English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Background  Next Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus  Legionella,  comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections.    Methods  A new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus  Legionella  using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples.    Results  Our results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high  in situ  specificity (>95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of  L. pneumophila  along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different  Legionella  species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including  L. pneumophila . The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of  Legionella  species, i. e. the complete  Legionella  microbiome, without the need for species-specific primers.    Conclusions  The developed NGS approach provides a new molecular surveillance tool to monitor all  Legionella  species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus-specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way.    Electronic supplementary material  The online version of this article (doi:10.1186/s12866-017-0987-5) contains supplementary material, which is available to authorized users.

bmc microbiology

Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems