Metagenomic 16S rDNA Illumina tags are a powerful alternative to amplicon sequencing to explore diversity and structure of microbial communities

Abstract

Sequencing of 16S rDNA polymerase chain reaction (PCR) amplicons is the most common approach for investigating environmental prokaryotic diversity, despite the known biases introduced during PCR. Here we show that 16S rDNA fragments derived from Illumina-sequenced environmental metagenomes (mi tags) are a powerful alternative to 16S rDNA amplicons for investigating the taxonomic diversity and structure of prokaryotic communities. As part of the Tara Oceans global expedition, marine plankton was sampled in three locations, resulting in 29 subsamples for which metagenomes were produced by shotgun Illumina sequencing (ca. 700 Gb). For comparative analyses, a subset of samples was also selected for Roche-454 sequencing using both shotgun (m454 tags; 13 metagenomes, ca. 2.4 Gb) and 16S rDNA amplicon (454 tags; ca. 0.075 Gb) approaches. Our results indicate that by overcoming PCR biases related to amplification and primer mismatch, mi tags may provide more realistic estimates of community richness and evenness than amplicon 454 tags. In addition, mi tags can capture expected beta diversity patterns. Using mi tags is now economically feasible given the dramatic reduction in high-throughput sequencing costs, having the advantage of retrieving simultaneously both taxonomic (Bacteria, Archaea and Eukarya) and functional information from the same microbial community.

An evaluation of the applicability of microarrays for monitoring toxic algae in Irish coastal waters

“…Entire genomes can now be sequenced using a new technology called Next Generation Sequencing (NGS) which can generate extensive transciptome data of prokaryotes and eukaryotes in a fast and cost effective way (Logares et al 2013;Zhan et al 2013;Kim et al 2014). There are a number of NGS platforms such as 454 pyrosequencing, illumina, SOLiD, HiSeq and MiSeq, PacBio RS all of which use multiple PCR amplification strategies which has been applied in diversity studies of microbial eukaryotes (Kim et al 2014).…”

“…This approach generates sequence data by matching a single nucleotide to its complementary base pair from a single strand of DNA fragment and detects which base was added at each step by the emission of light from the chemiluminescent enzymatic reaction of the DNA polymerase activity (Medlin and Kooistra 2010). The use of NGS technologies have rapidly increased in recent years and have provided many novel insights into taxonomy, phylogeny and evolutionary history of photosynthetic eukaryotes which can overcome traditional PCR biases related to amplification and primer mismatch, providing a more realistic estimation of community richness (Logares et al 2013;Kim et al 2014). NGS also has a great potential for future work in both discrimination and quantification of microalgae worldwide ).…”

“…Toxin results: Multi SPR and ELISA , high-throughput sequencing technologies (Logares et al, 2013;Zhan et al, 2013) and microarrays .…”

The 16S rRNA gene in the study of marine microbial communities

“…It comprises 356 datasets of sequences from the V6 region of the bacterial 16S rRNA gene obtained from studies of 40 different biomes, from pelagic and benthic regions to mangrove forests and sponge-associated bacteria (Amaral-Zettler et al 2010, Karsenti et al 2011, Zinger et al 2012. Based on studies of samples collected at 210 diverse oceanic sites and depths of 2000 m, the multinational consortium Tara Oceans has generated the Ocean Microbial Reference Gene Catalog comprising 35,650 taxonomically different organisms (Karsenti et al 2011, Logares et al 2014, Sunagawa et al 2015.…”

“…Comprende 356 bases de datos de secuencias de la región V6 del gen rRNA 16S bacteriano recabados de estudios de 40 biomas diferentes, desde regiones pelágicas y bentónicas hasta manglares y comunidades bacterianas asociadas a esponjas (Amaral-Zettler et al 2010, Karsenti et al 2011, Zinger et al 2012. Con base en estudios de muestras de recolectadas de 210 sitios oceánicos distintos y a profundidades de hasta 2000 m, el consorcio multinacional Tara Oceans ha generado un catálogo de genes de referencia (Ocean Microbial Reference Gene Catalog) de 35,650 microbios oceánicos taxonómicamente diferentes (Karsenti et al 2011, Logares et al 2014, Sunagawa et al 2015.…”