Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing

“Transcriptome analysis is rapid and comprehensive and has been constructed and annotated to assist in the identification of differentially expressed genes (DEGs) in different plant populations [33].”

“De novo assembly is an established technique for developing genome-level resources for non-model organisms, including plant species (Vera et al., 2008; Meyer et al., 2009; Der et al., 2011).”

“Large collections of EST sequences are very important for gene annotation and discovery [19], comparative genomics [20], development of molecular markers [16], and population genomics studies of genetic variation associated with adaptive traits [21].”

“The methods are now faster, have higher throughputs, and lower costs (Morozova and Marra, 2008; Vera et al., 2008) than other sequencing methods, and the results are now accepted by international programs such as the International Census of Marine Microbes (ICoMM, http://icomm.mbl.edu/).”

“Transcriptomes also include a large number of loci with known or predictable functions [22,23] and have been applied to comparative genomics [24], marker discovery [25], and population genomic studies [26].”

“…platform resulted in over 500000 high-quality reads, corresponding to a relatively high average coverage (>10×) of the assembled 14871 isotigs with a high average length of 1133 bp. Comparable to previous transcriptome sequencing studies [40,41], 88% of the reads could be assembled into contigs.”

“44, a freely available script designed to help run a de novo transcriptome annotation pipeline [49,64].”

“That ribosomal gene you find so interesting might be from your target organism, but it might also be from a pathogen, parasite, commensal microorganism or prey item (e.g. Vera et al. 2008).”

“organisms [28, 29].”

“Such populations represent less genetic diversity (6 to 20 SNPs per kilobase) and are sequenced to shallower depths (6-23X) [7-11].”

“Successful applications of these high-throughput sequencing technologies to transcriptome analysis have been reported [33-37].”

“…the genetic basis of adaptation is undergoing dramatic transformation owing to recent advances in molecular genetics and genomics that are redefining what constitutes a ‘model organism’ for intensive biological investigation (Hedges 2002; Ungerer et al. 2007; Vera et al. 2008; Thomson et al. 2010).”

“New molecular tools that are now available, especially high-throughput sequencing (Marioni et al., 2008; Vera et al., 2008) allowing for metagenomic analysis (Cox-Foster et al.”

“been expanded using next-generation sequencing (NGS) technologies [9,10].”

“…with genetic variation in a set of candidate genes that were selected on the basis of previous genomic (Saastamoinen et al. 2013) and transcriptomic (Vera et al. 2008; Kvist et al. 2013) studies, and which have a known role in the physiology or life history of this or other arthropod species.”

“Large collections of EST sequences have proven invaluable for gene annotation and discovery [9, 11], comparative genomics [12], development of molecular markers [13, 14], and for population genomic studies of genetic variation associated with adaptive traits [15].”

“[30 ] recently determined 608 053 ESTs of a butterfly Melitaea cinxia that has been intensively studied by ecologists.”

“In particular, the sequencing of transcriptomes results to be a costeffective analysis strategy, especially for nonmodel organisms (Vera et al. 2008; Elmer et al. 2010; Ekblom & Galindo 2011).”

“Moreover, such approaches allow at the same time pinpointing the genomic basis of ecologically relevant traits and their evolutionary history [15–19].”

“tional genomic studies, even if rich genetic resources are unavailable [14].”

“In the transcriptome analysis of another nonmodel organism, Melitaea cinxia (L.), Vera et al. (2008) noted a similar depth variation among contigs, while rarer transcripts were oft en hard to fi nd. Additional potential challenges arise in the investigation of the PCG genome because of the lack of…”

“With the next generation 454 pyrosequencing technology (Nordstrom et al. 2001; Vera et al. 2008) it is now possible to simultaneously evaluate multi-transcriptomic samples both qualitatively (gene discovery) and quantitatively (gene expression), and with the completion of genome sequencing, assembly and annotation of…”

“The Genome Sequencer FLX System (GS FLX), powered by 454 sequencing technology, was selected as the sequencing platform because this technology is more appropriate than other next-generation sequencing (NGS) platforms (such as Illumina Solexa and ABI SOLiD) for de novo transcriptome sequencing in nonmodel organisms (V…”

“Background Recent progress in the development of genomic techniques, including next generation sequencers, has greatly facilitated transcriptome analysis of ecologically important animals to reveal variations in mRNA expression patterns among closely related species and ecotypes within species [1-3].”

“ESTs typically remain uncharacterized following analysis via annotation pipelines (Coates et al., 2008; Vera et al., 2008).”

“The de novo transcriptome of a non-model organism was first published only eight years ago 55 and since then, rapidly emerging sequence technologies have been increasingly applied to the field of biomineralisation 14,56–58 .”

“Vera JC, et al have used RNA-seq to reveal the transcriptome of the Glanville fritillary butterfly, which makes RNA-seq especially attractive for non-model organisms with genomic sequences not known [12].”

“Total average GSS read length (264 nt) were longer than previous studies have reported in similar 454 sequencing runs for EST in non-model species including for Glanville fritillary (197 nt [19]), cichlid fishes (202 and 206 nt [25]) and European hake (206 nt [28]), but lower than for channel catfish (292 nt [29]), gi…”

“PipeMeta found 2,817 SNPs (2,095 were biallelic SNPs) in total from the 7,222 contigs, although 6,940 contigs contained no nucleotide substitution SNPs. SNPs that failed the criterion of having no more than two possible nucleotides were excluded from our analysis.”

“As the number of microarray platforms available for non-traditional model species is continually increasing [axolotl: [39], dolphin: [40], butterfly: [41,42], coral: [43], many fish species, for review: [44], crustaceans: [4547]], researchers focusing on these and related model systems will continue to benefit from th…”

“Next-generation sequencing techniques allow for the collection of mRNA sequences from small amounts of tissue in parallel across individuals and species [2], at a depth and coverage that allow de novo transcriptome assembly even in the absence of a closely related genome [10].”

“The NGS enables the derivation of a global gene or RNA expression profile that could lead to the discovery of genetic markers such as Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs), and candidate transcripts as markers for ecological fitness, quantitative trait loci (QTL) and so on [17,18].”

“Indeed, recent studies have demonstrated highly successful de novo assemblies of 454 EST data for organisms with few or no prior genomic resources (e.g. Novaes et al. 2008; Vera et al. 2008; Hahn et al. 2009; Kristiansson et al. 2009; Meyer et al. 2009; Schwarz et al. 2009; Parchman et al. 2010; Fraser et al. 2011; Ga…”

“This is a particularly useful method for research on non-model organisms, including those important in studying ecological and environmental questions [3].”

“…discovery (Clark et al. 2010), phylogenomic analysis (e.g. Smith et al. 2011; Hartmann et al. 2012), detecting selection in closely related species (Elmer et al. 2010), as well as for studies of differential gene expression (e.g. Vera et al. 2008; Siebert et al. 2011), among other applications.”

“Identification of genes from xenobiotic organisms in target species transcriptomes has been reported in many earlier studies (Miller et al., 2008; Vera et al., 2008; Hale et al., 2010; Liu et al., 2013b).”

“Glanville fritillary butterfly: [33]; guppy: [34]; big sagebrush: [35]; great tit: [36]; garter snake: [37]) and to produce important genomic resources useful for various downstream approaches such as targeted resequencing, or microsatellite and SNP genotyping.”

“The sequence information obtained from de-novo transcriptome sequencing does open up for development of species specific microarrays and taq man assays, and can be used as reference transcriptome for when determining expression profiles using RNA-sequencing (RNA-seq) [8] [9] [10] [11] [12] .”

“For transcriptome sequencing in non-model plants, the Roche 454 platform was initially preferred because it generates long reads (Vera et al., 2008; Meyer et al., 2009).”

“In particular, although laboratory organisms make excellent models for understanding links between genotype and phenotype, new sequencing approaches have opened the way to studying natural populations [3–6].”

“This approach is being applied in entomology for gene discovery [20], gene expression analysis of specific tissues and at different physiological conditions [21], to analyze single nucleotide polymorphisms (SNPs) [22], development studies of resistance to insecticides [23,24], understanding endosymbiosis interactions…”

“In particular, the growing number of transcriptome databases provides important insight into the functional genomics and gene expression profiles of species not typically targeted for investigation (Vera et al. 2008; Renaut et al. 2010; Pipes et al. 2013; Ekblom et al. 2014).”

“Additional sequencing effort and dedicated approach would be needed to warrant a comprehensive transcriptome analysis in A. subrufescens (Vera et al. 2008; Wall et al. 2009; Ward et al. 2012).”

“2009) is revolutionizing genetic studies of nonmodel organisms (Vera et al. 2008; Ekblom & Galindo 2011; Vijay et al. 2013).”

“For example, high-throughput sequencing of transcriptomes has been used to identify SNPs in the nonmodel Glanville fritillary butterfly (Melitaea cinxia; Vera et al. 2008), species in the fish genus Xiphophorus (Shen et al.”

“Furthermore, until recently, the construction of next-generation transcriptomes for non-model organisms has been limited to the 454 platform due to difficulties in assembling shorter reads from other platforms into contigs (Emrich et al. 2007; Vera et al. 2008; Schuster 2008).”

“A comparison of this study with recently published results, which employed 454 sequencing by Cheung et al. (2006) and Vera et al. (2008), showed that in this study, the proportion for high-quality readings was about 90%, which was superior to their reports.”

“Even many traditional non-model systems, those without substantial genomic resources, are now adding genomic tools to address a wide range of ecological questions [4-9].”

“NGS methods facilitate whole transcriptome sequencing at a fraction of the time and cost previously required for the Sanger method [11,12].”

“In fact, an attempt using 454-based RNA-sequencing, which generates longer reads than Illumina GA and AB SOLiD, to produce a de novo assembly of a non-model species transcriptome required the additional use of clone sequences, genomic data from a related model species and some human processing to reconstruct full sequ…”

“Whole-genome sequencing (WGS) generates a complete gene database of the species.”

“[94].”

“co-importance of variation in protein sequences and expression; (3) refinement of rudimentary molecular functions”

“For example, protocols now exist to develop assays for tens to hundreds of single nucleotide polymorphisms (SNPs) based on the in silico mining of transcriptome libraries (Vera et al. 2008).”

“For example, transcriptomic analyses using NGS have now been reported in many non-model insect species [43-48].”

“For transcriptome sequencing, the superior performance of high-throughput techniques have already been demonstrated in several studies, including the plant Medicago truncatula [25], the butterfly Melitaea cinxia [26], the tree Eucalyptus grandis [27] and the freshwater fish Micropterus salmoides [28].”

“With the introduction of next-generation sequencing technology, vast amounts of genomic data from lesser-used model species may now be collected in a short time and effort [9].”

“Obtaining large numbers of valuable EST sequences via NGS is important for gene annotation and discovery [28,29], comparative genomics [30], development of molecular markers [31,32], and population genomics studies of genetic variation linked to adaptive traits [33].”

“Recently, the next-generation of sequencing methods - for example, pyrosequencing - have improved and increased the sequencing reducing time and cost compared to the traditional Sanger method [47, 56].”

“De novo NGS transcriptome sequencing data of non-model lepidopteran species provide insights into the relation between the necessary sequencing effort when using 454 technology and the obtained transcriptome completeness: 3,500 contigs for 88,841 reads in Maruca vitrata [35], 30,000 contigs in Zygaena filipendulae for…”

“Massively parallel sequencing technologies, such as 454 pyrosequencing, remove many time consuming steps involved in Sanger sequencing, and have also facilitated transcriptome sequencing at a fraction of the time and reduced cost (Vera et al., 2008).”

“2010) and the Glanville fritillary butterfly Melitaea cinxia (Vera et al. 2008).”

“Because of the potential for high throughput, accuracy, and low cost, NGS is widely applied to analyze transcriptomes both qualitatively and quantitatively, and has been used successfully for de novo transcriptome sequencing and assembly in many non-model plants, such as pine, ginseng, Artermisia annua, common vetch,…”