Cross-Site Comparison of Ribosomal Depletion Kits for Illumina RNAseq Library Construction

Herbert, Zachary T.; Kershner, Jamie P.; Butty, Vincent; Thimmapuram, Jyothi; Choudhari, Sulbha; Alekseyev, Yuriy O.; Fan, Jun; Podnar, Jessica W.; Wilcox, Edward R.; Gipson, Jenny; Gillaspy, Allison F.; Jepsen, Kristen; BonDurant, Sandra Splinter; Morris, Krystalynne; Berkeley, Maura; LeClerc, Ashley; Simpson, Stephen D.; Sommerville, Gary; Grimmett, Leslie; Adams, Marie; Levine, Stuart S.

doi:10.1101/212639

Cited by 19 publications

(31 citation statements)

References 12 publications

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“…In contrast, bacteria do not typically poly-adenylate their mRNAs, and rRNA comprises 80% or more of the total RNA harvested from a given sample(7). To enrich for mRNA in RNA-seq samples, a general strategy involves the depletion of rRNAs by subtractive hybridization(8–10). This approach was at the heart of commercially available kits such as Ribo-Zero from Illumina, leading to RNA-seq data in which ~80-90% of the reads map to mRNAs.…”

Section: Introductionmentioning

confidence: 99%

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

Culviner

Guegler

Laub

2020

Preprint

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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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Section: Introductionmentioning

confidence: 99%

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

Culviner

Guegler

Laub

2020

Preprint

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“…106 These RG studies provide a broad foundation for cross-platform standardization, evaluation, and improvement of RNA-seq 107,108 and have led to other standards in genomics, metagenomics, and multiomics. 109…”

Section: Next-gen Sequencing Rgmentioning

confidence: 99%

A Review of the Scientific Rigor, Reproducibility, and Transparency Studies Conducted by the ABRF Research Groups

et al. 2020

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Shared research resource facilities, also known as core laboratories (Cores), are responsible for generating a significant and growing portion of the research data in academic biomedical research institutions. Cores represent a central repository for institutional knowledge management, with deep expertise in the strengths and limitations of technology and its applications. They inherently support transparency and scientific reproducibility by protecting against cognitive bias in research design and data analysis, and they have institutional responsibility for the conduct of research (research ethics, regulatory compliance, and financial accountability) performed in their Cores. The Association of Biomolecular Resource Facilities (ABRF) is a FASEB-member scientific society whose members are scientists and administrators that manage or support Cores. The ABRF Research Groups (RGs), representing expertise for an array of cutting-edge and established technology platforms, perform multicenter research studies to determine and communicate best practices and community-based standards. This review provides a summary of the contributions of the ABRF RGs to promote scientific rigor and reproducibility in Cores from the published literature, ABRF meetings, and ABRF RGs communications.

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“…Ingolia provided a method for depletion in the first RIBO-seq protocol published [5], but since then different kits have become available for prokaryotic rRNA removal, such as RiboZero (Illumina), RiboMinus (ThermoFisher), MICROBExpress (ThermoFisher) and others. There are publications available comparing the efficacy of rRNA removal for the different kits, thus this is not discussed further [43,44].…”

Section: Ribosomal Footprint Generationmentioning

confidence: 99%

Improving Bacterial Ribosome Profiling Data Quality

Glaub

Huptas

Neuhaus

et al. 2019

Preprint

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Ribosome profiling (RIBO-seq) in prokaryotes has the potential to facilitate accurate detection of translation initiation sites, to increase understanding of translational dynamics, and has already allowed detection of many unannotated genes. However, protocols for ribosome profiling and corresponding data analysis are not yet standardized. To better understand the influencing factors, we analysed 48 ribosome profiling samples from 9 studies on E. coli K12 grown in LB medium. We particularly investigated the size selection step in each experiment since the selection for ribosome-protected footprints (RPFs) has been performed at various read lengths. We suggest choosing a size range between 22-30 nucleotides in order to obtain protein-coding fragments. In order to use RIBO-seq data for improving gene annotation of weakly expressed genes, the total amount of reads mapping to protein-coding sequences and not rRNA or tRNA is important, but no consensus about the appropriate sequencing depth has been reached. Again, this causes significant variation between studies. Our analysis suggests that 20 million non rRNA/tRNA mapping reads are required for global detection of translated annotated genes. Further, we highlight the influence of drug induced ribosome stalling, causing bias at translation start sites. Drug induced stalling may be especially useful for detecting weakly expressed genes. These suggestions should improve both gene detection and the comparability of resulting ribosome profiling datasets.

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Cross-Site Comparison of Ribosomal Depletion Kits for Illumina RNAseq Library Construction

Cited by 19 publications

References 12 publications

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

A Review of the Scientific Rigor, Reproducibility, and Transparency Studies Conducted by the ABRF Research Groups

Improving Bacterial Ribosome Profiling Data Quality

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