31
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      A 16S rRNA gene sequencing and analysis protocol for the Illumina MiniSeq platform

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          High‐throughput sequencing of the 16S rRNA gene on the Illumina platform is commonly used to assess microbial diversity in environmental samples. The MiniSeq, Illumina's latest benchtop sequencer, enables more cost‐efficient DNA sequencing relative to larger Illumina sequencing platforms ( e.g., MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high‐throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed additional sequencing primers that enabled application of a dual‐index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples in four different sequencing runs as a quality control benchmark. We were able to recapture a realistic richness of the mock community in all sequencing runs, and identify meaningful differences in alpha and beta diversity in the environmental samples. Furthermore, rarefaction analysis indicated diversity in many environmental samples was close to saturation. These results show that the MiniSeq can produce similar quantities of high‐quality V4 reads compared to the MiSeq, yet is a cost‐effective option for any laboratory interested in performing high‐throughput 16S rRNA gene sequencing.

          Related collections

          Most cited references7

          • Record: found
          • Abstract: found
          • Article: not found

          Microbial population structures in the deep marine biosphere.

          The analytical power of environmental DNA sequences for modeling microbial ecosystems depends on accurate assessments of population structure, including diversity (richness) and relative abundance (evenness). We investigated both aspects of population structure for microbial communities at two neighboring hydrothermal vents by examining the sequences of more than 900,000 microbial small-subunit ribosomal RNA amplicons. The two vent communities have different population structures that reflect local geochemical regimes. Descriptions of archaeal diversity were nearly exhaustive, but despite collecting an unparalleled number of sequences, statistical analyses indicated additional bacterial diversity at every taxonomic level. We predict that hundreds of thousands of sequences will be necessary to capture the vast diversity of microbial communities, and that different patterns of evenness for both high- and low-abundance taxa may be important in defining microbial ecosystem dynamics.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            A renaissance for the pioneering 16S rRNA gene.

            Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the past quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata, and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: found
              Is Open Access

              Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer

              Background The miniaturised and portable DNA sequencer MinION™ has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinION™ system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences. Results Using R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90 % of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using ‘universal’ primers. Conclusions Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinION™ DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.
                Bookmark

                Author and article information

                Contributors
                w.orsi@lrz.uni-muenchen.de
                Journal
                Microbiologyopen
                Microbiologyopen
                10.1002/(ISSN)2045-8827
                MBO3
                MicrobiologyOpen
                John Wiley and Sons Inc. (Hoboken )
                2045-8827
                25 March 2018
                December 2018
                : 7
                : 6 ( doiID: 10.1002/mbo3.2018.7.issue-6 )
                : e00611
                Affiliations
                [ 1 ] Department of Earth and Environmental Sciences, Paleontology & Geobiology Ludwig‐Maximilians‐Universität München Munich Germany
                [ 2 ] GeoBio‐CenterLMU Ludwig‐Maximilians‐Universität München Munich Germany
                [ 3 ] SNSB ‐ Bayerische Staatssammlung für Paläontologie und Geologie Munich Germany
                Author notes
                [*] [* ] Correspondence

                William D. Orsi, Department of Earth and Environmental Sciences, Paleontology & Geobiology, Ludwig‐Maximilians‐Universität München, Munich, Germany.

                Email: w.orsi@ 123456lrz.uni-muenchen.de

                Author information
                http://orcid.org/0000-0002-6380-7421
                http://orcid.org/0000-0001-8704-1339
                http://orcid.org/0000-0002-4094-5637
                Article
                MBO3611
                10.1002/mbo3.611
                6291791
                29575567
                cebe4579-dc51-4ac4-8a62-2f9ad07cf04e
                © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 13 December 2017
                : 29 January 2018
                : 29 January 2018
                Page count
                Figures: 3, Tables: 2, Pages: 9, Words: 6625
                Funding
                Funded by: Deutsche Forschungsgemeinschaft
                Award ID: OR 417/1‐1
                Categories
                Original Research
                Original Articles
                Custom metadata
                2.0
                mbo3611
                December 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.5.4 mode:remove_FC converted:13.12.2018

                Microbiology & Virology
                16s rrna gene,high‐throughput sequencing,illumina,microbial diversity

                Comments

                Comment on this article