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Preparation of Amplicon Libraries for Metabarcoding of Marine Eukaryotes Using Illumina MiSeq: The Adapter Ligation Method

Authors:
Matthieu Leray at Smithsonian Tropical Research Institute
Matthieu Leray
Quiterie Haenel at Genedata AG
Quiterie Haenel
Sarah Bourlat at Research Museum Alexander Koenig
Sarah Bourlat
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Amplicon-based studies of marine microscopic eukaryotes, also referred to as metabarcoding studies, can be performed to analyze patterns of biodiversity or predator–prey interactions targeting the mitochondrial cytochrome oxidase 1 (CO1) or the small ribosomal subunit (SSU) markers. Because high-throughput sequencing (HTS) Illumina platforms provide millions of reads per run, hundreds of samples may be sequenced simultaneously. This protocol details the preparation of multiplexed amplicon libraries for Illumina MiSeq sequencing. We describe a strategy for sample multiplexing using a combination of tailed PCR primers and ligation of indexed adapters.
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Full-text available
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Data
Full-text available
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Environmental Status Assessment Using DNA Metabarcoding: Towards a Genetics Based Marine Biotic Index (gAMBI)
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Article
Full-text available
  • Sep 2013
  • BMC Ecol
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Article
Full-text available
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  • ECOL LETT
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Article
Full-text available
Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next generation sequencing technologies from 454 and Illumina. As these technologies advance it is critical to assess the strengths, weaknesses and overall suitability of these platforms for the interrogation of microbial communities. Here we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nt reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in their length (i.e. V34, V4, and V45) by re-sequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we re-processed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
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Environmental Monitoring: Inferring the Diatom Index from Next-Generation Sequencing Data
Article
  • Jun 2015
  • ENVIRON SCI TECHNOL
Diatoms are widely used as bio-indicators for the assessment of water quality in rivers and streams. Classically, the diatom biotic indices are based on the relative abundance of morphologically identified species weighted by their autoecological value. Obtaining such indices is time-consuming, costly and requires excellent taxonomic expertise, which is not always available. Here we tested the possibility to overcome these limitations by using a next-generation sequencing (NGS) approach to identify and quantify diatoms found in environmental DNA and RNA samples. We analysed 27 river sites in the Geneva area (Switzerland), in order to compare the values of the Swiss Diatom Index (DI-CH) computed either by microscopic quantification of diatom species or directly from NGS data. Despite gaps in the reference database and variations in relative abundance of analysed species, the diatom index shows a significant correlation between morphological and molecular data indicating similar biological quality status for the majority of sites. This proof-of-concept study demonstrates the potential of NGS approach for identification and quantification of diatoms in environmental samples, opening new avenues towards the routine application of genetic tools for bioassessment and biomonitoring of aquatic ecosystems.
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