De novo transcriptome assembly and polymorphism detection in the flowering plant Silene vulgaris (Caryophyllaceae)

Abstract

Members of the angiosperm genus Silene are widely used in studies of ecology and evolution, but available genomic and population genetic resources within Silene remain limited. Deep transcriptome (i.e. expressed sequence tag or EST) sequencing has proven to be a rapid and cost-effective means to characterize gene content and identify polymorphic markers in non-model organisms. In this study, we report the results of 454 GS-FLX Titanium sequencing of a polyA-selected and normalized cDNA library from Silene vulgaris. The library was generated from a single pool of transcripts, combining RNA from leaf, root and floral tissue from three genetically divergent European subpopulations of S. vulgaris. A single full-plate 454 run produced 959,520 reads totalling 363.6 Mb of sequence data with an average read length of 379.0 bp after quality trimming and removal of custom library adaptors. We assembled 832,251 (86.7%) of these reads into 40,964 contigs, which have a total length of 25.4 Mb and can be organized into 18,178 graph-based clusters or 'isogroups'. Assembled sequences were annotated based on homology to genes in multiple public databases. Analysis of sequence variants identified 13,432 putative single-nucleotide polymorphisms (SNPs) and 1320 simple sequence repeats (SSRs) that are candidates for microsatellite analysis. Estimates of nucleotide diversity from 1577 contigs were used to generate genome-wide distributions that revealed several outliers with high diversity. All of these resources are publicly available through NCBI and/or our website (http://silenegenomics.biology.virginia.edu) and should provide valuable genomic and population genetic tools for the Silene research community.

De novo transcriptome assembly and novel microsatellite marker information in Capsicum annuum varieties Saengryeg 211 and Saengryeg 213

“…Assembled contigs are organized into 'isogroups’ which represent all contigs from a given genetic locus. Within each isogroup, contigs can be connected in different permutations (termed 'isotigs’), each of which can be loosely thought of as a specific splice variant or allele (Sloan et al 2012; http://454.com/downloads/my454/documentation/gs-flx-plus/454SeqSys_SWManual-v2.6_PartC_May2011.pdf). …”

“…Recent advances in next generation sequencing (NGS) technologies have created unprecedented opportunities for generating genomic information in even previously uncharacterized systems (Logacheva et al 2011; Lu et al 2011; McDowell et al 2011; Sloan et al 2012). The process of whole genome sequencing, rapid identification and annotations of gene sequences (Mizrachi et al 2010; Garg et al 2011), novel and alternatively spliced genes (Roberts and Smith 2002) and the determination of the variations in nucleotide sequences provides new platform for gene expression analysis (Ameline-Torregrosa et al 2006; Kim et al 2006; Cohen et al 2010) and identification of regulatory elements, target critical genes and enormous polymorphic molecular markers motifs (Gore et al 2009; Tangphatsornruang et al 2009; You et al 2011; Zalapa et al 2012; Zhu et al 2012).…”

Transcriptome analysis revealed the dynamic oil accumulation in Symplocos paniculata fruit

“…It is widely used for exploring functional genes [11], constructing expression and transcriptional regulatory profiles [12], discovering molecular markers such as simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) [13, 14], and investigating comparative and evolutionary genomics [15, 16]. This technique is efficient to generate a large amount of genetic data.…”

De novo transcriptome assembly of Ipomoea nil using Illumina sequencing for gene discovery and SSR marker identification

“…This number (106,552) is lower than the predicted number of proteincoding genes (137,307) in the Blast2GO annotation, mainly attributable to the fact that many relatively short contigs (ORF ≤ 100aa) were annotated as well (Ramilowski et al 2013). Most of these assignments were based on marginal BLAST hits near the significance threshold and often involved low-complexity sequences, which are prone to producing false-positive hits (Sloan et al 2012). There are still contigs without significant matches to the existing databases, which could reflect either novel, specific genes of I. nil or non-coding RNAs or fragments of longer RNAs, etc.…”

“…Additionally, the cDNA library was constructed using pooled RNA samples from four representative tissue types. Since the aims of this project were to generate as many cDNA sequences as possible, pooling was a cost-effective strategy based on statistical and practical considerations (Sloan et al 2012;Xu et al 2012).…”