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Alberti, A. et al. Comparison of library preparation methods reveals their impact on interpretation of metatranscriptomic data. BMC Genomics (2014). doi:10.1186/1471-2164-15-912
The authors compared four library preparation methods for the interpretation of metatranscriptomic data: SMARTer Stranded RNA-Seq Kit (Takara Bio), Ovation Encore Complete Prokaryotic (NuGEN), Ovation RNA-Seq v2 (NuGEN), and TruSeq® Stranded Total RNA (Illumina). They found the SMARTer Stranded RNA-Seq Kit to be the best solution for smaller amounts of RNA, as it was the fastest, least biased, and most reproducible method for low-input mixed microbial samples.
de Oliveira, L. et al. Transcriptomic analysis of the red seaweed Laurencia dendroidea (Florideophyceae, Rhodophyta) and its microbiome. BMC Genomics 13, 487 (2012).
The authors performed simultaneous RNA-seq on seaweed and its microbiome and showed the importance of the red seaweed's microbiome in the breakdown of organic matter and production of nitrogen. The seaweed transcriptome also contained transcripts related to terpene biosynthesis, a building block for a variety of functionally active small molecules. The use of the SMARTer Stranded RNA-Seq Kit enabled interrogation of both the algae and the microbiome in the same preparation.
Hugerth, L. W. et al. Metagenome-assembled genomes uncover a global brackish microbiome. Genome Biol. 16, 279 (2015).
In this study, flow cells were attached to boreholes containing either modern marine or old saline waters of different origins and degrees of isolation from the light-driven surface of the earth. Using 16S rRNA gene sequencing, the authors showed that planktonic and attached populations were dissimilar and that gene frequencies in the metagenomes suggested that hydrogen-fed, carbon dioxide- and nitrogen-fixing populations were responsible for biofilm formation across the two aquifers. Metagenome libraries of extracted DNA from garnet grains were prepared using the ThruPLEX DNA-seq Kit with 96 dual indexes.
Hughes, L. C. et al. Transcriptomic differentiation underlying marine-to-freshwater transitions in the South American silversides Odontesthes argentinensis and O. bonariensis (Atheriniformes). Ecol. Evol. 7, 5258–5268 (2017).
The authors studied the transcriptome and microbiome differences in fish species that move from the ocean to inland rivers (marine to freshwater transitions). Few species are able to tolerate the extreme change in salt concentration between ocean water and fresh water. This study characterized the differences in transcriptional profiles and microbiome content of two species that make this remarkable transition. The team of researchers were able to simultaneously interrogate both the fish transcriptome and the microbiome of the fish gill in the same prep and sequencing experiment. The random priming approach of the SMARTer Stranded RNA-Seq Kit enabled this study.
Katrine, J. Antimicrobial resistance in selected environments in Norway: occurrence of antimicrobial resistant bacteria (ARB) and antimicrobial resistant genes (ARG) associated with wastewater treatment plants (WWTPs). (2017). www.genok.no
The authors investigated the prevalence of antibiotic-resistant bacteria and antibiotic-resistant genes in wastewater treatment plants in two locations in Norway using culture-based methods with molecular techniques and metagenomics studies. The authors aimed to provide a better understanding of the identification of the relationships between antimicrobial resistance (AMR) in the environment and the spread of AMR, not only from clinical settings to the environment but also from the environment to human and animal pathogens. Samples were immediately frozen to –20°C and kept frozen until DNA extraction. DNA samples were sent to the Norwegian Sequencing Centre (NSC) at the University of Oslo for metagenomic library preparation using the ThruPLEX DNA-Seq kit and sequencing on an Illumina MiSeq® PE300 platform.
Marynowska, M. et al. Optimization of a metatranscriptomic approach to study the lignocellulolytic potential of the higher termite gut microbiome. BMC Genomics 18, 681 (2017).
The authors used two different microbe transcript enrichment approaches to examine the microbiome content of the termite gut. They optimized a workflow to isolate RNA and prepare libraries from a very challenging sample type: the termite gut microbiome, which plays a critical role in breaking down complex molecules from plant matter. The SMARTer Stranded RNA-Seq Kit enabled use of low inputs of enriched RNA, with excellent transcriptome representation.
Sureshchandra, S. et al. Inflammatory determinants of pregravid obesity in placenta and peripheral blood. Front. Physiol. 9, 1089 (2018).
The researchers compared maternal cytokines and placental gene expression in lean and obese pregnant women in order to understand the biological underpinnings of a higher rate of complications in obese pregnant women. They looked at the levels of immune system signaling molecules (cytokines and growth factors), maternal gene expression, and placental microbiome gene expression. The study helped better connect the impact of obesity on poor pregnancy outcomes by linking expressed genes with the small molecules that act to signal immune responses. The study helped support the hypothesis that pregravid obesity is associated with a heightened systemic inflammation and dysregulation of nutrient transport for the fetus. For gene expression, they were able to simultaneously interrogate both the maternal gene expression profile and the microbiome transcriptome in a single library prep with the SMARTer Stranded RNA-Seq system. Maternal ribosomal RNA was depleted using a RiboGone kit.
Wu, X. et al. Potential for hydrogen-oxidizing chemolithoautotrophic and diazotrophic populations to initiate biofilm formation in oligotrophic, deep terrestrial subsurface waters. Microbiome 5, 37 (2017).
The authors reconstructed a large number of bacterioplankton genomes from a metagenomic time series from the Baltic Sea. Water samples were collected on 37 occasions between March and December of 2012, at 2-m depth, at the LMO, 10 km off the coast of Öland (Sweden), using a Ruttner sampler. All samples are referred to in the text and figures by their sampling date, in the format yymmdd. Samples were filtered successively at 3.0 μm and 0.22 μm. The 0.22-μm fraction was used for DNA extraction. The procedures for DNA extraction, phytoplankton counts, and measurement of chlorophyll a and other nutrients are described. DNA (2–10 ng) from each sample was prepared with the ThruPLEX DNA-Seq Kit according to the instructions of the manufacturer. Cleaning steps were performed with MyOne carboxylic acid-coated superparamagnetic beads (Invitrogen). Finished libraries were sequenced in SciLifeLab/NGI (Solna, Sweden) on an Illumina HiSeq® 2500. On average, 31.9 million paired-end reads of 2 × 100 bp were generated.
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