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Challenges and strategies in transcriptome assembly and differential gene expression quantification. A comprehensive in silico assessment of RNA-seq experiments
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
2013 (English)In: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 22, no 3, 620-634 p.Article in journal (Refereed) Published
Abstract [en]

Transcriptome Shotgun Sequencing (RNA-seq) has been readily embraced by geneticists and molecular ecologists alike. As with all high-throughput technologies, it is critical to understand which analytic strategies are best suited and which parameters may bias the interpretation of the data. Here we use a comprehensive simulation approach to explore how various features of the transcriptome (complexity, degree of polymorphism p, alternative splicing), technological processing (sequencing error e, library normalization) and bioinformatic workflow (de novo vs. mapping assembly, reference genome quality) impact transcriptome quality and inference of differential gene expression (DE). We find that transcriptome assembly and gene expression profiling (EdgeR vs. BaySeq software) works well even in the absence of a reference genome and is robust across a broad range of parameters. We advise against library normalization and in most situations advocate mapping assemblies to an annotated genome of a divergent sister clade, which generally outperformed de novo assembly (Trans-Abyss, Trinity, Soapdenovo-Trans). Transcriptome complexity (size, paralogs, alternative splicing isoforms) negatively affected the assembly and DE profiling, whereas the effects of sequencing error and polymorphism were almost negligible. Finally, we highlight the challenge of gene name assignment for de novo assemblies, the importance of mapping strategies and raise awareness of challenges associated with the quality of reference genomes. Overall, our results have significant practical and methodological implications and can provide guidance in the design and analysis of RNA-seq experiments, particularly for organisms where genomic background information is lacking.

Place, publisher, year, edition, pages
2013. Vol. 22, no 3, 620-634 p.
Keyword [en]
bioinformatics, comparative genomics, differential gene expression, RNA-seq, simulation, systems biology, transcriptome assembly
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-194741DOI: 10.1111/mec.12014ISI: 000313726300007OAI: oai:DiVA.org:uu-194741DiVA: diva2:606501
Available from: 2013-02-19 Created: 2013-02-19 Last updated: 2017-12-06Bibliographically approved
In thesis
1. The Genetics of Speciation and Colouration in Carrion and Hooded Crows
Open this publication in new window or tab >>The Genetics of Speciation and Colouration in Carrion and Hooded Crows
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A fundamental goal in biological research is to gain an understanding of the evolutionary processes and genetic elements that drive speciation. Genes responsible for reproductive isolation in young divergent lineages are particularly poorly known. In this thesis, the speciation genetics of carrion (Corvus (corone) corone) and hooded (C. (corone) cornix) crows were studied. These taxa differ strikingly in colouration and meet in a narrow hybrid zone in Europe, yet appear to be very similar genetically. A major component of reproductive isolation is social selection on colour differences.

First, we investigated the genetic basis of plumage divergence between carrion and hooded crows using a candidate gene approach. Nucleotide divergence was confirmed to be low, while there was no evidence for any of the sequenced genes to be associated with colour differences.

Second, we performed a simulation study to assess the performance of RNA-seq, a relatively novel approach that we later employed ourselves. We asked how variation in transcriptome complexity and bioinformatic workflow affected the accuracy of gene expression profiling. We generally found reassuring robustness and made a number of specific recommendations.

Third, we compared the corticosterone stress response of carrion and hooded crows. In accordance with the hypothesis that the degree of melanization and physiological traits are correlated due to pleiotropy, we found a higher stress response in hooded crows, and detected possibly associated gene expression in pituitary.

Fourth, we investigated genomic divergence by assembling a hooded crow reference genome followed by whole-genome resequencing of four European population samples. Northern European carrion crows were more similar to hooded crows than to Spanish carrion crows, pointing towards rampant introgression far beyond the hybrid zone. Nevertheless, several narrow genomic regions harboured high between-taxon divergence and were potentially associated with phenotypic traits.

Fifth, we compared whole-transcriptome gene expression profiles between crows, focusing on skin with developing feathers. We used a design that allowed to differentiate between taxon-specific, colour-specific and body patterning effects. Widespread underexpression of genes in the melanogenesis pathway was associated with grey colour, and we detected several genes that may contribute to colour divergence in this system.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1088
Keyword
evolutionary genetics, genomics, birds, next-generation sequencing, pigmentation, pigmentation genetics, eumelanin, social selection, gene expression, population genomics
National Category
Evolutionary Biology
Research subject
Biology with specialization in Evolutionary Genetics
Identifiers
urn:nbn:se:uu:diva-209243 (URN)978-91-554-8777-5 (ISBN)
Public defence
2013-11-29, Lindahlssalen, Evolutionary Biology Centre, Norbyvägen 18D, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2013-11-08 Created: 2013-10-15 Last updated: 2014-01-23

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Vijay, NagarjunPoelstra, Jelmer W.Wolf, Jochen B. W.

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