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

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.Conclusion: 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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Deploying new generation sequencing for the study of flesh color depletion in Atlantic Salmon (Salmo salar)

BMC Genomics ◽

10.1186/s12864-021-07884-9 ◽

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

BMC Biology ◽

10.1186/s12915-021-01053-w ◽

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.

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A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins

Journal of Virology ◽

10.1128/jvi.02750-15 ◽

2015 ◽

Vol 90 (3) ◽

pp. 1278-1289 ◽

Cited By ~ 13

Author(s):

Catrin Stutika ◽

Andreas Gogol-Döring ◽

Laura Botschen ◽

Mario Mietzsch ◽

Stefan Weger ◽

...

Keyword(s):

Life Cycle ◽

Fusion Protein ◽

Rna Sequencing ◽

Splice Variants ◽

Productive Infection ◽

Minus Strand ◽

Rna Seq ◽

Adeno Associated Virus ◽

Divergent Transcription ◽

Productive Replication

ABSTRACTAdeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5ˈ rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad.IMPORTANCENext-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAVin vivo, combinedin cellulaeandin silicostudies will help to forward the understanding of the unique, bipartite AAV life cycle.

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RNAtor: an Android-based application for biologists to plan RNA sequencing experiments

10.1101/095315 ◽

2016 ◽

Author(s):

Shruti Kane ◽

Himanshu Garg ◽

Neeraja M. Krishnan ◽

Aditya Singh ◽

Binay Panda

Keyword(s):

Rna Sequencing ◽

Mobile Applications ◽

Mobile Application ◽

Splice Variants ◽

Real Data ◽

Flow Cell ◽

Rna Seq ◽

Amount Of Information ◽

Novel Transcripts ◽

Google Play

AbstractRNA sequencing (RNA-seq) is a powerful technology for identification of novel transcripts (coding, non-coding and splice variants), understanding of transcript structures and estimation of gene and/or allelic expression. There are specific challenges that biologists face in determining the number of replicates to use, total number of sequencing reads to generate for detecting marginally differentially expressed transcripts and the number of lanes in a sequencing flow cell to use for the production of right amount of information. Although past studies attempted answering some of these questions, there is a lack of accessible and biologist-friendly mobile applications to answer these questions. Keeping this in mind, we have developed RNAtor, a mobile application for Android platforms, to aid biologists in correctly designing their RNA-seq experiments. The recommendations from RNAtor are based on simulations and real data.Availability and ImplementationThe Android version of RNAtor is available on Google Play Store and the code from GitHub (https://github.com/binaypanda/RNAtor).

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RNA-sequencing analysis of the effect of luteolin on methamphetamine-induced hepatotoxicity in rats: a preliminary study

PeerJ ◽

10.7717/peerj.8529 ◽

2020 ◽

Vol 8 ◽

pp. e8529

Author(s):

Dong Qu ◽

Kaikai Zhang ◽

Lijian Chen ◽

Qi Wang ◽

Huijun Wang

Keyword(s):

Fatty Acid ◽

Rna Sequencing ◽

Biochemical Analysis ◽

Fatty Acid Synthase ◽

Sequencing Analysis ◽

Protective Effects ◽

Rna Seq ◽

Oral Gavage ◽

Fatty Acid Binding ◽

Preliminary Study

In this study, RNA-sequencing (RNA-seq) was utilized to investigate the effects of luteolin on hepatotoxicity caused by methamphetamine (METH). The rats in METH group were administrated with METH (15 mg/kg, two times per day) via intraperitoneal (i.p.) injections for four consecutive days. The rats in luteolin + METH group were firstly administrated with luteolin (100 mg/kg, once a day) by oral gavage for 3 days before METH treatment. Lueolin attenuated the hepatotoxicity induced by METH via histopathological and biochemical analysis. The results of RNA-seq showed that luteolin could regulate 497 differentially expressed genes (DEGs), and the selected DEGs were mainly enriched in eight pathways, according to KEGG analysis. Furthermore, qRT-PCR was utilized to verify the results of RNA-seq. Six genes were selected as follows: liver enriched antimicrobial peptide 2 (Leap2), fatty acid synthase (Fasn), fatty acid binding protein 5 (Fabp5), patatin like phospholipase domain containing 3 (Pnpla3), myelin basic protein (Mbp) and calmodulin 3 (Calm3). Though because of the design flaws, the luteolin group has not been included, this study demonstrated that luteolin might exert hepato-protective effects from METH via modulation of oxidative phosphorylation, cytochrome P450 and certain signaling pathways.

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RNA sequencing by direct tagmentation of RNA/DNA hybrids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919800117 ◽

2020 ◽

Vol 117 (6) ◽

pp. 2886-2893 ◽

Cited By ~ 15

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.

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A systematic evaluation of single cell RNA-seq analysis pipelines

Nature Communications ◽

10.1038/s41467-019-12266-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 47

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.

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RNA-seq analysis identified glucose-responsive genes and YqfO as a global regulator in Bacillus subtilis

BMC Research Notes ◽

10.1186/s13104-021-05869-1 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yu Kanesaki ◽

Mitsuo Ogura

Keyword(s):

Bacillus Subtilis ◽

Rna Sequencing ◽

Metal Ion ◽

Close Relationships ◽

Rna Helicase ◽

Sigma Factors ◽

Rna Seq ◽

Global Regulator ◽

Bacterial Genomes ◽

Regulatory Effects

Abstract Objective We observed that the addition of glucose enhanced the expression of sigX and sigM, encoding extra-cytoplasmic function sigma factors in Bacillus subtilis. Several regulatory factors were identified for this phenomenon, including YqfO, CshA (RNA helicase), and YlxR (nucleoid-associated protein). Subsequently, the relationships among these regulators were analyzed. Among them, YqfO is conserved in many bacterial genomes and may function as a metal ion insertase or metal chaperone, but has been poorly characterized. Thus, to further characterize YqfO, we performed RNA sequencing (RNA-seq) analysis of YqfO in addition to CshA and YlxR. Results We first performed comparative RNA-seq to detect the glucose-responsive genes. Next, to determine the regulatory effects of YqfO in addition to CshA and YlxR, three pairs of comparative RNA-seq analyses were performed (yqfO/wt, cshA/wt, and ylxR/wt). We observed relatively large regulons (approximately 420, 780, and 180 for YqfO, CshA, and YlxR, respectively) and significant overlaps, indicating close relationships among the three regulators. This study is the first to reveal that YqfO functions as a global regulator in B. subtilis.

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Validation of methods for Low-volume RNA-seq

10.1101/006130 ◽

2014 ◽

Author(s):

Peter Acuña Combs ◽

Michael B Eisen

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Critical Evaluation ◽

Standard Protocol ◽

Library Preparation ◽

Rna Seq ◽

Simple Modification ◽

Input Sample ◽

Low Volume ◽

The Cost

Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published. However, we were concerned that the additional steps to deal with such minute quantities of input sample would introduce serious biases that would make analysis of the data using existing approaches invalid. In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in metrics of interest to us than a more standard protocol, but with at least two orders of magnitude less sample required. We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

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RNAtor: an Android-based application for biologists to plan RNA sequencing experiments

F1000Research ◽

10.12688/f1000research.11982.2 ◽

2017 ◽

Vol 6 ◽

pp. 997

Author(s):

Shruti Kane ◽

Himanshu Garg ◽

Neeraja M. Krishnan ◽

Aditya Singh ◽

Binay Panda

Keyword(s):

Rna Sequencing ◽

Mobile Applications ◽

Mobile Application ◽

Splice Variants ◽

Real Data ◽

Differentially Expressed ◽

Rna Seq ◽

Sample Analysis ◽

Allele Expression ◽

Novel Transcripts

RNA sequencing (RNA-seq) is a powerful technology that allows one to assess the RNA levels in a sample. Analysis of these levels can help in identifying novel transcripts (coding, non-coding and splice variants), understanding transcript structures, and estimating gene/allele expression. Biologists face specific challenges while designing RNA-seq experiments. The nature of these challenges lies in determining the total number of sequenced reads and technical replicates required for detecting marginally differentially expressed transcripts. Despite previous attempts to address these challenges, easily-accessible and biologist-friendly mobile applications do not exist. Thus, we developed RNAtor, a mobile application for Android platforms, to aid biologists in correctly designing their RNA-seq experiments. The recommendations from RNAtor are based on simulations and real data.

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