Dataset Information

Determining lineage-specific bacterial growth curves with a novel approach based on amplicon reads normalization using internal standard (ARNIS).

ABSTRACT: The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia coli cells added to samples just before biomass collection. This approach made it possible to reconstruct growth curves for 319 individual OTUs during the grazer-removal experiment conducted in a freshwater reservoir ?ímov. The high resolution data signalize significant functional heterogeneity inside the commonly investigated bacterial groups. For instance, many Actinobacterial phylotypes, a group considered to harbor slow-growing defense specialists, grew rapidly upon grazers' removal, demonstrating their considerable importance in carbon flow through food webs, while most Verrucomicrobial phylotypes were particle associated. Such differences indicate distinct life strategies and roles in food webs of specific bacterial phylotypes and groups. The impact of grazers on the specific growth rate distributions supports the hypothesis that bacterivory reduces competition and allows existence of diverse bacterial communities. It suggests that the community changes were driven mainly by abundant, fast, or moderately growing, and not by rare fast growing, phylotypes. We believe amplicon read normalization using internal standard (ARNIS) can shed new light on in situ growth dynamics of both abundant and rare bacteria.

SUBMITTER: Piwosz K

PROVIDER: S-EPMC6194029 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Determining lineage-specific bacterial growth curves with a novel approach based on amplicon reads normalization using internal standard (ARNIS).

Piwosz Kasia K Shabarova Tanja T Tomasch Jürgen J Šimek Karel K Kopejtka Karel K Kahl Silke S Pieper Dietmar H DH Koblížek Michal M

The ISME journal 20180706 11

The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia coli cells added to samples just before biomass ...[more]

PMID: 29980795

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Project description:High-throughput amplicon sequencing is a critical tool for studying microbiota; however, it results only in relative abundance data. Thus, changes in absolute abundance of microbiota cannot be determined, which hinders further microbiology research. We have therefore established a gradient internal standard absolute quantification (GIS-AQ) method to overcome this issue, which can simultaneously obtain the absolute abundances of bacteria and fungi. Deviations from the quantitative equations of microbes and internal standards were eliminated through calibration. Compared with traditional quantitative real-time PCR and microscopy quantifications, this method is reliable (R2average = 0.998; P < 0.001) and accurate (Pinternals versus microscopy > 0.05). The GIS-AQ method can be adapted to any amplicon primer choice (e.g., 336F/806R and ITS3/ITS4), rendering it applicable to ecosystem studies including food, soil, and water samples. Crucially, when using solid-state fermentation samples from various temporal dimensions, the results obtained from the relative and absolute abundance are different. The absolute abundance can be used to study the difference in communities between different samples, and the GIS-AQ method allows this to be done rapidly. Therefore, combining the absolute abundance with relative abundance can accurately reflect the microbiota composition.IMPORTANCE To solve the problem of amplicon sequencing cannot discern the microbiota absolute abundance, we proposed a gradient internal standard absolute quantification method. We used Chinese liquor fermentation as a model system to demonstrate the reliability and accuracy of the method. By comparing the relative and absolute abundances of microbiota in various temporal dimensions, we found dynamic changes in the absolute abundance of communities under various temporal dimensions from the relative abundance. Based on its design principle, this method can be widely applied to different ecosystems. Therefore, we believe that the GIS-AQ method can play an immeasurably useful role in microbiological research.

Dataset Information

Determining lineage-specific bacterial growth curves with a novel approach based on amplicon reads normalization using internal standard (ARNIS).

Publications

Determining lineage-specific bacterial growth curves with a novel approach based on amplicon reads normalization using internal standard (ARNIS).

Similar Datasets

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