scholarly journals A Comparison of mRNA Sequencing (RNA-Seq) Library Preparation Methods for Transcriptome Analysis

2021 ◽  
Author(s):  
Hiroki Ura ◽  
Sumihito Togi ◽  
Yo Niida
Keyword(s):  
Transcriptome Analysis ◽  
Expression Patterns ◽  
Full Length ◽  
Relative Advantage ◽  
Library Preparation ◽  
Rna Seq ◽  
Expression Levels ◽  
Mrna Sequencing ◽  
Advantages And Disadvantages ◽  
Better Than

Abstract Background mRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis. Results We observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated. Conclusions TruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.

scholarly journals Transcriptome Analysis Reveals HgCl2 Induces Apoptotic Cell Death in Human Lung Carcinoma H1299 Cells through Caspase-3-Independent Pathway

2021 ◽  
Vol 22 (4) ◽  
pp. 2006
Author(s):  
Mi Jin Kim ◽  
Jinhong Park ◽  
Jinho Kim ◽  
Ji-Young Kim ◽  
Mi-Jin An ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Transcriptome Analysis ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Expression Patterns ◽  
Nitrogen Compound ◽  
Apoptotic Cell Death ◽  
Mercury Chloride ◽  
Rna Seq

Mercury is one of the detrimental toxicants that can be found in the environment and exists naturally in different forms; inorganic and organic. Human exposure to inorganic mercury, such as mercury chloride, occurs through air pollution, absorption of food or water, and personal care products. This study aimed to investigate the effect of HgCl2 on cell viability, cell cycle, apoptotic pathway, and alters of the transcriptome profiles in human non-small cell lung cancer cells, H1299. Our data show that HgCl2 treatment causes inhibition of cell growth via cell cycle arrest at G0/G1- and S-phase. In addition, HgCl2 induces apoptotic cell death through the caspase-3-independent pathway. Comprehensive transcriptome analysis using RNA-seq indicated that cellular nitrogen compound metabolic process, cellular metabolism, and translation for biological processes-related gene sets were significantly up- and downregulated by HgCl2 treatment. Interestingly, comparative gene expression patterns by RNA-seq indicated that mitochondrial ribosomal proteins were markedly altered by low-dose of HgCl2 treatment. Altogether, these data show that HgCl2 induces apoptotic cell death through the dysfunction of mitochondria.

scholarly journals De novo gonad transcriptome analysis of the common littoral shrimp Palaemon serratus: novel insights into sex-related genes

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Inés González-Castellano ◽  
Chiara Manfrin ◽  
Alberto Pallavicini ◽  
Andrés Martínez-Lage
Keyword(s):  
Transcriptome Analysis ◽  
De Novo ◽  
Expression Patterns ◽  
Gonadal Development ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Illumina Hiseq ◽  
Specific Expression ◽  
Development Processes ◽  
The Common

Abstract Background The common littoral shrimp Palaemon serratus is an economically important decapod resource in some European communities. Aquaculture practices prevent the genetic deterioration of wild stocks caused by overfishing and at the same time enhance the production. The biotechnological manipulation of sex-related genes has the proved potential to improve the aquaculture production but the scarcity of genomic data about P. serratus hinders these applications. RNA-Seq analysis has been performed on ovary and testis samples to generate a reference gonadal transcriptome. Differential expression analyses were conducted between three ovary and three testis samples sequenced by Illumina HiSeq 4000 PE100 to reveal sex-related genes with sex-biased or sex-specific expression patterns. Results A total of 224.5 and 281.1 million paired-end reads were produced from ovary and testis samples, respectively. De novo assembly of ovary and testis trimmed reads yielded a transcriptome with 39,186 transcripts. The 29.57% of the transcriptome retrieved at least one annotation and 11,087 differentially expressed genes (DEGs) were detected between ovary and testis replicates. Six thousand two hundred seven genes were up-regulated in ovaries meanwhile 4880 genes were up-regulated in testes. Candidate genes to be involved in sexual development and gonadal development processes were retrieved from the transcriptome. These sex-related genes were discussed taking into account whether they were up-regulated in ovary, up-regulated in testis or not differentially expressed between gonads and in the framework of previous findings in other crustacean species. Conclusions This is the first transcriptome analysis of P. serratus gonads using RNA-Seq technology. Interesting findings about sex-related genes from an evolutionary perspective (such as Dmrt1) and for putative future aquaculture applications (Iag or vitellogenesis genes) are reported here. We provide a valuable dataset that will facilitate further research into the reproductive biology of this shrimp.

scholarly journals Zika infection of neural progenitor cells perturbs transcription in neurodevelopmental pathways

2016 ◽  
Author(s):  
Lynn Yi ◽  
Harold Pimentel ◽  
Lior Pachter
Keyword(s):  
Progenitor Cells ◽  
Transcriptome Analysis ◽  
Neural Progenitor Cells ◽  
Expression Patterns ◽  
Neural Progenitor ◽  
Neural Cells ◽  
Rna Seq ◽  
Transcriptional Dysregulation ◽  
Human Neural Progenitor Cells ◽  
Infection Pathways

AbstractBackgroundA recent study of the gene expression patterns of Zika virus (ZIKV) infected human neural progenitor cells (hNPCs) revealed transcriptional dysregulation and identified cell-cycle-related pathways that are affected by infection. However deeper exploration of the information present in the RNA-Seq data can be used to further elucidate the manner in which Zika infection of hNPCs affects the transcriptome, refining pathway predictions and revealing isoform-specific dynamics.Methodology/Principal FindingsWe analyzed data published by Tang et al. using state-of-the-art tools for transcriptome analysis. By accounting for the experimental design and estimation of technical and inferential variance we were able to pinpoint Zika infection affected pathways that highlight Zika’s neural tropism. The examination of differential genes reveals cases of isoform divergence.Conclusions/SignificanceTranscriptome analysis of Zika infected hNPCs has the potential to identify the molecular signatures of Zika infected neural cells. These signatures may be useful for diagnostics and for the resolution of infection pathways that can be used to harvest specific targets for further study.

scholarly journals Molecular cloning and expression profile of β-ketoacyl-acp synthase gene from tung tree (Vernicia fordii Hemsl.)

Genetika ◽  
2015 ◽  
Vol 47 (1) ◽  
pp. 143-159 ◽  
Author(s):  
Hongxu Long ◽  
Xiaofeng Tan ◽  
Fangfang Yan ◽  
Lin Zhang ◽  
Ze Li ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Full Length ◽  
Cloning And Expression ◽  
Tung Tree ◽  
Degenerate Primers ◽  
Expression Levels ◽  
Full Length Cdna ◽  
Vernicia Fordii

Tung tree (Vernicia fordii) is an important woody oil tree. Tung tree seeds contain 50-60% oil with approximately 80 mole ?-eleostearic acid (9 cis, 11 trans, 13 trans octadecatrienoic acid). Fatty acid synthesis is catalyzed by the concerted action of acetyl-CoA carboxylase and fatty acid synthase, a multienzyme complex including ?-ketoacyl-acyl-carrier-protein synthase (KAS). Little is known about KAS in tung tree. The objective of this study was to clone KAS genes and analyze their expression profiles in tung tree. A full-length cDNA encoding KAS III and a partial cDNA encoding KAS II were isolated from tung tree by PCR cloning using degenerate primers and rapid amplification of cDNA ends system. The full-length cDNA of VfKAS III was 1881 bp in length with an open reading frame of 1212 bp. VfKAS III genomic DNA was also isolated and sequenced, which contained 8 exons in 5403 bp length. The deduced VfKAS III protein shared approximately 80% identity with homologous KAS IIIs from other plants. Quantitative PCR analysis revealed that KAS II and KAS III were expressed in all of the tissues and organs tested but exhibited different expression patterns in tung tree. The expression levels of KAS II in young tissues were much lower than those in mature tissues, whereas the highest expression levels of KAS III were observed in young stem and young leaf. These results should facilitate further studies on the regulation of tung oil biosynthesis by KAS in tung tree.

scholarly journals Cloning, sequencing, and expression analysis of 32 NAC transcription factors (MdNAC) in apple

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8249
Author(s):  
Huifeng Li ◽  
Kun Ran ◽  
Qinglong Dong ◽  
Qiang Zhao ◽  
Song Shi
Keyword(s):  
Transcription Factors ◽  
Expression Patterns ◽  
Transcriptional Level ◽  
Rna Seq ◽  
Expression Levels ◽  
Nac Transcription Factors ◽  
Cdna Sequences ◽  
Link Type ◽  
Growth Development ◽  
Different Tissues

Background NAC transcription factors play important roles in the regulation of plant growth, development, abiotic and biotic stress responses. The transcriptional level of MdNACs in different tissues and under various biotic and abiotic stress treatments was determined to provide a solid foundation for studying the function of MdNACs. Methods Thirty-two full-length cDNA sequences of Md NACs were isolated by homologous comparison and RT-PCR confirmation, and the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. The prediction of subcellular locations of MdNAC proteins was performed using CELLO v.2.5, PSORT, and SoftBerry ProtComp 9.0. Expression levels of MdNACs were detected in 16 different tissues using an array. Expression patterns of MdNACs were detected in response to Alternaria alternata apple pathotype (AAAP) infection using RNA-seq, and the expression of MdNACs was analyzed under NaCl and mannitol treatments using RT-qPCR. Results The sequencing results produced 32 cDNAs (designated as MdNAC24-39, MdNAC54-65, and MdNAC67-70 with GenBank accession No. MG099861–MG099876, MG099891–MG099902, and MG099904–MG099907, respectively). Phylogenetic analysis revealed that MdNAC34 belonged to the ATAF group, MdNAC63 belonged to the AtNAC3 group, MdNAC24, MdNAC26-30, MdNAC32-33, MdNAC35, MdNAC37-39, MdNAC56-57, MdNAC59-62, MdNAC64-65, and MdNAC67-70 belonged to the NAM group, and MdNAC25, MdNAC36, MdNAC54-55, and MdNAC58 belonged to the VND group. Predictions of subcellular localization showed that MdNAC24-27, MdNAC29-30, MdNAC33-37, MdNAC39, MdNAC54-65, and MdNAC67-70 proteins were located in the nucleus, MdNAC28 proteins were located in the cytoplasm, MdNAC31-32 proteins were located in the nucleus and cytoplasm, and MdNAC38 proteins were located in the nucleus and plasma membrane. Array results indicated that 32 MdNACs were expressed in all examined tissues at various expression levels. RNA-seq results showed that expression levels of MdNAC26-28, MdNAC33-34, MdNAC60, MdNAC62-65, and MdNAC68 were induced, but MdNAC24, MdNAC32, and MdNAC58 were down-regulated in response to AAAP infection. Under salt treatment, MdNAC24, MdNAC27, MdNAC29, MdNAC34, MdNAC37, MdNAC39, MdNAC54, MdNAC59, and MdNAC63 transcription levels were induced. Under mannitol treatment, MdNAC32 and MdNAC54 transcription levels were induced, but MdNAC24, MdNAC28, MdNAC30, MdNAC33, MdNAC35, MdNAC37, MdNAC55, MdNAC56, MdNAC58, and MdNAC59 were down-regulated. Taken together, the results indicated that the cloned MdNAC genes were expressed constitutively in all examined tissues. These genes were up-regulated or down-regulated in response to AAAP infection and to salt or mannitol, which suggested they may be involved in the regulation of growth, development, and stress response in apple.

scholarly journals Identification of dominant transcripts in oxidative stress response by a full-length transcriptome analysis

2020 ◽  
Author(s):  
Akihito Otsuki ◽  
Yasunobu Okamura ◽  
Yuichi Aoki ◽  
Noriko Ishida ◽  
Kazuki Kumada ◽  
...  
Keyword(s):  
Stress Response ◽  
Target Genes ◽  
Transcriptome Profiling ◽  
Transcript Level ◽  
Full Length ◽  
Rna Seq ◽  
Short Read ◽  
Expression Levels ◽  
Long Read ◽  
Electrophilic Stress

Our body responds to environmental stress by changing the expression levels of a series of cytoprotective enzymes/proteins through multilayered regulatory mechanisms, including the KEAP1-NRF2 system. While NRF2 upregulates the expression of many cytoprotective genes, there are fundamental limitations in short-read RNA sequencing (RNA-Seq), resulting in confusion regarding interpreting the effectiveness of cytoprotective gene induction at transcript level. To precisely delineate isoform usage in the stress response, we conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) analyses of lymphoblastoid cells from three volunteers under normal and electrophilic stress-induced conditions. We first determined the first exon usage in KEAP1 and NFE2L2 (encoding NRF2) and found the presence of transcript diversity. We then examined changes in isoform usage of NRF2 target genes under stress conditions and identified a few isoforms dominantly expressed in the majority of NRF2 target genes. The expression levels of isoforms determined by Iso-Seq analyses showed striking differences from those determined by short-read RNA-Seq; the latter could be misleading in regards to the abundance of transcripts. These results support that transcript usage is tightly regulated to produce functional proteins under electrophilic stress. Our present study strongly argues that there are important benefits that can be achieved by long-read transcriptome sequencing.

scholarly journals Endocuticle is involved in caste differentiation of the lower termite

2021 ◽  
Author(s):  
Chenxu Ye ◽  
Zhuanzhuan Song ◽  
Taoyu Wu ◽  
Wenxiu Zhang ◽  
Noor us Saba ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Expression Patterns ◽  
Caste Differentiation ◽  
Developmental Pattern ◽  
Dynamic Changes ◽  
Expression Levels ◽  
Lower Termite ◽  
Primary Reproductives ◽  
Caste Development ◽  
Differentiation Pathways

Abstract Caste differentiation in termites is one of the most conspicuous examples of facultative polyphenism in animals. It is clear that specific cuticular formation occurs in hard exocuticles during caste differentiation. However, the developmental pattern of the soft endocuticle in the differentiation pathways of castes is unknown. To reveal whether the endocuticle is involved in caste differentiation, we compared the exocuticle and endocuticle thickness of individuals in 2 pathways (nymph line and worker line) of caste differentiation in the termite Reticulitermes aculabialis. The endocuticle protein genes were identified by transcriptome analysis and the expression patterns of these genes were confirmed in caste differentiation. We found that the endocuticle structure showed dynamic changes in 2 pathways, and the first difference in endocuticle structure occurred after larvae differentiation bifurcated into workers and nymphs. The thinning of the endocuticle was a significant event from nymphs developing into alates with the thickest exocuticle and thinnest endocuticle. The thickest endocuticle layers were found in the heads of the workers and the ultrastructure of the endocuticle in the heads was more complex than that in the thorax–abdomens. Six endocuticle protein genes were identified and annotated as endocuticle structural glycoproteins SgAbd-2, SgAbd-9, and Abd-5. The expression levels of endocuticle protein genes changed dramatically during caste development and the expression levels in neotenic reproductives (secondary reproductives) were significantly higher than those in alates (primary reproductives). These results reveal the roles of endocuticles in caste differentiation and adaptation to the environment.

scholarly journals Zebrafish retinal mRNA RNA-seq data processing

2020 ◽  
Author(s):  
Nicholas Owen ◽  
Mariya Moosajee
Keyword(s):  
Data Processing ◽  
Library Preparation ◽  
Rna Seq ◽  
Sequencing Data ◽  
Mrna Sequencing

Abstract This protocol details the step-by-step procedures followed to process zebrafish retinal mRNA sequencing data generated by the SMARTSeq2 library preparation protocols in the manuscript Richardson et al 2019 1.

scholarly journals Transcriptome analysis of the regulation of natamycin biosynthesis in Streptomyces natalensis HW-2 by fungal elicitor

2019 ◽  
Author(s):  
Dahong Wang ◽  
Wenhao Shen ◽  
Jiangfeng Yuan ◽  
Lanlan Wei ◽  
Ying Zhang
Keyword(s):  
Gene Expression ◽  
Transcriptome Analysis ◽  
Tricarboxylic Acid Cycle ◽  
Enrichment Analysis ◽  
Pentose Phosphate ◽  
Fungal Elicitor ◽  
Natural Food ◽  
Rna Seq ◽  
Expression Levels ◽  
Streptomyces Natalensis

Abstract Background Natamycin is a polyene macrolide polyketide antibiotics and used in 150 countries as a natural food preservative. Streptomyces natalensis is an important producer. Elicitation had been approved to be an effective method to improve the biosynthesis of secondary metabolites. Fungal elicitor from Penicillium chrysogenum AS 3.5163 showed inductive effect on the biosynthesis of natamycin in S. natalensis HW-2 fermentation. However, regarding the global gene expression of natamycin in response to fungal elicitor is not still reported. Results RNA-Seq analysis showed that there were 1265 differential expression genes (DEGs) at 40 h and 2196 DEGs at 80 h. The fungal elicitor had stronger effects on the transcription level of S. natalensis HW-2 at 80 h than that at 40 h. Gene Ontology (GO) enrichment analysis of DEGs showed significant enrichment in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the fungal elicitor mainly affected the expression levels of some genes about cellular process, metabolism and genetic information, especially in pentose phosphate pathway (PPP), glycolytic pathway (EMP) and tricarboxylic acid cycle (TCA). KEGG pathway showed that fungal elicitor had a greater influence on the metabolism of branched-chain amino acids (BCAAs). Among them, 23 DEGs associated with BCAAs metabolism were up-regulated or down-regulated. The supplementation experiment with BCAAs confirmed that 0.2 g/L of L-Ile and 0.5 g/L of L-Val increased natamycin yield by 17.6% and 37.8%, respectively. Fungal elicitor also up-regulated the transcriptional levels of most of the enzymes associated with the biosynthesis of natamycin and two important transcription regulators ( pimR and pimM ). To confirm the accuracy of RNA-Seq, the results of qPCR showed that these gene expression levels were in agreement with the transcription changes by RNA-Seq. Conclusion In this study, the change of transcriptional levels in S. natalensis HW-2 under treated with the fungal elicitor was firstly reported. The major finding of our comparative transcriptome analysis is that the fungal elicitor improves the supply of precursor, and alters the expression of natamycin related genes and regulator of secondary metabolism. From our results, we conclude that regulatory alterations are important factors for the enhancement of natamycin.

scholarly journals Comparative evaluation of full-length isoform quantification from RNA-Seq

2019 ◽  
Author(s):  
Dimitra Sarantopoulou ◽  
Soumyashant Nayak ◽  
Thomas G. Brooks ◽  
Nicholas F. Lahens ◽  
Gregory R. Grant
Keyword(s):  
Structural Parameters ◽  
Differential Expression Analysis ◽  
Full Length ◽  
Rna Seq ◽  
Rna Transcripts ◽  
Fundamental Difficulty ◽  
Isoform Quantification ◽  
Realistic Data ◽  
Naive Approach ◽  
Better Than

AbstractFull-length isoform quantification from RNA-Seq is a key goal in transcriptomics analyses and an area of active development. The fundamental difficulty stems from the fact that RNA transcripts are long, while RNA-Seq reads are typically short. We have generated realistic benchmarking data, and have performed a comprehensive comparative analysis of isoform quantification, including evaluating them on the level of differential expression analysis. Genome, transcriptome and pseudo alignment-based methods are included; and a naive approach is included to establish a baseline. Kallisto, RSEM, and Cufflinks exhibit the highest accuracy on idealized data, while on more realistic data they do not perform considerably better than the naive approach. We determine the effect of structural parameters, such as number of exons or number of isoforms, on accuracy. Overall, the tested methods show sufficient divergence from the truth to suggest that full-length isoform quantification should be employed selectively.

Sign in / Sign up

Export Citation Format

Share Document