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):  
Rna Sequencing ◽  
Rna Degradation ◽  
Read Coverage ◽  
Library Preparation ◽  
Rna Seq ◽  
Sample Handling ◽  
Freeze Thaw ◽  
Optimal Sample ◽  
The Impact ◽  
Do So

Abstract RNA-Seq is ubiquitous, but depending on the study, sub-optimal sample handling may be required, resulting in repeated freeze-thaw cycles. However, little is known about how each cycle impacts downstream analyses, due to a lack of study and known limitations in common RNA quality metrics, e.g., RIN, at quantifying RNA degradation following repeated freeze-thaws. Here we quantify the impact of repeated freeze-thaw on the reliability of RNA-Seq. To do so, we developed a method to estimate the relative noise between technical replicates independently of RIN. Using this approach we inferred the effect of both RIN and the number of freeze-thaw cycles on sample noise. We find that RIN is unable to fully account for the change in sample noise due to freeze-thaw cycles. Additionally, freeze-thaw is detrimental to sample quality and differential expression (DE) reproducibility, approaching zero after three cycles for poly(A)-enriched samples, wherein the inherent 3’ bias in read coverage is more exacerbated by freeze-thaw cycles, while ribosome-depleted samples are less affected by freeze-thaws. 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

2020 ◽  
Author(s):  
Benjamin P. Kellman ◽  
Hratch M. Baghdassarian ◽  
Tiziano Pramparo ◽  
Isaac Shamie ◽  
Vahid Gazestani ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Degradation ◽  
Read Coverage ◽  
Library Preparation ◽  
Rna Seq ◽  
Sample Handling ◽  
Freeze Thaw ◽  
Optimal Sample ◽  
The Impact ◽  
Do So

AbstractRNA-Seq is ubiquitous, but depending on the study, sub-optimal sample handling may be required, resulting in repeated freeze-thaw cycles. However, little is known about how each cycle impacts downstream analyses, due to a lack of study and known limitations in common RNA quality metrics, e.g., RIN, at quantifying RNA degradation following repeated freeze-thaws. Here we quantify the impact of repeated freeze-thaw on the reliability of downstream RNA-Seq analysis. To do so, we developed a method to estimate the relative noise between technical replicates independently of RIN. Using this approach we inferred the effect of both RIN and the number of freeze-thaw cycles on sample noise. We find that RIN is unable to fully account for the change in sample noise due to freeze-thaw cycles. Additionally, freeze-thaw is detrimental to sample quality and differential expression (DE) reproducibility, approaching zero after three cycles for poly(A)-enriched samples, wherein the inherent 3’ bias in read coverage is more exacerbated by freeze-thaw cycles, while ribosome-depleted samples are less affected by freeze-thaws. 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

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 Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Milda Mickutė ◽  
Kotryna Kvederavičiūtė ◽  
Aleksandr Osipenko ◽  
Raminta Mineikaitė ◽  
Saulius Klimašauskas ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Regulatory Networks ◽  
Library Preparation ◽  
Rna Seq ◽  
Basic Principles ◽  
Cofactor Binding ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Target Rna

Abstract Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

scholarly journals 3′ MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage

2020 ◽  
Vol 100 (10) ◽  
pp. 1345-1355 ◽  
Author(s):  
Stefaniya Boneva ◽  
Anja Schlecht ◽  
Daniel Böhringer ◽  
Hans Mittelviefhaus ◽  
Thomas Reinhard ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Storage Time ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Fresh Tissue ◽  
Long Term Storage ◽  
Ffpe Samples ◽  
The Impact ◽  
Term Storage

Abstract This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

scholarly journals Evaluating Alteration of Blood Transcriptome Affected by Storage Conditions Using RNA Sequencing

2020 ◽  
Author(s):  
Eun Jung Koh ◽  
So Yeon Yu ◽  
Seung Jun Kim ◽  
Eun-Il Lee ◽  
Seung Yong Hwang
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Whole Blood ◽  
Expression Patterns ◽  
Rna Degradation ◽  
Biological Research ◽  
P Value ◽  
Sample Handling ◽  
Rna Quality

Abstract BackgroundWhole blood is one of the most widely utilized human samples in biological research and is useful for analysing the mechanisms of diverse bio-molecular phenomena. However, owing to its fluidic properties, whole blood is relatively unstable in the frozen state compared to other biopsy samples. Because RNA is structurally unstable, sample damage can severely affect RNA quality, thereby reducing its usability. This study aimed to assess the quality of RNA prepared from blood stored at different temperatures and times prior to freezing, as well as the effect of freezer storage time. ResultsThe quality of the RNA derived from different blood samples was assessed by determining the RNA integrity number and RNA sequencing to identify genes (|fold-change (FC)| > 1.5, p-value < 0.05, false discovery rate (FDR) < 0.05) that were differentially expressed between the differently prepared RNA samples. We found that improper sample handling critically influenced both RNA quality and gene expression patterns. In particular, storing blood at room temperature over 12 h before freezing led to RNA degradation. Differential gene expression analysis revealed that expression of the CXCR1 gene was substantially reduced when using impaired RNA. ConclusionsThis study emphasizes the importance of proper sample management for obtaining reliable downstream application outcomes and suggests the CXCR1 gene as a candidate screening marker for RNA damage caused by improper sample handling.

scholarly journals RNA sequencing by direct tagmentation of RNA/DNA hybrids

2020 ◽  
Vol 117 (6) ◽  
pp. 2886-2893 ◽  
Author(s):  
Lin Di ◽  
Yusi Fu ◽  
Yue Sun ◽  
Jie Li ◽  
Lu Liu ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Single Cells ◽  
Transcriptome Profiling ◽  
Initial Material ◽  
Library Preparation ◽  
Rna Seq ◽  
Cdna Synthesis ◽  
Complementary Dna ◽  
Dna Analyses ◽  
Wide Range

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

scholarly journals A systematic evaluation of single cell RNA-seq analysis pipelines

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Beate Vieth ◽  
Swati Parekh ◽  
Christoph Ziegenhain ◽  
Wolfgang Enard ◽  
Ines Hellmann
Keyword(s):  
Best Practices ◽  
Sample Size ◽  
Single Cell ◽  
Differential Expression ◽  
Rna Sequencing ◽  
Systematic Evaluation ◽  
Library Preparation ◽  
Rna Seq ◽  
Rapid Spread ◽  
Single Cell Rna Sequencing

Abstract The recent rapid spread of single cell RNA sequencing (scRNA-seq) methods has created a large variety of experimental and computational pipelines for which best practices have not yet been established. Here, we use simulations based on five scRNA-seq library protocols in combination with nine realistic differential expression (DE) setups to systematically evaluate three mapping, four imputation, seven normalisation and four differential expression testing approaches resulting in ~3000 pipelines, allowing us to also assess interactions among pipeline steps. We find that choices of normalisation and library preparation protocols have the biggest impact on scRNA-seq analyses. Specifically, we find that library preparation determines the ability to detect symmetric expression differences, while normalisation dominates pipeline performance in asymmetric DE-setups. Finally, we illustrate the importance of informed choices by showing that a good scRNA-seq pipeline can have the same impact on detecting a biological signal as quadrupling the sample size.

scholarly journals Deploying new generation sequencing for the study of flesh color depletion in Atlantic Salmon (Salmo salar)

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Thu Thi Minh Vo ◽  
Tuan Viet Nguyen ◽  
Gianluca Amoroso ◽  
Tomer Ventura ◽  
Abigail Elizur
Keyword(s):  
Lipid Metabolism ◽  
Atlantic Salmon ◽  
Rna Sequencing ◽  
Metal Ion ◽  
Splice Variants ◽  
Library Preparation ◽  
Rna Seq ◽  
Flesh Color ◽  
Fatty Acid Binding ◽  
Farmed Atlantic Salmon

Abstract Background The flesh pigmentation of farmed Atlantic salmon is formed by accumulation of carotenoids derived from commercial diets. In the salmon gastrointestinal system, the hindgut is considered critical in the processes of carotenoids uptake and metabolism. In Tasmania, flesh color depletion can noticeably affect farmed Atlantic salmon at different levels of severity following extremely hot summers. In this study, RNA sequencing (RNA-Seq) was performed to investigate the reduction in flesh pigmentation. Library preparation is a key step that significantly impacts the effectiveness of RNA sequencing (RNA-Seq) experiments. Besides the commonly used whole transcript RNA-Seq method, the 3’ mRNA-Seq method is being applied widely, owing to its reduced cost, enabling more repeats to be sequenced at the expense of lower resolution. Therefore, the output of the Illumina TruSeq kit (whole transcript RNA-Seq) and the Lexogen QuantSeq kit (3’ mRNA-Seq) was analyzed to identify genes in the Atlantic salmon hindgut that are differentially expressed (DEGs) between two flesh color phenotypes. Results In both methods, DEGs between the two color phenotypes were associated with metal ion transport, oxidation-reduction processes, and immune responses. We also found DEGs related to lipid metabolism in the QuantSeq method. In the TruSeq method, a missense mutation was detected in DEGs in different flesh color traits. The number of DEGs found in the TruSeq libraries was much higher than the QuantSeq; however, the trend of DEGs in both library methods was similar and validated by qPCR. Conclusions Flesh coloration in Atlantic salmon is related to lipid metabolism in which apolipoproteins, serum albumin and fatty acid-binding protein genes are hypothesized to be linked to the absorption, transport and deposition of carotenoids. Our findings suggest that Grp could inhibit the feeding behavior of low color-banded fish, resulting in the dietary carotenoid shortage. Several SNPs in genes involving in carotenoid-binding cholesterol and oxidative stress were detected in both flesh color phenotypes. Regarding the choice of the library preparation method, the selection criteria depend on the research design and purpose. The 3’ mRNA-Seq method is ideal for targeted identification of highly expressed genes, while the whole RNA-Seq method is recommended for identification of unknown genes, enabling the identification of splice variants and trait-associated SNPs, as we have found for duox2 and duoxa1.

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