Dataset Information

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels.

ABSTRACT: The quantification of the total microbial content in metagenomic samples is critical for investigating the interplay between the microbiome and its host, as well as for assessing the accuracy and precision of the relative microbial composition which can be strongly biased in low microbial biomass samples. In the present study, we demonstrate that digital droplet PCR (ddPCR) can provide accurate quantification of the total copy number of the 16S rRNA gene, the gene usually exploited for assessing total bacterial abundance in metagenomic DNA samples. Notably, using DNA templates with different integrity levels, as measured by the DNA integrity number (DIN), we demonstrated that 16S rRNA copy number quantification is strongly affected by DNA quality and determined a precise correlation between quantification underestimation and DNA degradation levels. Therefore, we propose an input DNA mass correction, according to the observed DIN value, which could prevent inaccurate quantification of 16S copy number in degraded metagenomic DNAs. Our results highlight that a preliminary evaluation of the metagenomic DNA integrity should be considered before performing metagenomic analyses of different samples, both for the assessment of the reliability of observed differential abundances in different conditions and to obtain significant functional insights.

SUBMITTER: Manzari C

PROVIDER: S-EPMC7660251 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels.

Manzari Caterina C Oranger Annarita A Fosso Bruno B Piancone Elisabetta E Pesole Graziano G D'Erchia Anna Maria AM

Microbial genomics 20201001 10

The quantification of the total microbial content in metagenomic samples is critical for investigating the interplay between the microbiome and its host, as well as for assessing the accuracy and precision of the relative microbial composition which can be strongly biased in low microbial biomass samples. In the present study, we demonstrate that digital droplet PCR (ddPCR) can provide accurate quantification of the total copy number of the 16S rRNA gene, the gene usually exploited for assessing ...[more]

PMID: 32749951

Similar Datasets

Project description:The invasive American bullfrog (Lithobates catesbeianus) imperils freshwater biodiversity worldwide. Effective management hinges on early detection of incipient invasions and subsequent rapid response, as established populations are extremely difficult to eradicate. Although environmental DNA (eDNA) detection methods provide a highly sensitive alternative to conventional surveillance techniques, extensive testing is imperative to generate reliable output. Here, we tested and compared the performance of two primer/probe assays to detect and quantify the abundance of bullfrogs in Western Europe in silico and in situ using digital droplet PCR (ddPCR). Although both assays proved to be equally target-specific and sensitive, one outperformed the other in ddPCR detection resolution (i.e., distinguishing groups of target-positive and target-negative droplets), and hence was selected for further analyses. Mesocosm experiments revealed that tadpole abundance and biomass explained 99% of the variation in eDNA concentration. Because per individual eDNA emission rates did not differ significantly among tadpoles and juveniles, and adults mostly reside out of the water, eDNA concentration can be used as an approximation of local bullfrog abundance in natural populations. Seasonal eDNA patterns in three colonized ponds showed parallel fluctuations in bullfrog eDNA concentration. An increase in eDNA concentration was detected in spring, followed by a strong peak coinciding with the breeding season (August, September or October), and continuously low eDNA concentrations during winter. With this study, we report the validation process required for appropriately implementing eDNA barcoding analyses in lentic systems. We demonstrate that this technique can serve as a solid and reliable tool to detect the early stages of bullfrog invasions and to quantify temporal changes in abundance that will be useful in coordinating large-scale bullfrog eradication programs and evaluating their efficiency.

Project description:BackgroundSystemin has been extensively studied since it was discovered and is described as a peptidic hormone in tomato plants and other Solanaceae. Jasmonic acid and systemin are proposed to act through a positive feed-back loop with jasmonic acid, playing synergistic roles in response to both wounding and insect attack. Despite its biological relevance, most studies regarding the function of systemin in defence have been studied via PROSYSTEMIN (PROSYS) gene expression, which encodes the propeptide prosystemin that is later cleaved to systemin (SYS). Interestingly, hardly any studies have been based on quantification of the peptide.ResultsIn this study, a simple and accurate method for systemin quantification was developed to understand its impact on plant metabolism. The basal levels of systemin were found to be extremely low. To study the role of endogenous systemin on plant metabolism, systemin was quantified in a transgenic line overexpressing the PROSYS gene (PS+) and in a silenced antisense line (PS-). We evaluated the relevance of systemin in plant metabolism by analysing the metabolomic profiles of both lines compared to wildtype plants through untargeted metabolomic profiling. Compounds within the lignan biosynthesis and tyrosine metabolism pathways strongly accumulated in PS+ compared to wild-type plants and to plants from the PS- line. The exogenous treatments with SYS enhanced accumulation of lignans, which confirms the role of SYS in cell wall reinforcement. Unexpectedly, PS+ plants displayed wild-type levels of jasmonic acid (JA) but elevated accumulation of 12-oxo-phytodienoic acid (OPDA), suggesting that PS+ should not be used as an over-accumulator of JA in experimental setups.ConclusionsA simple method, requiring notably little sample manipulation to quantify the peptide SYS, is described. Previous studies were based on genetic changes. In our study, SYS accumulated at extremely low levels in wild-type tomato leaves, showed slightly higher levels in the PROSYSTEMIN-overexpressing plants and was absent in the silenced lines. These small changes have a significant impact on plant metabolism. SA and OPDA, but not JA, were higher in the PROSYS-overexpressing plants.

Dataset Information

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels.

Publications

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets