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

High-throughput sequencing techniques were used to analyze the diversity of respiratory microbiota in health and disease. Despite the extensive data available on the respiratory tract bacterial microbiota, the remnants of the less studied fungal components. This is partly due to the technical problems associated with fungal metagenomics analysis. In this study, we compared two protocols of DNA extraction and amplification of two targets for the combined fungal bacteria and fungi that are targeted amplicon sequencing analysis of sputum microbiota.Six breathing, selected randomly from a sample routine Mondor Hospital (Créteil, France) and treated anonymously, were tested after a routine culture of bacteria and fungi.

Two of spiked Fumigati Aspergillus and Aspergillus Nigri (105 conidia / ml). After the mechanical lysis, DNA was extracted using the automatic extraction of QIAsymphony® (AQE) or users PowerSoil® MoBio extraction (MPE). DNA yield and purity compared. DNA extracted from sputum spiked into the target (i) real-time PCR for detection of Aspergillus DNA and (ii) a combination of primary targeting barcode metagenomic analysis for ITS1 and ITS2 fungi, and bacteria V1-V2 and V3-V4 region of the 16S. amplicon libraries were prepared using reagents MiSeq V3 kit on the Illumina platform. Data were analyzed using software PyroMIC © and Shaman, and compared with culture results.AQE extraction provides a higher yield of the (ratio AQE / MPE DNA = 4.5 [1.3 to 11]) DNA in a shorter period of time.

Results of Aspergillus DNA is detected by qPCR were similar to sputum spiked regardless of the extraction protocol. extraction quite an impact on biodiversity or the relative abundance of bacterial communities using targeted amplicon sequencing (2/43 taxa impact). For fungi, the relative abundance of major taxa 4/11 affected and AQE results were closer to the culture results. V1-V2 or V3-V4 and ITS1 or ITS2 target is judged diversity major taxa of bacteria and fungi, but ITS2 and V3-V4 detected smaller taxa.Our results show the importance of joint extraction of DNA for bacteria and fungi targeted metagenomics respiratory samples. Extraction protocol may affect the results of the DNA and the relative abundance of a few bacteria, but more fungal taxa. For the analysis of fungi, ITS2 allow detection of a large number of taxa small compared with ITS1.

ACTG1 human mutation associated with high frequency hearing loss, and patients with mutations in this gene are good candidates for the electric acoustic stimulation. To better understand the genetic etiology of hearing loss cases, massively parallel DNA sequencing performed on a 7048 Japanese probands related hearing loss. Among the 1,336 patients with autosomal dominant hearing loss, we identified 15 probands (1.1%) with 13 ACTG1 potentially pathogenic variant. Six new variant and seven were reported previously.

We collect and analyze detailed clinical features of these patients. The average level of development of pure-tone hearing worsening of the average for the four frequency is 1.7 dB / year of age 0-50 years, and all individuals over 60 years old has a severe hearing loss. To better understand the mechanisms underlying causes of disease, the intracellular localization of the wild-type and mutant gamma-actins checked using NIH / 3T3 fibroblast cell line

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