Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species

AbstractThe profiling of gene expression by RNA-sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is ribosomal RNA (rRNA), it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued. Here, we report the development a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin beads then depletes rRNA from a complex, total RNA sample such that ~75-80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold-change measurements in RNA-seq experiments between our method and the previously available Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or metagenomic sample of interest.ImportanceThe ability to examine global patterns of gene expression in microbes through RNA-sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of ribosomal RNA from total RNA samples. Otherwise, rRNA would comprise upwards of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from messenger RNA or requiring high total read depth. A commonly used, kit for rRNA subtraction from Illumina was recently discontinued. Here, we report the development of a ‘do-it-yourself’ kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligos enable sufficient rRNA depletion to produce RNA-seq data with 75-80% of reads comming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

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Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species

10.1101/2020.09.19.294595 ◽

2020 ◽

Author(s):

Hanwen Gu ◽

Yu H. Sun ◽

Xin Zhiguo Li

Keyword(s):

Gene Expression ◽

Deep Sequencing ◽

Cost Effective ◽

Ribosome Profiling ◽

Avian Species ◽

Alternative Methods ◽

Model Organisms ◽

Probe Design ◽

Library Construction ◽

Rrna Depletion

ABSTRACTDeep sequencing of RNAs has greatly aided the study of the transcriptome, enabling comprehensive gene expression profiling and the identification of novel transcripts. While mRNAs are of the greatest interest in gene expression studies as they encode for proteins, mRNAs make up only 3-5% of total RNAs, with the majority comprising ribosomal RNAs (rRNAs). Therefore, applications of deep sequencing to RNA face the challenge of how to efficiently enrich mRNA species prior to library construction. Traditional methods extract mRNAs using oligo-dT primers targeting the poly-A tail on mRNAs; however, this approach is not comprehensive as it does not account for mRNAs lacking the poly-A tail or other lncRNAs that we may be interested in. Alternative methods deplete rRNAs, but such approaches require species-specific probes and the commercially available kits are costly and have only been developed for a limited number of model organisms. Here we describe a quick, cost-effective method for depleting rRNAs using custom-designed oligos. We use chickens as an example species for probe design, and the same approach also apply to mice. Using this protocol, we have not only removed the rRNAs from total RNAs for RNA-seq library construction but also depleted rRNA fragments from ribosome-protected fragments for ribosome profiling. Currently, this is the only rRNA depletion-based method for avian species; this method thus provides a valuable resource for both the scientific community and the poultry industry.

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A Simple, Cost-Effective, and Robust Method for rRNA Depletion in RNA-Sequencing Studies

mBio ◽

10.1128/mbio.00010-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 9

Author(s):

Peter H. Culviner ◽

Chantal K. Guegler ◽

Michael T. Laub

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Bacterial Species ◽

Extended Period ◽

Cost Effective ◽

Rna Seq ◽

Robust Method ◽

Total Rna ◽

Metagenomic Sample ◽

Rrna Depletion

ABSTRACT The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ∼75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest. IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a “do-it-yourself” kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

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Evaluation of protocols for rRNA depletion-based RNA sequencing of nanogram inputs of mammalian total RNA

PLoS ONE ◽

10.1371/journal.pone.0224578 ◽

2019 ◽

Vol 14 (10) ◽

pp. e0224578 ◽

Cited By ~ 2

Author(s):

Simon Haile ◽

Richard D. Corbett ◽

Steve Bilobram ◽

Karen Mungall ◽

Bruno M. Grande ◽

...

Keyword(s):

Rna Sequencing ◽

Total Rna ◽

Rrna Depletion

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Faculty Opinions recommendation of Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717991048.793473278 ◽

2013 ◽

Author(s):

Karla Neugebauer

Keyword(s):

Human Brain ◽

Rna Sequencing ◽

Total Rna

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Monitoring the microbiome for food safety and quality using deep shotgun sequencing

npj Science of Food ◽

10.1038/s41538-020-00083-y ◽

2021 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Kristen L. Beck ◽

Niina Haiminen ◽

David Chambliss ◽

Stefan Edlund ◽

Mark Kunitomi ◽

...

Keyword(s):

Rna Sequencing ◽

Environmental Changes ◽

Pet Food ◽

Analysis Pipeline ◽

Multiple Sequence ◽

Food Ingredients ◽

Total Rna ◽

Specific Species ◽

Ingredient Composition ◽

Food Safety And Quality

AbstractIn this work, we hypothesized that shifts in the food microbiome can be used as an indicator of unexpected contaminants or environmental changes. To test this hypothesis, we sequenced the total RNA of 31 high protein powder (HPP) samples of poultry meal pet food ingredients. We developed a microbiome analysis pipeline employing a key eukaryotic matrix filtering step that improved microbe detection specificity to >99.96% during in silico validation. The pipeline identified 119 microbial genera per HPP sample on average with 65 genera present in all samples. The most abundant of these were Bacteroides, Clostridium, Lactococcus, Aeromonas, and Citrobacter. We also observed shifts in the microbial community corresponding to ingredient composition differences. When comparing culture-based results for Salmonella with total RNA sequencing, we found that Salmonella growth did not correlate with multiple sequence analyses. We conclude that microbiome sequencing is useful to characterize complex food microbial communities, while additional work is required for predicting specific species’ viability from total RNA sequencing.

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Ribo-ODDR: Oligo design pipeline for experiment-specific rRNA depletion in ribo-seq

Bioinformatics ◽

10.1093/bioinformatics/btab171 ◽

2021 ◽

Author(s):

Ferhat Alkan ◽

Joana Silva ◽

Eric Pintó Barberà ◽

William J Faller

Keyword(s):

Ribosome Profiling ◽

Supplementary Information ◽

Experimental Conditions ◽

Computational Framework ◽

Rna Translation ◽

Rrna Depletion ◽

Selection For ◽

Nucleotide Resolution ◽

User Friendly ◽

Oligo Design

Abstract Motivation Ribosome Profiling (Ribo-seq) has revolutionized the study of RNA translation by providing information on ribosome positions across all translated RNAs with nucleotide-resolution. Yet several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of rRNA fragments. Various strategies can be employed to tackle this issue, including the use of commercial rRNA depletion kits. However, as they are designed for more standardized RNAseq experiments, they may perform suboptimally in Ribo-seq. In order to overcome this, it is possible to use custom biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos. Results Here, we first show that a major confounding issue is that the rRNA fragments generated via Ribo-seq vary significantly with differing experimental conditions, suggesting that a “one-size-fits-all” approach may be inefficient. Therefore we developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We experimentally show that Ribo-ODDR designed oligos outperform commercially available kits and lead to a significant increase in rRNA depletion in Ribo-seq. Availability Ribo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR Supplementary information Supplementary data are available at Bioinformatics online.

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