Of contigs and quagmires: next‐generation sequencing pitfalls associated with transcriptomic studies

DeWoody, J. Andrew; Abts, Kendra C.; Fahey, Anna L.; Ji, Yanzhu; Kimble, Steven J. A.; Marra, Nicholas J.; Wijayawardena, Bhagya K.; Willoughby, Janna R.

doi:10.1111/1755-0998.12107

Cited by 21 publications

(15 citation statements)

References 70 publications

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“…The problem that this creates in subsequent analysis and annotation of new transcriptomes, is that of self-propagation, in that the best BLAST hits remain hypothetical unknowns with regard to functional annotation (DeWoody et al 2013).…”

Section: Transcriptomics and Ancestral Reconstructionmentioning

confidence: 99%

Ancestral reconstruction of tick lineages

Mans

Castro

Pienaar

et al. 2016

Ticks and Tick-borne Diseases

101

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Section: Transcriptomics and Ancestral Reconstructionmentioning

confidence: 99%

Ancestral reconstruction of tick lineages

Mans

Castro

Pienaar

et al. 2016

Ticks and Tick-borne Diseases

101

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“…In-depth reviews regarding assembly tools, metrics and pitfalls in dealing with transcriptome assemblies and analyses have also been previously provided by several groups [37][38][39][40]. Many transcriptome assemblies have relied on paired-end Illumina sequencing to achieve sequencing depth to obtain a comprehensive transcriptome.…”

Section: Discussionmentioning

confidence: 99%

Transcriptome Survey of a Marine Food Fish: Asian Seabass (Lates calcarifer)

Thevasagayam

Sridatta

Jiang

et al. 2015

JMSE

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The Asian seabass (or barramundi; Lates calcarifer) is a marine teleost and a popular food fish in Southeast Asia and Australia. To date, comprehensive genome and OPEN ACCESS J. Mar. Sci. Eng. 2015, 3 383 transcriptome sequence information has not been available for this species in public repositories. Here, we report a comprehensive de novo transcriptome assembly of the Asian seabass. These data will be useful for the development of molecular tools for use in aquaculture of Asian seabass as well as a resource for genome annotation. The transcriptome was obtained from sequences generated from organs of multiple individuals using three different next-generation sequencing platforms (454-FLX Titanium, SOLiD 3+, and paired-end Illumina HiSeq 2000). The assembled transcriptome contains >80% of the expected protein-coding loci, with 58% of these represented by a predicted full-length cDNA sequence when compared to the available Nile tilapia RefSeq dataset. Detailed descriptions of the various steps involved in sequencing and assembling a transcriptome are provided to serve as a helpful guide for transcriptome projects involving de novo assembly of short sequence reads for non-model teleosts or any species of interest.

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“…Previously, there have been cursory surveys of MHC diversity in the D. spectabilis genome [36]. However, genomic resources in this group are limited and those that do exist (e.g., a low-coverage genome in D. ordii [65]) are poorly annotated and too shallow for effective use in sequence assembly across the Heteromyidae [66]. Thus, we conducted RNA-sequencing to de novo assemble transcriptomes that we used to quantify gene expression and to test for divergent selection between the tropical and temperate species.…”

Section: Discussionmentioning

confidence: 99%

Transcriptomic characterization of the immunogenetic repertoires of heteromyid rodents

Marra

DeWoody

2014

BMC Genomics

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BackgroundWhen populations evolve under disparate environmental conditions, they experience different selective pressures that shape patterns of sequence evolution and gene expression. These may be manifested in genetic and phenotypic differences such as a diverse immunogenetic repertoire in species from tropical latitudes that have greater and/or different parasite burdens than more temperate species. To test this idea, we compared the transcriptomes of one tropical species (Heteromys desmarestianus) and two species from temperate latitudes (Dipodomys spectabilis and Chaetodipus baileyi) from the Heteromyidae. We did so in a search for positive selection on sequences and/or differential expression, while controlling for phylogenetic history in our choice of species.ResultsWe identified 127,812 contigs and annotated 34,878 of these, identifying immune genes associated with interleukins, cytokines, and the production of mast cells. We identified 632 genes that were upregulated in H. desmarestianus (8.7% of genes tested) and 492 (6.7%) that were downregulated. Gene ontology terms including “immune response” were associated with 31 (4.9%) of the 632 upregulated genes. We found preliminary evidence for positive selection on three genes (Palmitoyltransferase ZDHHC5 Ubiquitin-conjugating enzyme E2 N, Krueppel-like factor 10, and Spindle and kinetochore-associated protein 1) along the H. desmarestianus lineage.ConclusionsOverall our findings pinpoint genes in species from disparate environments that are on different evolutionary trajectories in terms of expression levels and/or nucleotide sequence. Our data indicate there are significant differences in the expression of genes among the spleen transcriptomes of these species and that a number of these differentially expressed genes do not show the same pattern of differential expression in another tissue type. This points to the possibility of expression differences between these species specific to the spleen transcriptome.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-929) contains supplementary material, which is available to authorized users.

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Of contigs and quagmires: next‐generation sequencing pitfalls associated with transcriptomic studies

Cited by 21 publications

References 70 publications

Ancestral reconstruction of tick lineages

Ancestral reconstruction of tick lineages

Transcriptome Survey of a Marine Food Fish: Asian Seabass (Lates calcarifer)

Transcriptomic characterization of the immunogenetic repertoires of heteromyid rodents

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