Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing

“[29,33,35,42]).”

“Recent advances in genomics, in particular next generation sequencing, mean that generating sequence data for non-model organisms has become accessible both in terms of timescale and price [5,6].”

“Pyrosequencing with 454 technology provides a method for measuring transcript levels in non-model organisms where DNA microarrays are not easily available ( Vera et al. 2008 ).”

“Indeed, SNP marker development through NGS of transcriptome data has consequently become more and more common in nonmodel species over the past few years (see for example [36–38]).”

“…biologists, who previously relied on microarray analysis for those data (e.g. Toth et al. 2007; Marioni et al. 2008; Sultan et al. 2008; Vera et al. 2008), and for researchers investigating animals for which microarrays were a long way from being available or optimized (e.g. Gilad et…”

“As a result, because of the parasite’s potential and highly significant negative impact on ecosystems and fisheries, it appears crucial that the entire genome be sequenced or transcriptomes (Vera et al., 2008) of the parasite at various lie history stages should be documented and compared between clades.”

“This approach has been broadly used in ecological genomics (Vera et al. 2008; Babik et al. 2010; Jeukens et al. 2010; Wolf et al. 2010; Salem et al. 2012).”

“Results from early RNAseq experiments have already identified many previously unknown expressed transcripts, and have also been used to detect novel non-coding RNA molecules (Lister et al., 2008; Vera et al., 2008).”

“blies was performed in transcriptome data for various life history stages of the Glanville fritillary butterfly (Melitaea cinxia) (Vera et al., 2008).”

“In addition, this approach has also allowed the analysis of gene expression profiling using quantitative (q) PCR, microarrays or the quantification of short cDNA reads [29–31].”

“We also tested whether duplicated zebra finch b-defensin genes where evolving according to different selection pressures compared to unduplicated genes and investigated tissue specific expression patterns of this gene cluster using a digital transcriptomic approach [27,28].”

“[32]].”

“Predicted protein domain organisation of IRs with associated Pfam predicted domains (adapted from [23,54]).”

“Recently developed high-throughput sequencing technologies offer a new approach (RNASeq) for characterizing transcriptomes through high-throughput sequencing, mapping and quantification [20-31].”

“The emergence of next-generation sequencing technologies has substantially helped advance plant genome research, particularly for non-model plant species [14].”

“However, with the advent of next generation sequencing techniques, for example 454 sequencing for non-model organisms (Vera et al., 2008) and rapid SNP discovery (Baird et al.”

“The criteria that we used for SNP mining (at least 3× occurrence of the minority allele and at least 6× read coverage) are more stringent than those typically used (2× occurrence of the minority allele, and 4× or 5× read coverage) [11,31,32].”

“zation of non-model plant transcriptomes [47,48].”

“Primmer (2009) reviewed examples in the Atlantic Salmon (Vasemägi et al. 2005a, b), the California Condor (Romanov et al. 2006) and the Glanville Fritillary butterfly (Vera et al. 2008; Wheat at all, this issue).”

“The ability to sequence genomes of non-model organisms with little or no preliminary sequence information represents a major step in comparative genomics and evolutionary biology (Vera et al. 2008).”

“From these simulated data, they applied six different sampling schemes: (1) Trapper sampling, 360 individuals drawn randomly across the entire simulated landscape, (2) Research sampling, individuals drawn in 10 clusters of 36, i.”

“Therefore, with regard to the rapid progress in the field of nextgeneration sequencing, which allows the development of new, deeper, and faster analyses of forensically-relevant processes of development, without being limited to model organisms [25–28], we analyzed the transcriptome of C.”

“Moreover, some transcriptome studies were made in order to provide ESTs datasets of model and non-model insects [9,17–19] and have become an effective way of assessing gene expression levels.”

“According to contemporary approaches to Roche 454 transcriptome analyses, we encapsulated intraspecific, tissue and temporal variability in the transcriptome capture experiment (Vera et al. 2008).”

“In addition, previous studies [30,31,33] have considered singletons to be a valid source for gene discovery whereas the 96,000 singletons (4805 annotated) obtained in the present study were not included in further analysis.”

“…more data become available across larger numbers of nonmodel organisms (Wakeley 2004; Anderson 2007) with the advent of new sequencing technologies (Vera et al. 2008; Wheat 2009) and increasing analytical advances for exploring a larger number of specified histories (Lopes et al. 2009; Wegmann et…”

“be due to conserved motifs in different genes, or the presence of variants of single genes created by alternative splicing or allelic differences within the population of individuals sequenced [8].”

“, transcriptome sequencing, custom oligonucleotide microarray fabrication, and large-scale SNP discovery (157)].”

“…entomology, this method is being increasingly used to gain insight into different insect genomes, from both agriculturally important nonmodel and non-whole genome sequenced insects (Vera et al., 2008; Pauchet et al., 2009, 2010a,b; Mittapalli et al., 2010; O’Neil et al., 2010; Price et al., 2011).”

“Recent studies have demonstrated the success of 454 de novo sequencing, assembly and analysis of transcriptomes in non-model organisms with no prior genomic resources [30-33].”

“This new promise has created an intense demand for tools that will enable the de novo assembly of genomic information (Vera et al., 2008; Zerbino & Birney, 2008; Birol et al., 2009; Hahn et al., 2009; Guttman et al., 2010; Kumar & Blaxter, 2010; Robertson et al., 2010; Wang et al., 2010).”

“Our study, as well as those by Vera et al. (2008) and Parchman et al. (2010), highlights the usefulness of NGS for rapid and cost-effective SSRs discovery.”

“The poor annotation efficiency was comparable to those reported in other de novo transcriptome sequencing studies based on the NGS platforms [22,33–36].”

“This type of sequencing can result in high transcriptome coverage depth and facilitates the de novo assembly of transcriptomes from species where full genomes do not exist [17,18].”

“Previous reports of ESTs from non-model, yet ecologically and economically important organisms have been successfully sequenced and annotated using next generation sequencing [19-21].”

“The robustness of these technologies has been demonstrated in profiling the transcriptomes of not only model animals [7, 8] but also non-model species [9, 10].”

“We used assembly parameters that are similar to those reported in other transcriptome assemblies (e.g. Renaut et al. 2010; Vera et al. 2008), including an estimate of 100x fixed coverage, an estimated transcriptome length of 20 megabases, gap penalty = 25, match size = 19, mismatch penalty = 25,…”

“Roche 454 pyrosequencing has in the recent past revolutionized functional genomic studies in non-model organisms, particularly in insects wherein little to no genetic information is available (Francischetti et al., 2007; Morozova and Marra, 2008; Margulies et al., 2005; Vera et al., 2008; Pauchet et al., 2009; Mittapa…”

“RNA-seq is not limited to detecting transcripts for organisms with existing genomic sequences; it also can be used to sequence nonmodel organisms that lack of genomic information [21-25].”

“These technologies have proven effective for expanding the available sequence information not only for model species [36,37] but also for non-model species [38,39] such as Ophrys, the large genome size of which (,10 Gbp [40]) makes transcriptome sequencing a good choice for gene discovery.”

“RNA samples were hybridized to custom-made microarrays (Vera et al. 2008).”

“As an alternative to whole-genome sequencing, one could focus on narrowing the initial template by, for example, using a normalized cDNA library to generate transcriptome data, and thereby increase the useable output (Vera et al. 2008).”

“The possibility of sequencing transcriptomes at single-base resolution has opened a wide frontier of applications in transcriptomics research: transcriptome profiling of non-model organisms [5,6], novel transcripts discovery [7], quantification of allele-specific gene expression [8], investigation of * Correspondence:…”

“The average length of the unigenes was 709 bp, which was longer than those assembled in previous studies, for example, with butterfly (197 bp) [28], sweet potato (581 bp) [15], Salvia miltiorrhiza (331 bp) [19], and safflower (446 bp) [29].”

“异、基因家族中相似基因以及可变剪接造成的不同 转录本的表达 ([29]), 同时不存在传统微阵列杂交的荧 光模拟信号带来的交叉反应和背景噪音问题, 能覆 盖信号超高的动态变化范围。(2)高灵敏度:能够检 测到细胞中少至几个拷贝的稀有转录本。(3)任意物 种的全基因组分析:无需预先设计特异性探针, 因 此无需了解物种基因信息, 能够直接对任何物种进 行转录组分析 , 这对非模式生物的研究尤为重要 , 例如Wang等 ([30])、Xiang等 [31] 和 Vera等 ([32])利用 RNA-Seq技术分别对白粉虱、海鲈鱼和蝴蝶转录组 进行了研究。同时能够检测未知基因, 发现新的转 录本, 并精确地识别可变剪切位点及cSNP, U…”

“Thus, the identification and characterization of expressed genes in the form of expressed sequence tags (ESTs) from a full-length cDNA library represents an attractive and valuable alternative to whole genome sequencing (Bouck and Vision 2007; Emrich et al. 2007; Vera et al. 2008).”

“While such approaches may become widely used within the next few years, genetic markers have so far mostly been developed through sequencing the parts of the genome that were expressed at the time of sampling (e.g. Vera et al. 2008).”

“299 as microarrays and subtraction cDNA libraries (Shendure, 2008; Vera et al., 2008; Weber et al., 2007).”

“Gene expression studies have huge potential for non-model species by using 454 sequencing to quickly sequence thousands of transcripts: these sequences are used to direct the synthesis of oligonucleotide microarrays (Vera et al., 2008).”

“Still, this methodology would also work well when addressing a non-model species without any prior genome information [10].”

“We can now generate sequence data for 100s of loci in dozens of individuals from non-model species relatively quickly (Shendure and Ji 2008; Meyer et al. 2008; Vera et al. 2008).”

“funestus [24], Aedes aegypti [25], Bactrocera dorsalis [26], Melitaea cinxia [27], Zygaena filipendulae [28], Chrysomela tremulae [29], Aphis glycines [30], Cimex lectularius [31], Manduca sexta [32,33], Laodelphax striatellus [34], Stomoxys calcitrans [35], Dermacentor variabilis [35], Erynnis propertius and Papilio…”

“Also, many EST libraries of a wide range of plant species have been constructed for genes involved in plant growth and differentiation [46,50], biochemical pathways [47,48], secondary metabolism [51,52] as well as responses to environmental stresses and pathogen attack [53].”

“[90] Vera JC, Wheat CW, Fescemyer HW, et al.”

“[9] Vera JC, Wheat CW, Fescemyer HW, et al.”

“In addition, this sequencing method allows the de novo assembly and annotation of expressed genes [13], which makes it highly suitable for non-model organisms.”

“Sequencing of cDNAs and subsequent assembly of these data into transcripts is an effective means to discover novel genes [106], and has been greatly facilitated among non-model organisms by recent advances in high throughput RNA sequencing (RNA-seq) [107].”

“To date, the transcriptomes of 68 species from 7 insect orders have been sequenced [41–43], and the transcriptome and expression profile data of some lady beetles such as Cryptolaemus montrouzieri Mulsant and Propylaea japonica (Thunberg) have been analyzed [44, 45].”

“This proportion of contigs with BLAST hits is similar to previous transcriptome studies [47-49], in which contigs without significant hits may consist of untranslated transcripts, chimeras or assembly artifacts.”

“BLASTx in the order of 30% of the sequences is common when using NGS technologies to characterize the transcriptome of nonmodel species (Novaes et al. 2008; Vera et al. 2008; Parchman et al. 2010).”

“Next-generation sequencers would facilitate gen- ome scan analyses by using sequenced RAD (Baird et al. 2008; Hohenlohe et al. 2010; Rowe et al. 2011; Roesti et al. 2012) and transcriptome analyses by RNA-sequencing in non-model organisms (Vera et al. 2008).”

“3% [53]) with a subset getting annotated.”

“In particular, sequencing complete transcriptomes [2] or complexity-reduced fractions of genomes [3] has enabled the identification of genome-wide molecular markers such as single nucleotide polymorphisms (SNPs) and microsatellites (SSRs).”

“These methods are quickly becoming integrated into ecological studies and conservation applications (Vera et al. 2008; Abdelkrim et al. 2009; Allentoft et al. 2009; Rasmussen and Noor 2009; Castoe et al. 2010).”

“The average length found in the present research was also larger than those of other non-model organisms ranging from 197 to 707 bp, [21-25].”

“[1,8,30,31]) have used 454 pyrosequencing because of the longer read length (>200 bp) produced from this type of sequencer, several recent studies have successfully performed de novo assembly of transcriptome data from Illumina reads ranging from 35 to 100 bp in length [3,32-35].”

“Transcriptome sequencing facilitates functional genomic studies, including global gene expression, novel gene discovery, and assembly of full-length genes [13-16].”

“Highthroughput sequencing is well suited for large scale EST discovery, providing a tool for gene discovery in nonmodel crops to evaluate the changes in gene expression to abiotic or biotic stresses [39-41].”

“Although most of these studies are based on the long-read sequence data using 454 pyrosequencing or employ a hybrid approach, the de novo assembly of transcriptomes using short reads (Illumina or SOLiD) has also received attention because of its relatively low cost [17,44–46].”

“In addition, many studies have used de novo assembly of such data to produce genome-level resources for nonmodel organisms [35-38].”

“Among the currently used high-throughput techniques, pyrosequencing is a useful method for de novo transcriptome analysis of non-model species (Novaes et al. 2008; Vera et al. 2008; Parchman et al. 2010).”

“, 2007) or microarrays (Vera et al., 2008).”

“Thus, high throughput transcriptome sequencing represents a common starting point for large scale sequencing projects for a broad taxonomic range of organisms [18-20].”

“Therefore, next-generation sequencing has become the gold-standard technology for use for in gene discovery and high-throughput identification of molecular markers in nonmodel organisms (Vera et al. 2008).”

“Roche’s 454 GSFLX and Illumina/Solexa Genome Analyzer have been mainly used in de novo transcriptome assembly (Bai et al. 2011; Ge et al. 2011; Su et al. 2011; Vera et al. 2008; Wang et al. 2010a, b; Xue et al. 2010).”