A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery

Fauver, Joseph R.; Akter, Shamima; Morales, Aldo Ivan Ortega; Black, William C.; Rodríguez, Américo D.; Stenglein, Mark D.; Ebel, Gregory D.; Weger-Lucarelli, James

doi:10.1016/j.virol.2018.12.020

“…Another option for highly fragmented RNA may be an enzymatic depletion method where rRNA is bound by tiling DNA probes and then subjected to RNAse H digestion. The RNAse H treatment is sometimes preceded by a reverse transcriptase step to extend the DNA/rRNA hybrids and is always followed by a DNAse treatment to remove the DNA probes (Fauver et al 2019;Morlan et al 2012). These steps lead to a longer depletion protocol and possibly more opportunities for RNA degradation or non-specific RNA targeting than Ribo-Pop.…”

Section: Discussionmentioning

confidence: 99%

Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion

Thompson

¹

,

Kiourlappou

²

,

Davis

³

2020

RNA

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The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises the vast majority of a total RNA sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-minute protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable "open source" design strategy would enable virtually any lab to pursue full transcriptome sequencing in their organism of interest with minimal time and resource investment.

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“…Another option for highly fragmented RNA may be an enzymatic depletion method where rRNA is bound by tiling DNA probes and then subjected to RNAse H digestion. The RNAse H treatment is sometimes preceded by a reverse transcriptase step to extend the DNA/rRNA hybrids and is always followed by a DNAse treatment to remove the DNA probes 23,24 . These steps lead to a longer depletion protocol and possibly more opportunities for RNA degradation or non-specific RNA targeting than Ribo-Pop.…”

Section: Discussionmentioning

confidence: 99%

Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion

Thompson

¹

,

Kiourlappou

²

,

Davis

³

2020

Preprint

View full text Add to dashboard Cite

The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises the vast majority of a total RNA sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20minute protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable "open source" design strategy would enable virtually any lab to pursue full transcriptome sequencing in their organism of interest with minimal time and resource investment. FIGURE 1. Short anti-sense oligos effectively deplete ribosomal RNA Oligonucleotides targeting the Drosophila small rRNA (18S) were individually tested for their ability to deplete the 18S transcript from larval total RNA. The percent of remaining 18S was quantified by qPCR. Values are derived from 18S normalized to Act5c, in turn normalized to a non-depleted sample (no probe control). Values are the averages of three replicates of the depletion experiment, each using a different sample of larval RNA. A) Outline of the single-probe depletion assay. A 3′ biotinylated probe targeting a specific site in the 18S is added to total RNA and subjected to hybridization. The target is captured with streptavidin beads and the remaining target is measured from the supernatant. B) The percent of 18S rRNA remaining for all tested oligos of size 26 -32 nt, arranged 5′ to 3′ by target site. Error bars are standard deviation between separate hybridization experiments, each performed with a different RNA sample. C) Performance comparison between the initial set of low Tm probes (probes #1 -#11, Tm < 71 ˚C) targeting the left side of the 18S transcript and the second set of high Tm probes (probes #21 -30, Tm > 71 ˚C). Twosided t-test p = 0.12. Probe #2 and probe #29 are outliers, possibly for structural reasons (see figure 2). D) Correlation between the predicted Tm of the probe/target hybrid and the percent of remaining target for the 30 ~30mer probes tested in the single-probe depletion assay (p = 0.01, Spearman's correlation).

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“…Although most RNA-Seq protocols include the depletion of rRNA before sequencing, moderate up to high amounts of rRNA can still be found in samples [ 18 , 19 ]. In particular, this is true for non-model organisms where library preparation kits are not optimized [ 20 ]. Therefore, we have decided to perform an rRNA cleaning step by default, which can be deactivated by the user.…”

Section: Materials and Methodsmentioning

confidence: 99%

RNAflow: An Effective and Simple RNA-Seq Differential Gene Expression Pipeline Using Nextflow

Lataretu

¹

,

Hölzer

²

2020

Genes

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RNA-Seq enables the identification and quantification of RNA molecules, often with the aim of detecting differentially expressed genes (DEGs). Although RNA-Seq evolved into a standard technique, there is no universal gold standard for these data’s computational analysis. On top of that, previous studies proved the irreproducibility of RNA-Seq studies. Here, we present a portable, scalable, and parallelizable Nextflow RNA-Seq pipeline to detect DEGs, which assures a high level of reproducibility. The pipeline automatically takes care of common pitfalls, such as ribosomal RNA removal and low abundance gene filtering. Apart from various visualizations for the DEG results, we incorporated downstream pathway analysis for common species as Homo sapiens and Mus musculus. We evaluated the DEG detection functionality while using qRT-PCR data serving as a reference and observed a very high correlation of the logarithmized gene expression fold changes.

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A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery

Cited by 23 publications

References 55 publications

Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion

Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion

Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion

RNAflow: An Effective and Simple RNA-Seq Differential Gene Expression Pipeline Using Nextflow

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