Modified RNA-seq method for microbial community and diversity analysis using rRNA in different types of environmental samples

Yan, Yonggang; Zou, Bin; Zhu, Ting; Hozzein, Wael N.; Quan, Zhe‐Xue

doi:10.1371/journal.pone.0186161

Cited by 12 publications

(14 citation statements)

References 72 publications

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“…We modified metatranscriptome method which requires an immediate adaptor ligation step at the 5′ end of the RNA before reverse-transcription. This modified method is capable of constructing metatranscriptome library from total nucleic acid, and the little requirement for RNA quantities enables it can be used for various environmental samples ( Yan et al, 2017 ). The accuracy of this modified method was confirmed with mock communities, although random primers non-randomly hybridize to RNA template and the partial 16S rRNA sequences which only cover the regions of V1–V2 may cause some limitations ( Yan et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

“…A preliminary experiment had previously been designed and conducted to confirm that residual genomic DNA has no effect on library construction and data analysis even if the quantity of inputted DNA was several times higher than that of the RNA ( Yan et al, 2017 ). Therefore, each metatranscriptome library was constructed directly using 20 ng total RNA without a residual DNA removal step.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we developed a modified metatranscriptomic method that requires an immediate adaptor ligation step at the 5′ end of the RNA before it undergoes reverse-transcription. Using this method, we could obtain more 16S rRNA sequences of same regions (V1–V2) without the interference of DNA in order to analyze OTU-based microbial communities and diversity ( Yan et al, 2017 ). In addition, the construction of a total nucleic acid library based on diverse environmental samples, especially low-biomass samples with low RNA yields, was shown to be feasible ( Yan et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

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Microbial Communities and Diversities in Mudflat Sediments Analyzed Using a Modified Metatranscriptomic Method

et al. 2018

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Intertidal mudflats are land–sea interaction areas and play important roles in global nutrient cycles. However, a comprehensive understanding of microbial communities in these mudflats remains elusive. In this study, mudflat sediment samples from the Dongtan wetland of Chongming Island, the largest alluvial island in the world, were collected. Using a modified metatranscriptomic method, the depth-wise distributions of potentially active microbial communities were investigated based on small subunit ribosomal RNA (SSU rRNA) sequences. Multiple environmental factors were also measured and analyzed in conjunction with the prokaryotic composition profiles. A prokaryotic diversity analysis based on the metatranscriptome datasets revealed two or threefold higher diversity indices (associated with potentially active microbes participating in biogeochemical processes in Dongtan) compared with the diversity indices based on 16S rRNA gene amplicons. Bacteria were numerically dominant relative to archaea, and the potentially active prokaryotic taxa were mostly assigned to the bacterial phyla Chloroflexi, Acidobacteria, and Bacteroidetes and the classes Delta- and Gamma-proteobacteria, along with the archaeal lineages phylum Bathyarchaeota and the order Thermoplasmatales. The total nitrogen and carbon content of the sediment samples were environmental factors that significantly affected the depth-wise distributions of both bacterial and archaeal communities. Furthermore, the activity of potentially active taxa (including the prevalent order Desulfobacterales and family Anaerolineaceae) appeared to be significantly underestimated by PCR-based methods, notably at the DNA level, and indicates that using normal PCR amplification of DNA limits the study of potential microbial activity. This is the first study of potentially active microbial communities in depth-wise sediments from Dongtan. The improved knowledge of microbial communities in Dongtan provides a foundation for exploring biogeochemical cycling and microbial functions.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Microbial Communities and Diversities in Mudflat Sediments Analyzed Using a Modified Metatranscriptomic Method

et al. 2018

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“…Meta-transcriptomics using RNA-Sequencing is a powerful tool for interrogating transcribed genes in a sample [14] and can be utilised for microbial classification based on specific RNA transcripts [15].…”

Section: Introductionmentioning

confidence: 99%

MinION Sequencing of colorectal cancer tumour microbiomes – a comparison with amplicon-based and RNA-Sequencing

Taylor

Pearson

Miller

et al. 2019

Preprint

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Recent evidence suggests a role for the gut microbiome in the development and progression of colorectal cancer. In this study we compare MinION sequencing with meta-transcriptomics and amplicon-based sequencing for microbiome analysis of colorectal tumour tissue samples. DNA and RNA were extracted from 11 colorectal tumour samples. RNA-Sequencing, 16S rRNA amplicon sequencing and MinION genomic sequencing was carried out and resulting data used as input for taxonomic classification using Kraken2. Taxonomic concordance between the three platforms at different taxonomic levels was tested on a per sample basis. The average number of reads per sample using RNA-Sequencing was more than 129 times that generated using MinION sequencing. However, the average read length of MinION sequences was more than 13 times that of RNA or 16S rRNA amplicon sequencing. 16S sequencing was less informative beyond the genus level, and both RNA-Sequencing and MinION sequencing could detect more phyla and genera in the same samples, compared to 16S sequencing. Long-read sequences generated using MinION sequencing can compensate for low numbers of reads for bacterial classification. MinION sequencing can discriminate between bacterial strains and plasmids and shows potential as a tool for microbiome sequencing of colorectal cancers in a clinical setting.

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“…S.2). When testing a new approach for 16S rRNA transcript sequencing based on ligation of an adapter to the end of the gene prior to RT with random hexamers,Yan et al, 2017, found errors in the observed ratios of their RNA mock communities of up to 3-fold compared to the expected proportions. These results are comparable to those found in this study.…”

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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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Modified RNA-seq method for microbial community and diversity analysis using rRNA in different types of environmental samples

Cited by 12 publications

References 72 publications

Microbial Communities and Diversities in Mudflat Sediments Analyzed Using a Modified Metatranscriptomic Method

Microbial Communities and Diversities in Mudflat Sediments Analyzed Using a Modified Metatranscriptomic Method

MinION Sequencing of colorectal cancer tumour microbiomes – a comparison with amplicon-based and RNA-Sequencing

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

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