Directional High-Throughput Sequencing of RNAs Without Gene-Specific Primers

Mäki, Anita; Tiirola, Marja

doi:10.2144/btn-2018-0082

Cited by 11 publications

(13 citation statements)

References 14 publications

(14 reference statements)

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“…In addition, since sequencing results rely on robust genetic markers, sequences can be retained, and their affiliation can be refined later when more reference sequences are available in databases. Here, we applied the recently developed HTS method (Mäki and Tiirola, 2018) for SSU rRNA sequencing of phytoplankton samples. In this study, SSU rRNA was chosen as the target gene because it is an established molecular marker for phylogenetic identification of microbial communities (Woese, 1987;Vinje et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…The library preparation was done as described by Mäki and Tiirola (2018). Briefly, RNA extractions were run in a precast 1% agarose E-Gel EX gel (Invitrogen, US) and 16S/18S rRNA fragments were cut from the gel using a flamed scalpel and then purified using the Zymoclean Gel RNA Recovery Kit (Zymo Research, US).…”

Section: Rna Extraction Library Preparation and Sequencingmentioning

confidence: 99%

“…In addition to poly(A) tail use, the RNA-based HTS library construction may also be based on ligation of sequencing adapters to enable library amplification (Linnarsson, 2010). In a novel library construction method, ligation of the RNA oligo (M13) to the 5′-end of the rRNAs enabled simultaneous primer-independent HTS of rRNAs from prokaryotic and eukaryotic species (Mäki and Tiirola, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we compared the results of the phytoplankton community composition obtained by the novel HTS method (Mäki and Tiirola, 2018) with conventional light microscopy, and evaluated the coverage of the HTS method. The major advantage of simultaneous sequencing of 16S and 18S rRNA is that it allows the detection of all phytoplankton taxa, i.e., prokaryotic cyanobacteria and eukaryotic phytoplankton without bias due to copy number variation and unequal primer match.…”

Section: Introductionmentioning

confidence: 99%

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Consistency of Targeted Metatranscriptomics and Morphological Characterization of Phytoplankton Communities

et al. 2020

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The composition of phytoplankton community is the basis for environmental monitoring and assessment of the ecological status of aquatic ecosystems. Community composition studies of phytoplankton have been based on time-consuming and expertise-demanding light microscopy analyses. Molecular methods have the potential to replace microscopy, but the high copy number variation of ribosomal genes and the lack of universal primers for simultaneous amplification of prokaryotic and eukaryotic genes complicate data interpretation. In this study, we used our previously developed directional primerindependent high-throughput sequencing (HTS) approach to analyze 16S and 18S rRNA community structures. Comparison of 83 boreal lake samples showed that the relative abundances of eukaryotic phytoplankton at class level and prokaryotic cyanobacteria at order level were consistent between HTS and microscopy results. At the genus level, the results had low correspondence, mainly due to lack of sequences in the reference library. HTS was superior to identify genera that are extensively represented in the reference databases but lack specific morphological characteristics. Targeted metatranscriptomics proved to be a feasible method to complement the microscopy analysis. The metatranscriptomics can also be applied without linking the sequences to taxonomy. However, direct indexing of the sequences to their environmental indicator values needs collections of more comprehensive sample sets, as long as the coverage of molecular barcodes of eukaryotic species remains insufficient.

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Section: Discussionmentioning

confidence: 99%

Section: Rna Extraction Library Preparation and Sequencingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Consistency of Targeted Metatranscriptomics and Morphological Characterization of Phytoplankton Communities

et al. 2020

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“…A small number of studies investigating primer-free approaches to characterise 16S rRNA transcripts revealed better accuracy (Mäki and Tiirola, 2018) and sensitivity (Hoshino and Inagaki, 2013) with primer-free approaches for amplicon sequencing than PCR of the cDNA.…”

Section: Introductionmentioning

confidence: 99%

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

Cholet

Ijaz

Smith

2020

Preprint

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SummaryRT-Q-PCR, and RT-PCR amplicon sequencing, provide a convenient, target-specific, high-sensitivity approach for gene expression studies and are widely used in environmental microbiology. Yet, the effectiveness and reproducibility of the reverse transcription step has not been evaluated. Therefore, we tested a combination of four commercial reverse transcriptases with two priming techniques to faithfully transcribe 16S rRNA and amoA transcripts from marine sediments. Both enzyme and priming strategy greatly affected quantification of the exact same target with differences of up to 600-fold. Furthermore, the choice of RT system significantly changed the communities recovered. For 16S rRNA, both enzyme and priming had a significant effect with enzyme having a stronger impact than priming. Inversely, for amoA only the change in priming strategy resulted in significant differences between the same sample. Specifically, more OTUs and better coverage of amoA transcripts diversity were obtained with GS priming indicating this approach was better at recovering the diversity of amoA transcripts. Moreover, sequencing of RNA mock communities revealed that, even though transcript α diversities (i.e. OTU counts within a sample) can be biased by the RT, the comparison of β diversities (i.e. differences in OTU counts between samples) is reliable as those biases are reproducible between environments.Originality-Significance StatementIs the complementary DNA (cDNA) produced after Reverse Transcription (RT) a faithful representation of the starting RNA? This is a fundamental and important question for transcriptomic-based studies in environmental microbiology that aim to quantify and/or examine the diversity of transcripts via RT approaches. Yet little is known about the reliability and reproducibility of this step. Here, we evaluated the effect of the two main components of the RT reaction – the retro transcriptase enzyme and priming strategy (gene specific vs random priming), on the quantification and diversity of cDNA. We found that both have a significant impact. We further provide evidence to enable informed choices as to the enzyme and priming combinations to improve the performance of RT-PCR approaches. Taken together, this work will improve the reliability and reproducibility of transcript-based studies in environmental microbiology.

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Construction of Metatranscriptomic Libraries for 5′ End Sequencing of rRNAs for Microbiome Research

Tiirola

Mäki

2021

Microbial Systems Biology

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Directional High-Throughput Sequencing of RNAs Without Gene-Specific Primers

Cited by 11 publications

References 14 publications

Consistency of Targeted Metatranscriptomics and Morphological Characterization of Phytoplankton Communities

Consistency of Targeted Metatranscriptomics and Morphological Characterization of Phytoplankton Communities

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

Construction of Metatranscriptomic Libraries for 5′ End Sequencing of rRNAs for Microbiome Research

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