Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction

Herbert, Zachary T.; Kershner, Jamie P.; Butty, Vincent L.; 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:10.1186/s12864-018-4585-1

Cited by 46 publications

(23 citation statements)

References 12 publications

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“…In the alternative strategy, the RNA:DNA hybrids are degraded by RNAse H so that the targeted sequences are no longer available for subsequent applications such as polyA+ selection. A cross-site comparative study with commercially available rRNA depletion kits including kits based on the capture of rRNA by complementary oligonucleotides coupled to beads and also kits based on the hybridization of rRNA with antisense DNA oligonucleotides followed by degradation of RNA:DNA hybrids with RNase H, showed that although there were differences between the underlying rRNA depletion chemistries, all tested kits were able to successfully remove a significant amount of rRNA in library preparations [16]. All kits were able to remove ribosomal RNA to below 20%, but in comparison, the kits that degraded the rRNA by RNase H treatment showed more consistent results than kits that used the bead-based capture method for rRNA depletion.…”

Section: Introductionmentioning

confidence: 99%

“…All kits were able to remove ribosomal RNA to below 20%, but in comparison, the kits that degraded the rRNA by RNase H treatment showed more consistent results than kits that used the bead-based capture method for rRNA depletion. Furthermore, Adiconis et al [17] and Herbert et al [16] found that the RNase H-mediated method performed best for rRNA depletion in case of low quality RNA.…”

Section: Introductionmentioning

confidence: 99%

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Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

et al. 2019

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Background In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. Results Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins ( CSN1S1, CSN1S2, CSN2 and CSN3 ) and whey proteins ( LALBA and PAEP ) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli ( E. coli ) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challenge-associated biological signaling pathway patterns. Conclusions The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity. Electronic supplementary material The online version of this article (10.1186/s12864-019-5781-3) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

et al. 2019

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“…However, the set of oligonucleotides can also be easily optimized for a different species or set of species using the open-source software developed here and available on GitHub. In principle, inclusion of multiple oligonucleotides per RNA species to be depleted (11,8, and 2 for the 23S, 16S, and 5S rRNAs, respectively) should enable depletion of even partially degraded rRNA, though here we did not test various levels of RNA degradation. If unwanted RNA species are observed in a final sample, additional oligonucleotides could be designed and added to deplete them.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, bacteria do not typically polyadenylate 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)(9)(10). This approach was at the heart of commercially available kits such as Ribo-Zero from Illumina, leading to RNA-seq data in which ϳ80 to 90% of the reads map to mRNAs.…”

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confidence: 99%

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

Culviner

Guegler

Laub

2020

mBio

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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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“…Together, these observations support the existence of a complex dialogue between the nuclear and mitochondrial genomes, far more involved and dynamic than previously estimated. Because of the sheer amount of rRNA sequences in cellular transcriptomes (usually 95% of total RNAs), rRNA depletion of both nuclear and mitochondria‐encoded sequences has been adopted as a cost‐effective measure to analyze gene expression regulation . In addition, any repetitive sequences remaining in RNA‐sequencing datasets are usually eliminated from downstream analyses as well.…”

Section: What Does a Dual‐genome System Mean For Health?mentioning

confidence: 99%

MOTS‐c: A Mitochondrial‐Encoded Regulator of the Nucleus

Benayoun

Lee

2019

BioEssays

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Mitochondria are increasingly being recognized as information hubs that sense cellular changes and transmit messages to other cellular components, such as the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, and lysosomes. Nonetheless, the interaction between mitochondria and the nucleus is of special interest because they both host part of the cellular genome. Thus, the communication between genome‐bearing organelles would likely include gene expression regulation. Multiple nuclear‐encoded proteins have been known to regulate mitochondrial gene expression. On the contrary, no mitochondrial‐encoded factors are known to actively regulate nuclear gene expression. MOTS‐c (mitochondrial open reading frame of the 12S ribosomal RNA type‐c) is a recently identified peptide encoded within the mitochondrial 12S ribosomal RNA gene that has metabolic functions. Notably, MOTS‐c can translocate to the nucleus upon metabolic stress (e.g., glucose restriction and oxidative stress) and directly regulate adaptive nuclear gene expression to promote cellular homeostasis. It is hypothesized that cellular fitness requires the coevolved mitonuclear genomes to coordinate adaptive responses using gene‐encoded factors that cross‐regulate the opposite genome. This suggests that cellular gene expression requires the bipartite split genomes to operate as a unified system, rather than the nucleus being the sole master regulator.

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Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction

Cited by 46 publications

References 12 publications

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

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

MOTS‐c: A Mitochondrial‐Encoded Regulator of the Nucleus

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