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

Abstract

BackgroundThe 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.MethodsFive 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.ResultsAlthough 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.ConclusionsNone 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.Electronic supplementary materialThe online version of this article (10.1186/s40248-018-0166-z) contains supplementary material, which is available to authorized users.

Age and the aging process significantly alter the small bowel microbiome

“…Microbial recovery from small intestinal luminal contents is made challenging by the viscosity of small intestinal mucus, small sample volumes, and low microbial biomass. To overcome this, we developed and validated techniques that allowed for greater recovery of small intestinal microbes, as well as improved DNA isolation and library preparation for 16S rRNA sequencing (Leite et al, 2019), including treating small intestinal aspirates with dithiothreitol (DTT), which can reduce the disulfide bonds linking mucin subunits and which has previously been used to liquefy sputum samples for DNA extraction (Oriano et al, 2019). These validated techniques were also used in the present study, allowing us to Cell Reports 36, 109765, September 28, 2021 7 Article ll OPEN ACCESS explore how small intestinal microbial populations change with age.…”

Deciphering the Ecology of Cystic Fibrosis Bacterial Communities: Towards Systems-Level Integration

“…Finally, there are a number of methodological factors to consider when assessing influences on any microbiota evaluation. For example, different methods for extraction [55,56] and sequencing of DNA [57] can yield different microbiota profiles, with effect sizes often similar to those of the biological factors being studied [58]. Respiratory sample type (sputa, swabs, bronchoalveolar lavage fluid, protected brush sampling, lung explant) can also influence calculated community structure, not only because they may reflect different airway locations, but also because different sampling methods tend to be used in the various life and disease stages of patients with CF (e.g., swabs are used most often in children, sputum tends to be produced at later stages, and lung explants usually reflect end-stage disease) [43,59].…”

Optimizing microbiome sequencing for small intestinal aspirates: validation of novel techniques through the REIMAGINE study

“…Although standards have been set for the assessment of the stool microbiome, these standards have not been assessed for small intestinal fluid assessment. Mucous in general is a viscous fluid that can trap bacteria in its matrix and previous studies performed with sputum samples have shown that treating this viscosity has an impact on the microbial assessment [5,16]. However, until now, no studies have investigated the impact on microbial assessment and DNA recovery in aspirates collected from small bowel.…”

“…Viscosity of the small bowel mucous could impede or affect DNA isolation. One possible remedy would be to reduce viscosity prior to DNA isolation by treating the aspirates with dithiothreitol (DTT), which is commonly used to reduce the disulfide bonds between cysteine residues of proteins and can also reduce the disulfide bonds linking mucin subunits in mucus, improving bacterial recovery and DNA extraction methods [5]. DTT has previously been used to liquefy sputum samples for DNA extraction [5].…”