Modeling RNA degradation for RNA-Seq with applications

RNA sequencing (RNA-seq) is a powerful approach for measuring gene expression levels in cells and tissues, but it relies on high-quality RNA. We demonstrate here that statistical adjustment using existing quality measures largely fails to remove the effects of RNA degradation when RNA quality associates with the outcome of interest. Using RNA-seq data from molecular degradation experiments of human primary tissues, we introduce a method—quality surrogate variable analysis (qSVA)—as a framework for estimating and removing the confounding effect of RNA quality in differential expression analysis. We show that this approach results in greatly improved replication rates (>3×) across two large independent postmortem human brain studies of schizophrenia and also removes potential RNA quality biases in earlier published work that compared expression levels of different brain regions and other diagnostic groups. Our approach can therefore improve the interpretation of differential expression analysis of transcriptomic data from human tissue.

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Using pan RNA-seq analysis to reveal the ubiquitous existence of 5’ end and 3’ end small RNAs

10.1101/444117 ◽

2018 ◽

Author(s):

Xiaofeng Xu ◽

Haishuo Ji ◽

Zhi Cheng ◽

Xiufeng Jin ◽

Xue Yao ◽

...

Keyword(s):

Gene Expression ◽

Steady State ◽

Small Rnas ◽

Gene Expression Regulation ◽

Regulation Of Gene Expression ◽

Cost Effective ◽

Rna Degradation ◽

Point Of View ◽

Rna Seq ◽

Dsrna Cleavage

AbstractIn this study, we used pan RNA-seq analysis to reveal the ubiquitous existence of 5’ end and 3’ end small RNAs. 5’ and 3’ sRNAs alone can be used to annotate mitochondrial with 1-bp resolution and nuclear non-coding genes and identify new steady-state RNAs, which are usually from functional genes. Using 5’, 3’ and intronic sRNAs, we revealed that the enzymatic dsRNA cleavage and RNAi could involve in the RNA degradation and gene expression regulation of U1 snRNA in human. The further study of 5’, 3’ and intronic sRNAs help rediscover double-stranded RNA (dsRNA) cleavage, RNA interference (RNAi) and the regulation of gene expression, which challenges the classical theories. In this study, we provided a simple and cost effective way for the annotation of mitochondrial and nuclear non-coding genes and the identification of new steady-state RNAs, particularly long non-coding RNAs (lncRNAs). We also provided a different point of view for cancer and virus, based on the new discoveries of dsRNA cleavage, RNAi and the regulation of gene expression.

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Gene expression estimates: Influence of sequencing library construction, fish sampling methods, and tissue harvesting time

10.22541/au.161142130.04599849/v1 ◽

2021 ◽

Author(s):

Nickolas Moreno ◽

Leif Howard ◽

Scott Relyea ◽

James Dunnigan ◽

Matthew Boyer ◽

...

Keyword(s):

Gene Expression ◽

Sampling Methods ◽

Cutthroat Trout ◽

Rna Degradation ◽

Model Organisms ◽

Rna Seq ◽

Gene Expression Variation ◽

Oncorhynchus Clarkii ◽

Expression Variation ◽

Study Gene Expression

RNA sequencing (RNA-Seq) is becoming a popular method for measuring gene expression in non-model organisms, including wild populations sampled in the field. While RNA-Seq can be used to measure gene expression variation among wild-caught individuals and can yield important biological insights into organismal function, technical variables may also influence gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish species, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq methods: 3’ RNA-Seq and whole mRNA-Seq. We evaluated the effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 minutes after euthanasia on estimated gene expression in blood, gill, muscle, and liver. We found higher RNA degradation in the liver compared to the other tissues. There were fewer expressed genes in blood compared to gill and muscle. We found no difference in gene expression among sampling methods or due to a delay in tissue collection. However, we detected fewer genes with 3’ RNA-Seq than with whole mRNA-Seq and found statistically significant differences in gene expression between 3’ RNA-Seq and whole mRNA-Seq. The magnitude and direction of these differences does not appear to be dependent on gene type or length. Our findings indicate that RNA-Seq is robust to the technical variables related to the field sampling techniques tested here but varies based on the tissue sampled and the RNA-Seq library used. This study advances understanding of usefulness of RNA-Seq to study gene expression variation in evolution, ecology, and conservation.

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DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate

10.1101/2020.09.15.299180 ◽

2020 ◽

Author(s):

Makoto Kashima ◽

Mari Kamitani ◽

Yasuyuki Nomura ◽

Hiromi Hirata ◽

Atsushi J. Nagano

Keyword(s):

Reverse Transcription ◽

Large Scale ◽

Field Experiments ◽

Rna Extraction ◽

Rna Degradation ◽

Individual Variability ◽

Rna Seq ◽

Tissue Samples ◽

Comparable Amount ◽

Chemical Screening

AbstractUsing current mRNA quantification methods such as RT-qPCR and RNA-Seq, it is very difficult to examine thousands of tissue samples due to cost and labor of RNA extraction and quantification steps. Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. We showed that appreciate concentration of DTT prevented RNA degradation but not RT in the lysates of several plants’ tissues, yeast, and zebrafish larvae. Using the buffer, direct reverse transcription on the lysates could produce comparable amount of cDNA with that synthesized from purified RNA. Furthermore, we established DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq) method. DeLTa-Seq is a cost-effective, high-throughput and highly-precise quantification method for the expressions of hundreds of genes. It enables us to conduct large-scale studies using thousands of samples such as chemical screening, field experiments and studies focusing on individual variability.

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Abstract 20232: Haploinsufficiency of MYBPC3 in the Development of Hypertrophic Cardiomyopathy

Circulation ◽

10.1161/circ.130.suppl_2.20232 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

David Barefield ◽

Mohit Kumar ◽

Joshua Gorham ◽

Jonathan Seidman ◽

Christine Seidman ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Rna Degradation ◽

Rna Seq ◽

Reduced Ejection Fraction ◽

Force Development ◽

Protein Levels ◽

Maximal Force ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Truncation Mutant

Introduction: Mutations in MYBPC3, encoding cardiac myosin binding protein-C (cMyBP-C), account for ~40% of hypertrophic cardiomyopathy (HCM) cases. MYBPC3 mutations are usually encode truncated proteins and are not found in tissue and are typically heterozygous (Het) in humans. Reduced protein levels occur in human HCM patients with these mutations, suggesting haploinsufficiency. However, it is unknown if cMyBP-C reduction causes or results from hypertrophy. Hypothesis: To test whether haploinsufficiency occurs following cardiac stress and if heterozygous MYBPC3 mice had worsened disease progression. Methods & Results: Transverse aortic constriction (TAC) was performed on 3 month old wild type (WT) and Het MYBPC3 truncation mutant mice which were allowed to hypertrophy for 4 or 12 weeks. Het TAC mice showed increased hypertrophy 12 weeks post-TAC compared to WT TAC controls. Het TAC hearts showed reduced ejection fraction compared to WT TAC at 4 and 12 weeks. MYBPC3 transcript levels were significantly reduced in sham and TAC Het hearts. cMyBP-C levels decreased in Het sham and TAC at 4 weeks but returned to baseline levels at 12 weeks. Het TAC myocytes showed higher Ca2+ sensitivity at 4 weeks, and impaired maximal force development. Het sham and TAC skinned cardiomyocytes showed reduced length dependent increases in Ca2+ sensitivity and maximal force development. RNA-Seq shows no alterations in proteasome of RNA-degradation pathways which have been suggested to play a role in the pathology of these mutations. Overexpression of WT cMyBP-C in the presence of truncated MYBPC3 rescued the decline in force observed in Het myocytes in the absence of stress. Conclusions: Heterozygous MYBPC3 truncation mutant carriers develop more profound hypertrophy and dysfunction following stress. Also, increased MYBPC3 expression reverses myocyte deficits in force generation in the presence of truncated alleles.

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RNA-seq: impact of RNA degradation on transcript quantification

BMC Biology ◽

10.1186/1741-7007-12-42 ◽

2014 ◽

Vol 12 (1) ◽

pp. 42 ◽

Cited By ~ 199

Author(s):

Irene Gallego Romero ◽

Athma A Pai ◽

Jenny Tung ◽

Yoav Gilad

Keyword(s):

Rna Degradation ◽

Rna Seq ◽

Transcript Quantification

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Stochastic Analysis of the RT-PCR Process in Single-Cell RNA-Seq

Mathematics ◽

10.3390/math9192515 ◽

2021 ◽

Vol 9 (19) ◽

pp. 2515

Author(s):

Aarón Vázquez-Jiménez ◽

Osbaldo Resendis-Antonio

Keyword(s):

Single Cell ◽

Stochastic Approach ◽

Analytical Framework ◽

Reaction Rates ◽

Rna Degradation ◽

Minimal Amount ◽

Rna Seq ◽

Rt Pcr ◽

Biochemical Processes ◽

Degradation Rates

The single-cell RNA-seq allows exploring the transcriptome for one cell at a time. By doing so, cellular regulation is pictured. One limitation is the dropout events phenomenon, where a gene is observed at a low or moderate expression level in one cell but not detected in another. Dropouts obscure legitimate biological heterogeneity leading to the description of a small fraction of the meaningful relations. We used a stochastic approach to model the Reverse Transcription Polymerase Chain Reaction (RT-PCR) kinetic, in which we contemplated the temperature profile, RT-PCR duration, and reaction rates. By studying the underlying biochemical processes of RT-PCR, using a computational and analytical framework, we show a minimal amount of RNA to avoid dropout events. We further use this fact to characterize the limits in the dispersion reduction. Dispersion asymptotically decreases as the RNA initial value increases. Despite always being a basal dispersion, their decreasing speed is modulated mainly by the degradation rates, particularly for the RNA. We concluded that the critical step into the RT-PCR is the RT phase due to the fragile nature of the RNA. We propose that limiting RNA degradation might ensure that the portraited transcriptional landscape is unbiased by technical error.

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A framework for RNA quality correction in differential expression analysis

10.1101/074245 ◽

2016 ◽

Cited By ~ 2

Author(s):

Andrew E. Jaffe ◽

Ran Tao ◽

Alexis L. Norris ◽

Marc Kealhofer ◽

Abhinav Nellore ◽

...

Keyword(s):

Human Brain ◽

Differential Expression ◽

Expression Analysis ◽

Differential Expression Analysis ◽

Rna Degradation ◽

Rna Seq ◽

Human Brain Tissue ◽

Postmortem Human Brain ◽

Expression Levels ◽

Rna Quality

AbstractRNA sequencing (RNA-seq) is a powerful approach for measuring gene expression levels in cells and tissues, but it relies on high-quality RNA. We demonstrate here that statistical adjustment employing existing quality measures largely fails to remove the effects of RNA degradation when RNA quality associates with the outcome of interest. Using RNA-seq data from a molecular degradation experiment of human brain tissue, we introduce the quality surrogate variable (qSVA) analysis framework for estimating and removing the confounding effect of RNA quality in differential expression analysis. We show this approach results in greatly improved replication rates (>3x) across two large independent postmortem human brain studies of schizophrenia. Finally, we explored public datasets to demonstrate potential RNA quality confounding when comparing expression levels of different brain regions and diagnostic groups beyond schizophrenia. Our approach can therefore improve the interpretation of differential expression analysis of transcriptomic data from the human brain.

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

10.21203/rs.3.rs-67621/v2 ◽

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.

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