carsten S. Jacobsen |
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| Rok vydání: | 2019 |
| Předmět: |
DNA
Bacterial Lysis Science Microbial Consortia DIVERSITY SOFTWARE Real-Time Polymerase Chain Reaction DNA sequencing BACILLUS-SUBTILIS BIOMASS law.invention Matrix (chemical analysis) 03 medical and health sciences chemistry.chemical_compound law BIOAUGMENTATION DNA Bacterial/chemistry Environmental DNA MICROBIAL COMMUNITIES Food science Soil Microbiology Polymerase chain reaction 030304 developmental biology 2. Zero hunger 0303 health sciences Multidisciplinary 030306 microbiology 16S RIBOSOMAL-RNA Extraction (chemistry) PROFILES ACIDS DNA extraction TRANSFORMATION Enhancer Elements Genetic Microbial Consortia/genetics chemistry 13. Climate action Medicine RNA extraction DNA |
| Zdroj: | Gobbi, A, Santini, R G, Filippi, E, Ellegaard-Jensen, L, Jacobsen, C S & Hansen, L H 2019, ' Quantitative and qualitative evaluation of the impact of the G2 enhancer, bead sizes and lysing tubes on the bacterial community composition during DNA extraction from recalcitrant soil core samples based on community sequencing and qPCR ', PLOS ONE, vol. 14, no. 4, e0200979 . https://doi.org/10.1371/journal.pone.0200979 PLoS ONE, Vol 14, Iss 4, p e0200979 (2019) PLOS ONE Gobbi, A, Santini, R G, Filippi, E, Ellegaard-Jensen, L, Jacobsen, C S & Hansen, L H 2019, ' Quantitative and qualitative evaluation of the impact of the G2 enhancer, bead sizes and lysing tubes on the bacterial community composition during DNA extraction from recalcitrant soil core samples based on community sequencing and qPCR ', PLoS ONE, vol. 14, no. 4, e0200979 . https://doi.org/10.1371/journal.pone.0200979 |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0200979 |
| Popis: | Soil DNA extraction encounters numerous challenges that can affect both yield and purity of the recovered DNA. Clay particles lead to reduced DNA extraction efficiency, and PCR inhibitors from the soil matrix can negatively affect downstream analyses when applying DNA sequencing. Further, these effects impede molecular analysis of bacterial community compositions in lower biomass samples, as often observed in deeper soil layers. Many studies avoid these complications by using indirect DNA extraction with prior separation of the cells from the matrix, but such methods introduce other biases that influence the resulting microbial community composition.To address these issues, a direct DNA extraction method was applied in combination with the use of a commercial product, the G2 DNA/RNA Enhancer®, marketed as being capable of improving the amount of DNA recovered after the lysis step. The results showed that application of G2 increased DNA yields from the studied clayey soils from layers between 1.00 and 2.20 m below ground level.Importantly, the use of G2 did not introduce bias, as it did not result in any significant differences in the biodiversity of the bacterial community measured in terms of alpha and beta diversity and taxonomical composition.Finally, this study considered a set of customised lysing tubes for evaluating possible influences on the DNA yield. Tubes customization included different bead sizes and amounts, along with lysing tubes coming from two suppliers. Results showed that the lysing tubes with mixed beads allowed greater DNA recovery compared to the use of either 0.1 or 1.4 mm beads, irrespective of the tube supplier.These outcomes may help to improve commercial products in DNA/RNA extraction kits, besides raising awareness about the optimal choice of additives, offering opportunities for acquiring a better understanding of topics such as vertical microbial characterisation and environmental DNA recovery in low biomass samples. |
| Databáze: | OpenAIRE |
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