Next-generation transcriptome assembly

Martin, Jeffrey A.; Wang, Zhong

doi:10.1038/nrg3068

Cited by 1,090 publications

(963 citation statements)

References 72 publications

Supporting

Mentioning

942

Contrasting

Unclassified

Order By: Relevance

“…RNA sequencing (RNA-seq) has been successfully used to define the transcriptome and to find novel transcripts absent from the reference genome (Martin and Wang 2011). Compared to genome sequencing, RNA-seq is more economical, especially in the exploration of the complex maize genome containing more than 85% repetitive sequences (Schnable et al 2009).…”

Section: Introductionmentioning

confidence: 99%

Maize pan-transcriptome provides novel insights into genome complexity and quantitative trait variation

Jin

Liu

et al. 2015

Preprint

View full text Add to dashboard Cite

Variation in gene expression contributes to the diversity of phenotype. The construction of the pan-transcriptome is especially necessary for species with complex genomes, such as maize. However, knowledge of the regulation mechanisms and functional consequences of the pan-transcriptome is limited. In this study, we identified 13,382 nuclear expression presence and absence variation candidates (ePAVs, expressed in 5%~95% lines; based on the reference genome) by re-analyzing the RNA sequencing data from the kernels (15 days after pollination) of 368 maize diverse inbreds. It was estimated that only ~1% of the ePAVs are explained by DNA sequence presence and absence variations (PAV). The ePAV genes tend to be regulated by distant eQTLs when compared with non-ePAV genes (called here core expression genes, expressed in more than 95% lines). When the expression presence/absence status was used as the "genotype" to perform genome-wide association study, 56 (0.42%) ePAVs were significantly associated with 15 agronomic traits and 1,967 (14.74%) with 526 metabolic traits, measured from the mature kernels. While the above was majorly based on the reference genome, by using a modified 'assemble-then-align' strategy, 2,355 high confidence novel sequences with a total length of 1.9Mb were found absent in the current B73 reference genome (v2). Ten randomly selected novel sequences were validated with genomic PCR. A simulation analysis suggested that the pan-transcriptome of the maize whole kernel is approaching a maximum value of 63,000 genes. Two novel validated sequences annotated as NBS_LRR like genes were found to associate with flavonoid content and their homologs in rice were also found to affect flavonoids and disease-resistance. Novel sequences absent in the present reference genome might be functionally important and deserve more attentions. This study provides novel perspectives and resources to discover maize quantitative trait variations and help us to better understand the kernel regulation networks, thus enhancing maize breeding.

show abstract

Section: Introductionmentioning

confidence: 99%

Maize pan-transcriptome provides novel insights into genome complexity and quantitative trait variation

Jin

Liu

et al. 2015

Preprint

View full text Add to dashboard Cite

show abstract

“…As normalized expression intensity with RPKM is dependent on the length of the gene, we would underestimate the expression level of the gene in the case of single cell cDNAs with short length (under 1 kb on average at most) if the gene has a greater length. Sequencing reads were assigned across isoforms using a modified expectation/maximization algorithm (Martin and Wang, 2011). Based on the evidence that single cell cDNA was short in length in this study (Kurimoto et al, 2006; Fig.…”

Section: Resultsmentioning

confidence: 99%

A simple and novel method for RNA‐seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase

Brouilette

Kuersten

Mein

et al. 2012

Developmental Dynamics

View full text Add to dashboard Cite

Background: Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus, a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. Results and Conclusions: Here, we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus, this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs. Developmental Dynamics 241:1584-1590, 2012. V C 2012 Wiley Periodicals Inc.

show abstract

“…Reference-based approaches are more sensitive and robust to sequencing errors, but depend on the quality of the reference genome and the evolutionary distance to the target organism (Chang et al 2014). In contrast, de novo methods can reconstruct novel and trans-spliced transcripts and are not affected by misassemblies in the reference genome (Martin and Wang 2011). In order to take advantage of both methods and increase the robustness of the transcriptome assemblies of our non-model species, we used both approaches complementarily.…”

Section: Read Processing and Assemblymentioning

confidence: 99%

Genomics of Adaptation to Multiple Concurrent Stresses: Insights from Comparative Transcriptomics of a Cichlid Fish from One of Earth’s Most Extreme Environments, the Hypersaline Soda Lake Magadi in Kenya, East Africa

et al. 2015

View full text Add to dashboard Cite

The Magadi tilapia (Alcolapia grahami) is a cichlid fish that inhabits one of the Earth's most extreme aquatic environments, with high pH ( -10), salinity (-60 % of seawater), high temperatures ( -40 °C), and fluctuating oxygen regimes. The Magadi tilapia evolved several unique behavioral, physiological, and anatomical adaptations, some of which are constituent and thus retained in freshwater conditions. We conducted a transcriptomic analysis on A. grahami to study the evolutionary basis of tolerance to multiple stressors. To identify the adaptive regulatory changes associated with stress responses, we massively sequenced gill transcriptomes (RNAseq) from wild and freshwater-acclimated specimens of A. grahami. As a control, corresponding transcriptome data from Oreochromis leucostictus, a closely related freshwater species, were generated. We found expression differences in a large number of genes with known functions related to osmoregulation, energy metabolism, ion transport, and chemical detoxification. Over-representation of metabolism-related gene ontology terms in wild individuals compared to laboratory-acclimated specimens suggested that freshwater conditions greatly decrease the metabolic requirements of this species. Twenty-five genes with diverse physiological functions related to responses to water stress showed signs of divergent natural selection between the Magadi tilapia and its freshwater relative, which shared a most recent common ancestor only about four million years ago. The complete set of genes responsible for urea excretion was identified in the gill transcriptome of A. grahami, making it the only fish species to have a functional ornithine-urea cycle pathway in the gills a major innovation for increasing nitrogenous waste efficiency.

show abstract

Next-generation transcriptome assembly

Cited by 1,090 publications

References 72 publications

Maize pan-transcriptome provides novel insights into genome complexity and quantitative trait variation

Maize pan-transcriptome provides novel insights into genome complexity and quantitative trait variation

A simple and novel method for RNA‐seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase

Genomics of Adaptation to Multiple Concurrent Stresses: Insights from Comparative Transcriptomics of a Cichlid Fish from One of Earth’s Most Extreme Environments, the Hypersaline Soda Lake Magadi in Kenya, East Africa

Contact Info

Product

Resources

About