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      Microbial single-cell omics: the crux of the matter

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          Abstract

          Abstract

          Single-cell genomics and transcriptomics can provide reliable context for assembled genome fragments and gene expression activity on the level of individual prokaryotic genomes. These methods are rapidly emerging as an essential complement to cultivation-based, metagenomics, metatranscriptomics, and microbial community-focused research approaches by allowing direct access to information from individual microorganisms, even from deep-branching phylogenetic groups that currently lack cultured representatives. Their integration and binning with environmental ‘omics data already provides unprecedented insights into microbial diversity and metabolic potential, enabling us to provide information on individual organisms and the structure and dynamics of natural microbial populations in complex environments. This review highlights the pitfalls and recent advances in the field of single-cell omics and its importance in microbiological and biotechnological studies.

          Key points

          • Single-cell omics expands the tree of life through the discovery of novel organisms, genes, and metabolic pathways.

          • Disadvantages of metagenome-assembled genomes are overcome by single-cell omics.

          • Functional analysis of single cells explores the heterogeneity of gene expression.

          • Technical challenges still limit this field, thus prompting new method developments.

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          Most cited references88

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          Opportunities and challenges in long-read sequencing data analysis

          Long-read technologies are overcoming early limitations in accuracy and throughput, broadening their application domains in genomics. Dedicated analysis tools that take into account the characteristics of long-read data are thus required, but the fast pace of development of such tools can be overwhelming. To assist in the design and analysis of long-read sequencing projects, we review the current landscape of available tools and present an online interactive database, long-read-tools.org, to facilitate their browsing. We further focus on the principles of error correction, base modification detection, and long-read transcriptomics analysis and highlight the challenges that remain.
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            Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification.

            We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and phi29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.
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              Microbial community gene expression in ocean surface waters.

              Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species-including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition-and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction ( approximately 50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.
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                Author and article information

                Contributors
                kaster@kit.edu
                Journal
                Appl Microbiol Biotechnol
                Appl. Microbiol. Biotechnol
                Applied Microbiology and Biotechnology
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                0175-7598
                1432-0614
                26 August 2020
                26 August 2020
                2020
                : 104
                : 19
                : 8209-8220
                Affiliations
                [1 ]GRID grid.7892.4, ISNI 0000 0001 0075 5874, Institute for Biological Interfaces 5, , Karlsruhe Institute of Technology, ; Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany
                [2 ]GRID grid.7892.4, ISNI 0000 0001 0075 5874, Institute for Applied Biosciences, , Karlsruhe Institute of Technology, ; Fritz-Haber-Weg 4, D-76131 Karlsruhe, Germany
                Author information
                http://orcid.org/0000-0002-6361-9551
                Article
                10844
                10.1007/s00253-020-10844-0
                7471194
                32845367
                c42072bf-3713-42e5-9216-9abee2807352
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 15 April 2020
                : 8 August 2020
                : 17 August 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: 320579085
                Award Recipient :
                Categories
                Mini-Review
                Custom metadata
                © Springer-Verlag GmbH Germany, part of Springer Nature 2020

                Biotechnology
                targeted cell sorting,amplification,bioinformatics,bacteria,archaea
                Biotechnology
                targeted cell sorting, amplification, bioinformatics, bacteria, archaea

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