Combined bacterial and fungal targeted amplicon sequencing of respiratory samples: Does the DNA extraction method matter?

Cécile Angebault, Mathilde Payen, Paul-Louis Woerther, Christophe Rodriguez, Françoise Botterel, Cécile Angebault, Mathilde Payen, Paul-Louis Woerther, Christophe Rodriguez, Françoise Botterel

Abstract

Background: High-throughput sequencing techniques are used to analyse the diversity of the respiratory microbiota in health and disease. Although extensive data are available regarding bacterial respiratory microbiota, its fungal component remains poorly studied. This is partly due to the technical issues associated with fungal metagenomics analyses. In this study, we compared two DNA extraction protocols and two fungal amplification targets for combined bacterial and fungal targeted amplicon sequencing analyses of the respiratory microbiota.

Methods: Six sputa, randomly selected from routine samples in Mondor Hospital (Creteil, France) and treated anonymously, were tested after bacterial and fungal routine culture. Two of which were spiked with Aspergillus Fumigati and Aspergillus Nigri (105 conidia/mL). After mechanical lysis, DNA was extracted using automated QIAsymphony® extraction (AQE) or manual PowerSoil® MoBio extraction (MPE). DNA yield and purity were compared. DNA extracted from spiked sputa was subjected to (i) real-time PCR for Aspergillus DNA detection and (ii) combined metagenomic analyses targeting barcoded primers for fungal ITS1 and ITS2, and bacterial V1-V2 and V3-V4 16S regions. Amplicon libraries were prepared using MiSeq Reagent V3 kit on Illumina platform. Data were analysed using PyroMIC© and SHAMAN software, and compared with culture results.

Results: AQE extraction provided a higher yield of DNA (AQE/MPE DNA ratio = 4.5 [1.3-11]) in a shorter time. The yield of Aspergillus DNA detected by qPCR was similar for spiked sputa regardless of extraction protocol. The extraction moderately impacted the diversity or relative abundances of bacterial communities using targeted amplicon sequencing (2/43 taxa impacted). For fungi, the relative abundances of 4/11 major taxa were impacted and AQE results were closer to culture results. The V1-V2 or V3-V4 and ITS1 or ITS2 targets assessed similarly the diversity of bacterial and fungal major taxa, but ITS2 and V3-V4 detected more minor taxa.

Conclusion: Our results showed the importance of DNA extraction for combined bacterial and fungal targeted metagenomics of respiratory samples. The extraction protocol can affect DNA yield and the relative abundances of few bacterial but more fungal taxa. For fungal analysis, ITS2 allowed the detection of a greater number of minor taxa compared with ITS1.

Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: FB received grants from Astellas, and payment for lectures from Merck. CA received a travel grant from MSD France in 2017. MP, PLW, CR declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Workflow of the study.
Fig 1. Workflow of the study.
A- Comparison of two DNA extraction protocols (automated QIAsymphony extraction [AQE] using DSP DNA midi kit and manual PowerSoil® MoBio extraction [MPE]) in terms of quantity and quality of DNA extracted from 6 sputa (6 patients, P1, P2, P3, P4, P5, P6). B- Comparison of bacterial and fungal diversity detected in two sputa (2 patients, P1, P2) spiked with 105 conidia of Aspergillus section Fumigati and Aspergillus section Nigri using (i) culture method and (ii) 16S/ITS targeted amplicon sequencing (DNA extracted using AQE or MPE protocols; amplification targets V1-V2 and V3-V4 16S for bacteria, and ITS2 and ITS2 for fungi; MiSeq® Illumina, 2x300 v3 kit).
Fig 2. Taxa plots summarizing the relative…
Fig 2. Taxa plots summarizing the relative abundances of fungal genera or sections identified in the respiratory samples of 2 patients (P1 and P2) using culture and ITS targeted amplicon sequencing.
The sputa of P1 and P2 were spiked with 105 CFU/ml of Aspergillus section Nigri and Aspergillus section Fumigati. For ITS targeted amplicon sequencing analysis, DNA was extracted from the respiratory samples using 2 protocols: the automated QIAsymphony extraction [AQE] with DSP DNA midi kit, and the manual PowerSoil® MoBio extraction [MPE]; and amplified by ultra-deep sequencing (MiSeq®, Illumina) using two ITS targets (ITS1 and ITS2). Methods used the same starting quantities of samples and the final volumes were equal. Metagenomic analysis revealed 5 extremely low-represented genera (overall relative abundance < 0.1% for each taxon) which were gathered in “other genera” category: Rhizophlyctis, Schizophyllum, Hanseniaspora, Penicillium, and Inocutis.
Fig 3. Taxa plots summarizing the relative…
Fig 3. Taxa plots summarizing the relative abundances of bacterial genera identified in the respiratory samples of 2 patients (P1 and P2) using 16S targeted amplicon sequencing.
DNA was extracted using 2 protocols: the automated QIAsymphony extraction [AQE] with DSP DNA midi kit, and the manual PowerSoil® MoBio extraction [MPE]; and amplified by ultra-deep sequencing (MiSeq®, Illumina) using two 16S targets (V1-V2 and V3-V4).
Fig 4
Fig 4
Boxplot of log2 abundances of bacterial (A) and fungal (B) taxa at genus or section level detected with significantly different abundances (P-value < 0.05) according to the employed extraction protocol. DNA was extracted either by manual PowerSoil® MoBio extraction -MPE- [dark blue] or automated QIAsymphony extraction -AQE- using DSP DNA midi kit [light blue].

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