Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing

“Further, unlike the shorter reads generated using the Illumina or SOLiD sequencers, the length of the 454 reads allows for interpretation of sequences generated from species lacking a genome sequence or extensive transcriptome sequences for comparison (e.g., [66]).”

“…genome assembly Reference Wasp (Polistes metricus) 454 X X X Toth et al., 2007 Glanville fritillary butterfly (Melitaea cinxia) 454 X X X X Vera et al., 2008; Wheat, 2010 Garden pea (Pisum sativum) 454 X Bräutigam et al., 2008 Bigmouth bass (Micropterus salmoides) 454 X Garcia-Reyero et…”

“As genomic data become available for non-model organisms (Vera et al., 2008), comparative phylogeographic studies will allow identification of different locus-specific divergent selection patterns (Ellegren and Sheldon, 2008; Luikart et al., 2003; MacCallum and Hill, 2006) between pairs of…”

“…it has also been widely documented in laboratory studies of responses to environmental temperature and water availability (e.g. Bennett et al. 1992, Cavicchi et al. 1995, Bull et al. 2000, Gibbs et al. 2003, Bubliy & Loeschcke 2005, Driessen et al. 2007, Sørensen et al. 2007, Vera et al. 2008).”

“, adaptor trimming and size selection), an improvement over the 85–90% that passed similar quality filters in previous studies [17,18,23].”

“However, most of these studies are based on the long-read sequence data using 454 pyrosequencing or employing hybrid approach.(15,16) Although the efforts have been made to develop tools for de novo assembly using shortread sequence data, their use in transcriptome assembly has not been well demonstrated yet.”

“…are several groups generating Expressed Sequence Tag (EST) libraries for diverse butterfly taxa; e.g., Pieris rapae (http://www. ice.mpg.de/tmo/research/InsectGenome.htm), Heliconius melpomene (Jiggins et al., 2005), Bicyclus anynana (Beldade et al., 2006), and Melitaea cinxia (Vera et al., 2008).”

“These sequences also produced longer unigenes (mean = 581 bp) than those assembled in previous studies for example, butterfly (197 bp) [60], Eucalyptus grandis (247 bp) [11], coral larval (440 bp) [17], lodegpole pine (500 bp) [10]) and whitefly (clusters: 372 bp; singletons: 265 bp) [27]).”

“, 197 bp, [5]; 247 bp, [7]; 440 bp, [16]).”

“This large number of reads with paired-end information produced much longer unigenes (mean: 629 bp) than those in previous studies [35,37,49,51-53].”

“Since RNA-seq is not limited to detecting transcripts that correspond to existing genomic sequences, it is particularly attractive for non-model organisms with genomic sequences that are yet to be determined [24-27].”

“This finding provides corroborating evidence in support of the high number of ESTs in a study of a butterfly species for which there was no predicted gene homologue in Bombyx mori (Vera et al. 2008).”

“…reads have been considered difficult (Schuster 2008), and researchers working on non-model organisms have often turned to the more expensive longer sequence reads (250–450 bp) obtained by the 454 Life Sciences (Roche) technology (Margulies et al. 2005; e.g., Vera et al. 2008; Hale et al. 2009).”

“Next generation sequencing methods continue to reduce the development cost of genomic tools for nonmodel organisms and have yielded promising results in wild species (Toth et al. 2007; Vera et al. 2008).”

“At present, similar resources are available for few insect– plant systems (Whiteman and Jander 2010), though this is changing with the advent of genomic technologies (Hudson 2008, Vera et al. 2008, Gompert et al. 2010, Renn and Siemens 2010).”

“The frequency of annotated isotigs is consistent with the 20 to 40% values previously reported for de novo transcriptome assemblies of eukaryotes [20,21,31,32].”

“A large number of studies have been performed on these two kinds of sequencers [32-38].”

“Sequencing error levels are low (,1%) and arise primarily from homopolymer runs [7], but tend to be resolved in cases where there is sufficient coverage depth to allow the assembly of overlapping reads [8].”

“…, Heliconius erato , and Melitaea cinxia [11]; TGICL Pythium ultimum [31]; Zoarces viviparous [32]; Medicago truncatula [33] SeqMan Melitaea cinxia [34]; Cochliomyia hominivorax [35]; Pinus contorta [36] stackPACK Arabidopsis thaliana [1] Table 2 Features of assembly programmes compared in this…”

“2007), genome sequencing of ecologically important models (Vera et al. 2008) as well as accurate gene expression analysis (Torres et al.”

“Nextgeneration sequencing technologies offer unprecedented access to whole genome sequences, whole population sequencing and whole transcriptome sequences (Hudson 2008; Vera et al. 2008).”

“It is thus not surprising that the first RNA-seq studies in nonmodel organisms used transcriptome information obtained by sequencing from a single individual or a pool of individuals to construct microarrays for quantifying individual gene expression (Vera et al. 2008).”

“High Levels of Gene Novelty in H. asinina and P. maxima Nacre-Forming Gene Sets Both P. maxima and H. asinina EST data sets are comprised largely of sequences with no associated GO terms, similar to other nonmodel invertebrate EST data sets (Leu et al. 2007; Vera et al. 2008; Takahashi et al. 2009).”

“The assembly result indicated that the mean length of unigenes was 592 bp, which was longer than the results shown in previous studies [24,33,43,55,56,58].”

“Interestingly, however, these singletons can also represent rare transcripts of genes expressed at low levels [39], and therefore constitute an interesting source of genomic data.”

“, 197 bp [31]; 440 bp [33]; 500 bp [34]; 422 bp [35]; 300 bp [36]; 521 bp [37]; 547 bp [38]).”

“organisms [10, 11], novel transcripts discovery [12], Measuring differential gene expression with RNA-seq 131”

“In an example of this integrated technique, Vera et al. (2008) used high-throughput RNAseq and de novo assembly of the Glanville fritillary butterfly (Melitaea cinxia) transcriptome from 80 individuals across eight populations.”

“These technologies have shown great potential for expanding sequence databases of not only model species [14–18] but also non-model organisms [19–24].”

“2009), transcriptome characterization of non-model species (Vera et al. 2008) and high-throughput surveying of microbial ecosystem diversity (Edwards et al.”

“Another strategy for reducing genome complexity is to sequence the transcriptome of a pool of individuals (Barbazuk et al. 2007; Toth et al. 2007; Vera et al. 2007; Collins et al. 2008; Novaes et al. 2008; Renaut et al. 2010).”

“The light quality triggers an elongation response and allows for competitive growth in plants from open habitats, whereas plants from shaded environments do not respond as strongly, since they will never grow taller than the canopy plants (Dudley and Schmitt 1995, Donohue et al. 2000, Weinig 2000).”

“Transcriptome sequencing facilitates functional genomic studies, including global gene expression, novel gene discovery, assembly of full-length genes, and single nucleotide polymorphism (SNP) discovery [9,13].”

“The advent of massively parallel sequencing technologies holds much promise for speeding up the progress in understanding the genetic basis of (ecological) speciation (Noor & Feder 2006; Ellegren 2008b; Vera et al. 2008; Gilad et al. 2009).”

“Some recent studies have indicated that short reads from 454 GS FLX pyrosequencing may be effectively assembled and used to characterise the gene space in various organisms [16,21,32,33].”

“…of high throughput sequencing projects is to reveal sequence variation such as copy number variants, insertion-deletions (indels) or single nucleotide polymorphisms (SNPs) by sequencing pools of genetically heterogeneous individuals (Barbazuk et al. 2007; Vera et al. 2008; Wiedmann et al. 2008).”

“the transcriptome will be identified (Vera et al. 2008), or from genomic DNA such that SNPs from all over the genome will be reported.”

“Transcriptomics gives access to large numbers of genes at relatively low cost, plus information about gene expression levels [27–29], with potential applications for SNP discovery and speciation genomics [30–32].”

“Taking into consideration the number, length, coverage and quality of assembled gene models, as well as their digital expression profiles, this dataset surpasses several previous de novo transcriptome assemblies using Illumina [17,18] or 454 technology [13-16].”

“2007), the silkworm genome aided analysis of Glanville fritillary butterfly ESTs (Vera et al. 2008) and chicken and zebra finch genomes inform transcriptome research on non-model bird species (Kunstner et al.”

“A large proportion of the reads assembled into contigs in case of field pea (87%), which is comparable to the values observed in other studies (Glanville fritillary butterfly, 91% [46]; Eucalyptus grandis, 88% [47]; Acropora millepora larvae, 90% [48]).”

“Importantly, these new technologies promise to be very useful for genome analysis in nonmodel organisms (Vera et al. 2008; see also Ellegren 2008; Hudson 2008).”

“Mean length of lake sturgeon reads after removing primers, linkers, and substandard bases was 190 bp, in between the 111 bp and 209 bp reported in similar non-model 454 studies [9,10].”

“A total of 47 968 single sequence reads (i.e. not assembled into contigs) generated from the 454 sequencing were trimmed with PipeMeta (Vera et al. 2008) and assembled with the GS De Novo Assembler (Roche Diagnostics Corporation, Indianapolis, IN), keeping the default settings and a minimum sequence length of 45 base…”

“…unigenes sampled in the M. sexta midgut transcriptome (6593) was lower than the predicted ~9000 unigenes obtained using a similar method on the butterfly Melitaea cinxia (Vera et al., 2008), probably because we sampled a single M. sexta tissue rather than whole Me. cinxia adults and larvae.”

“The 454/GS-Titanium sequencing platform, used in this study, has been used to address this issue and lead to successful assembly of the 454 pyrosequencing data, which make this technology suitable for de novo transcriptome sequencing of non-model species such as aquaculture species [7,30].”

“Following the pioneering study on the transcriptome of the Glanville fritillary (Vera et al. 2008b), a manuscript describing the full genome is currently in”

“[8–10], with only a very few examples of de novo transcriptome sequencing using the Illumina platform occurring in the literature (e.”

“At present, exploration of the transcribed subset of the genome seems most promising (Vera et al. 2008).”

“A popular target for NGS is species transcriptome, which offers direct access to the coding sequences of many genes, plus information on their relative expression levels (Vera et al. 2008; Elmer et al. 2010; Renaut et al. 2010; Wolf et al. 2010).”

“…identified SNPs and the underlying response to selection on metabolic rate remains unclear, Babik et al.’s (2010) study exemplifies the potential offered by new generation sequencing technologies for studying BMR in animals whose genome sequences are not available (see also Vera et al. 2008).”

“Screening of a large number of loci spread throughout the genome (Vera et al. 2008; Meyer et al. 2009) will greatly improve the accuracy and power to infer population demographic history (Gilad et al.”

“Similarly, the first de novo transcriptome assembly paper based on the first generation of 454 sequencing technology achieved relatively good assembly performance, with a large number of positively identified contigs in BLAST comparisons versus several insect databases, and reads that were on average only 110bp long […”

“The annotation rate in our study is also comparable to those (20,40%) reported in the previous de novo transcriptome sequencing studies for non-model organisms [26,27,33,34,35].”

“Recently, 454 sequencing was applied for the identification of gene-derived SNPs in a number of species such as eucalyptus grandis [18], pine tree [19], butterfly [20], lake sturgeon [21] and coral [22].”

“related reference genome (Vera et al., 2008).”

“A method for non-model organisms using 454 pyrosequencing data was recently published [ 3 ], highlighting how next-generation sequencing enables transcriptome analysis from any species.”

“2007), the Glanville fritillary butterfly (Vera et al. 2008), and a paper wasp (Toth et al.”

“To which extent this goal has been achieved? As demonstrated in previous studies [54], the use of nextgeneration sequencing technology yielded a number of expressed sequences unattainable until only recently.”

“3 x coverage; [26]), Flooded or Rose gum (245 nt; [32]) or Shore Pine (306 nt at 3.”

“It has been used for the de novo characterization of the transcriptome of the Glanville fritillary butterfly (Vera et al. 2008) and the Eucalyptus grandis genome (Novaes et al.”

“For example, Vera et al. (2008) generated a de novo assembly of the transcriptome of a non-traditional eukaryote model organism (Melitaea cinxia, a butterfly), for which genomic data was not previously available.”

“An example of this is provided in this issue (Vera et al. 2008) by the first large-scale attempt toward genome sequencing of an ecologically important model, based on the new ‘454- sequencing technology’.”

“g.[1-5]) and have focused on single species.”

“Development of such resources for Sarcophaga would greatly benefit the study of flesh flies as has been done with other non-traditional model insects such as Polistes wasps [19], Glanville fritillary butterflies [20], and tobacco hornworm moths [21].”

“This was similar to other non-model invertebrate EST data sets [74,75,76,77].”

“Next generation 454 EST sequencing has been shown to be a very efficient, cost-effective approach for transcriptome analysis of non-model species and for the discovery of rare and novel transcripts [16-21].”

“age has only been used in sequencing of transcriptomes [47,48],”

“The recent introduction of ‘next-generation’ or massive parallel sequencing technologies (Rothberg & Leamon 2008) in molecular ecology (Vera et al. 2008) holds promise that this will eventually come about (Bonneaud et al. 2008; Ellegren 2008b; Hudson 2008).”

“In addition to its high capability of capturing transcripts from the target species, high throughput transcriptome sequencing using the next-generation technology had enabled capture of expressed sequences from xenobiotic species that are commensal with the species of study [18,19].”

“…advances in sequencing have resulted in high-throughput techniques that permit extensive genetic surveys of non-model organisms (Toth et al., 2007; Vera et al., 2008), and transcripts of highly expressed receptors may be captured by sequencing RNA from antennal tissues (Ramsdell et al., 2010;…”