Comparison of different conditions for DNA extraction in sputum - a pilot study

Martina Oriano, Leonardo Terranova, Antonio Teri, Samantha Sottotetti, Luca Ruggiero, Camilla Tafuro, Paola Marchisio, Andrea Gramegna, Francesco Amati, Fabrizio Nava, Elisa Franceschi, Lisa Cariani, Francesco Blasi, Stefano Aliberti, Martina Oriano, Leonardo Terranova, Antonio Teri, Samantha Sottotetti, Luca Ruggiero, Camilla Tafuro, Paola Marchisio, Andrea Gramegna, Francesco Amati, Fabrizio Nava, Elisa Franceschi, Lisa Cariani, Francesco Blasi, Stefano Aliberti

Abstract

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.

Keywords: DNA extraction; Microbiome; Microbiota; Sequencing; Sputum.

Conflict of interest statement

The two IRB approvals for bronchiectasis and CF are the number #660, October 14th, 2014, and the 594_2016 bis, October 11th, 2016, respectively; both of them granted from the Ethical Committee Milano Area B. Written consent for the use of both clinical and biological data has been given by enrolled patients and following Hospital IRB approval.Not applicable.The authors declare that they have no competing interests. FB is Editor-in-Chief of Multidisciplinary Respiratory Medicine, whereas SA is Associate Editor of Multidisciplinary Respiratory Medicine.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Comparison between median (IQR) levels of DNA extraction yield across 18 evaluated conditions
Fig. 2
Fig. 2
Comparison between median (IQR) levels of 16 s rRNA gene Real-Time PCR across 18 evaluated conditions
Fig. 3
Fig. 3
Comparison between median levels (IQR) of Real-Time PCR for S. aureus in sputum samples and across the 18 evaluated conditions
Fig. 4
Fig. 4
Comparison of median levels of Alpha diversity across the 18 evaluated conditions expressed as Shannon index and relative effective number of species (ENOS) (a and b), and Simpson index and relative ENOS (c and d)
Fig. 5
Fig. 5
Relative abundances of bacterial genera in each of the 5 sputum samples across the 18 evaluated conditions

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