scholarly journals Computational discovery of hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity Numbers

2019 ◽  
Author(s):  
Paschalis Natsidis ◽  
Philipp H. Schiffer ◽  
Irepan Salvador-Martínez ◽  
Maximilian J. Telford
Keyword(s):  
Ribosomal Rna ◽  
Convergent Evolution ◽  
Animal Species ◽  
Great Majority ◽  
Computational Approach ◽  
28S Rrna ◽  
Rna Seq ◽  
Ribosomal Rnas ◽  
Rna Integrity ◽  
Computational Discovery

ABSTRACTIn some eukaryotes, a ‘hidden break’ has been described in which the 28S ribosomal RNA molecule is cleaved into two subparts. The break is common in protostome animals (arthropods, molluscs, annelids etc.) but a break has also been reported in some vertebrates and non-metazoan eukaryotes. We present a new computational approach to determine the presence of the hidden break in 28S rRNAs using mapping of RNA-Seq data. We find a homologous break is present across protostomes although has been lost in a small number of taxa. We show that rare breaks in vertebrate 28S rRNAs are not homologous to the protostome break. A break is found in just 4 out of 331 species of non-animal eukaryotes studied and three of these are located in the same position as the protostome break suggesting a striking instance of convergent evolution. RNA Integrity Numbers (RIN) rely on intact 28s rRNA and will be consistently underestimated in the great majority of animal species with a break.

scholarly journals Computational discovery of hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity Numbers

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Paschalis Natsidis ◽  
Philipp H. Schiffer ◽  
Irepan Salvador-Martínez ◽  
Maximilian J. Telford
Keyword(s):  
Ribosomal Rna ◽  
Convergent Evolution ◽  
Animal Species ◽  
Great Majority ◽  
Computational Approach ◽  
28S Rrna ◽  
Rna Seq ◽  
Ribosomal Rnas ◽  
Rna Integrity ◽  
Computational Discovery

AbstractIn some eukaryotes, a ‘hidden break’ has been described in which the 28S ribosomal RNA molecule is cleaved into two subparts. The break is common in protostome animals (arthropods, molluscs, annelids etc.), but a break has also been reported in some vertebrates and non-metazoan eukaryotes. We present a new computational approach to determine the presence of the hidden break in 28S rRNAs using mapping of RNA-Seq data. We find a homologous break is present across protostomes although it has been lost in a small number of taxa. We show that rare breaks in vertebrate 28S rRNAs are not homologous to the protostome break. A break is found in just 4 out of 331 species of non-animal eukaryotes studied and, in three of these, the break is located in the same position as the protostome break suggesting a striking instance of convergent evolution. RNA Integrity Numbers (RIN) rely on intact 28S rRNA and will be consistently underestimated in the great majority of animal species with a break.

scholarly journals Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

2020 ◽  
Author(s):  
Benjamin Kellman ◽  
Hratch Baghdassarian ◽  
Tiziano Pramparo ◽  
Isaac Shamie ◽  
Vahid Gazestani ◽  
...  
Keyword(s):  
Differential Expression ◽  
Rna Sequencing ◽  
Ribosomal Rna ◽  
Rna Degradation ◽  
Library Preparation ◽  
Rna Seq ◽  
Rna Integrity ◽  
Freeze Thaw ◽  
The Impact ◽  
Do So

Abstract Background: Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging.Results: Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3’ shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation.Conclusion: The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.

scholarly journals Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

2021 ◽  
Author(s):  
Benjamin Kellman ◽  
Hratch Baghdassarian ◽  
Tiziano Pramparo ◽  
Isaac Shamie ◽  
Vahid Gazestani ◽  
...  
Keyword(s):  
Differential Expression ◽  
Rna Sequencing ◽  
Ribosomal Rna ◽  
Rna Degradation ◽  
Library Preparation ◽  
Rna Seq ◽  
Rna Integrity ◽  
Freeze Thaw ◽  
The Impact ◽  
Do So

Abstract Background: Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging.Results: Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3’ shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation.Conclusion: The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.

scholarly journals Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Benjamin P. Kellman ◽  
Hratch M. Baghdassarian ◽  
Tiziano Pramparo ◽  
Isaac Shamie ◽  
Vahid Gazestani ◽  
...  
Keyword(s):  
Differential Expression ◽  
Rna Sequencing ◽  
Ribosomal Rna ◽  
Rna Degradation ◽  
Library Preparation ◽  
Rna Seq ◽  
Rna Integrity ◽  
Freeze Thaw ◽  
The Impact ◽  
Do So

Abstract Background Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging. Results Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3′ shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation. Conclusion The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility. Graphical abstract

scholarly journals Insects’ RNA Profiling Reveals Absence of “Hidden Break” in 28S Ribosomal RNA Molecule of Onion Thrips,Thrips tabaci

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Rosaline Wanjiru Macharia ◽  
Fidelis Levi Ombura ◽  
Erick Onyango Aroko
Keyword(s):  
Ribosomal Rna ◽  
Thrips Tabaci ◽  
28S Rrna ◽  
Onion Thrips ◽  
Rna Integrity ◽  
Rna Profiling ◽  
Insect Order ◽  
Banding Profile ◽  
Rrna Sequences

With an exception of aphids, insects’ 28S rRNA is thought to harbor a “hidden break” which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect’s RNA, the “hidden break” is absent in the 28S rRNA of onion thrips,Thrips tabaci. On the other hand, presence of “hidden break” was depicted in whiteflies’ 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect’s 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species.

Phylogeny of hymenolepidids (Cestoda: Cyclophyllidea) from mammals: sequences of 18S rRNA and COI genes confirm major clades revealed by the 28S rRNA analyses

2021 ◽  
Vol 95 ◽  
Author(s):  
B. Neov ◽  
G.P. Vasileva ◽  
G. Radoslavov ◽  
P. Hristov ◽  
D.T.J. Littlewood ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
18S Rrna ◽  
Phylogenetic Analyses ◽  
The Other ◽  
Rrna Genes ◽  
Host Switching ◽  
28S Rrna ◽  
Mitochondrial Cytochrome ◽  
Rapid Radiation ◽  
18S Rrna Genes

Abstract The aim of the study is to test a hypothesis for the phylogenetic relationships among mammalian hymenolepidid tapeworms, based on partial (D1–D3) nuclear 28S ribosomal RNA (rRNA) genes, by estimating new molecular phylogenies for the group based on partial mitochondrial cytochrome c oxidase I (COI) and nuclear 18S rRNA genes, as well as a combined analysis using all three genes. New sequences of COI and 18S rRNA genes were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analyses confirmed the major clades identified by Haukisalmi et al. (Zoologica Scripta 39: 631–641, 2010): Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade. While the Ditestolepis clade is associated with soricids, the structure of the other three clades suggests multiple evolutionary events of host switching between shrews and rodents. Two of the present analyses (18S rRNA and COI genes) show that the basal relationships of the four mammalian clades are branching at the same polytomy with several hymenolepidids from birds (both terrestrial and aquatic). This may indicate a rapid radiation of the group, with multiple events of colonizations of mammalian hosts by avian parasites.

An Easy, Cost‐Effective, and Scalable Method to Deplete Human Ribosomal RNA for RNA‐seq

2021 ◽  
Vol 1 (6) ◽  
Author(s):  
Amber Baldwin ◽  
Adam R. Morris ◽  
Neelanjan Mukherjee
Keyword(s):  
Ribosomal Rna ◽  
Cost Effective ◽  
Rna Seq

scholarly journals SARS-CoV-2 convergent evolution as a guide to explore adaptive advantage

2021 ◽  
Author(s):  
Jiri Zahradnik ◽  
Jaroslav Nunvar ◽  
Gideon Schreiber
Keyword(s):  
Receptor Binding ◽  
Convergent Evolution ◽  
Great Majority ◽  
Viral Population ◽  
Binding Motif ◽  
Furin Cleavage ◽  
S Protein ◽  
Adaptive Mutations ◽  
Furin Cleavage Site ◽  
Protein Receptor

Much can be learned from 1.2 million sequences of SARS-CoV-2 generated during the last 15 months. Out of the overwhelming number of mutations sampled so far, only few rose to prominence in the viral population. Many of these emerged recently and independently in multiple lineages. Such a textbook example of convergent evolution at the molecular level is not only curiosity but a guide to uncover the basis for adaptive advantage behind these events. Focusing on the extent of the convergent evolution in the spike (S) protein, our report confirms that the most concerning SARS-CoV-2 lineages carry the heaviest burden of convergent S-protein mutations, suggesting their fundamental adaptive advantage. The great majority (21/25) of S-protein sites under convergent evolution tightly cluster in three functional domains; N-terminal domain, receptor-binding domain, and Furin cleavage site. We further show that among the S-protein receptor-binding motif mutations, ACE2 affinity-improving substitutions are favored. While the probed mutation space in the S protein covered all amino-acids reachable by single nucleotide changes, substitutions requiring two nucleotide changes or epistatic mutations of multiple-residues have only recently started to emerge. Unfortunately, despite their convergent emergence and physical association, most of these adaptive mutations and their combinations remain understudied. We aim to promote research of current variants which are currently understudied but may become important in the future.

scholarly journals Biogenesis and Function of Small Non-Coding RNAs Derived from Eukaryotic Ribosomal RNA

2018 ◽  
Author(s):  
Marine Lambert ◽  
Abderrahim Benmoussa ◽  
Patrick Provost
Keyword(s):  
Ribosomal Rna ◽  
Potential Role ◽  
A Priori ◽  
Biological Significance ◽  
Rna Seq ◽  
New Class ◽  
Scientific Mind ◽  
Non Coding Rnas ◽  
And Function ◽  
Rrna Sequences

The advent of RNA-sequencing (RNA-Seq) technologies has markedly improved our knowledge and expanded the compendium of small non-coding RNAs, most of which derive from the processing of longer RNA precursors. In this review article, we will discuss about the biogenesis and function of small non-coding RNAs derived from eukaryotic ribosomal RNA (rRNA), called rRNA fragments (rRFs), and their potential role(s) as regulator of gene expression. This relatively new class of ncRNAs remained poorly investigated and underappreciated until recently, due mainly to the a priori exclusion of rRNA sequences—because of their overabundance—from RNA-Seq datasets. The situation surrounding rRFs resembles that of microRNAs (miRNAs), which used to be readily discarded from further analyses, for more than five decades, because we could not believe that RNA of such a short length could bear biological significance. As if we had not yet learned our lesson not to restrain our investigative, scientific mind from challenging widely accepted beliefs or dogmas, and from looking for the hidden treasures in the most unexpected places.

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