Abstract
BackgroundNext 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.MethodsA 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.ResultsOur 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.ConclusionsThe 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.
Highlights
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
Natural aquatic environments are the major reservoir for Legionella species, with aerosol-generating man-made water systems such as cooling towers, drinking water supply systems (DWSS) and recreational waters being the main sources of human exposure to Legionella species [5]
Sequence accuracy and quality of generated Next Generation Sequencing (NGS) libraries In order to minimise error rate, validation of our deep sequencing approach was performed with proofreading DNA polymerase KAPA HiFi and compared with the extensively used HotStarTaq DNA polymerase
Summary
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. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections. In medical and environmental diagnostics the prime interest often lays in pathogenic or potentially pathogenic microorganisms We will call this part of the microbiome “patho-microbiome” and it can consist of viruses, bacteria and lower eukaryotes. Despite the importance of assessing the total pathomicrobiome, the methodologies to address all major pathogenic microorganisms are very complex [1]. To make such an enterprise more amenable to a straight forward methodological approach, we have chosen the genus Legionella as a subject, which is relevant for medical and environmental microbiologists and taxonomically well defined. L. longbeachae infections are commonly linked with the presence of the bacteria in potting soils, instead of freshwater environments, with a high frequency of reported cases in Australia [6]
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