TY - JOUR
T1 - Comparison of different conditions for DNA extraction in sputum - A pilot study
AU - Oriano, Martina
AU - Terranova, Leonardo
AU - Teri, Antonio
AU - Sottotetti, Samantha
AU - Ruggiero, Luca
AU - Tafuro, Camilla
AU - Marchisio, Paola
AU - Gramegna, Andrea
AU - Amati, Francesco
AU - Nava, Fabrizio
AU - Franceschi, Elisa
AU - Cariani, Lisa
AU - Blasi, Francesco
AU - Aliberti, Stefano
PY - 2019/1/31
Y1 - 2019/1/31
N2 - Background: The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis. Methods: Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and S. aureus and, then, microbiome was evaluated. Results: Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for S. aureus was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT. Conclusions: None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield.
AB - Background: The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis. Methods: Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and S. aureus and, then, microbiome was evaluated. Results: Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for S. aureus was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT. Conclusions: None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield.
KW - DNA extraction
KW - Microbiome
KW - Microbiota
KW - Sequencing
KW - Sputum
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U2 - 10.1186/s40248-018-0166-z
DO - 10.1186/s40248-018-0166-z
M3 - Article
AN - SCOPUS:85060873981
VL - 14
JO - Multidisciplinary Respiratory Medicine
JF - Multidisciplinary Respiratory Medicine
SN - 1828-695X
IS - 1
M1 - 6
ER -