Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing

“2010), and butterflies (Vera et al. 2008).”

“Using cDNA template for population genetics, however, would require cDNA normalization, might result in a decrease in the proportion of variable sites and the number of markers evolving neutrally, and creates assembly problems arising from alternative splicing of mRNA (Vera et al. 2008).”

“Several studies have demonstrated the feasibility of NGS for identifying SNPs for population studies and gene sequences for use as phylogenetic markers [18-35].”

“Indeed, a variant of this strategy was used successfully when validating the de novo assembly of the Glanville fritillary butterfly's transcriptome [45].”

“This was one of the first non-laboratory organisms studied using high-throughput sequencing approaches [64], which demonstrates not only the feasibility but also the usefulness of obtaining genomic data from wild populations.”

“For this reason, there have been a number of recent efforts to push forward butterfly genomics [13,14], including construction of large EST collections [15–17], and genetic linkage maps [18–21] for a few target species.”

“The researchers used 454 pyrosequencing to study hundreds of thousands of expressed sequence tags (ESTs) in samples from a metapopulation in Finland (Vera et al. 2008), and integrated the genomic information both with field data on individual dispersal and with physiological parameters related to flight.”

“expression [18–22].”

“For our first goal, we selected GSFLX pyrosequencing based on cost effectiveness, rapidly improving accuracy, and technological improvement to the point that read lengths are suitable for de novo assembly of sizeable contigs even in the absence of genome-based templates (Cheung et al., 2006; Wicker et al., 2006; Novae…”

“The use of 454-pyrosequencing is a fast and efficient approach for gene discovery and enrichment of transcriptomes in non-model organisms [20].”

“Sequencing from multiple developmental stages has been an important strategy for maximizing exon coverage as well as complete transcript recovery (Vera et al. 2008).”

“SmartScreener [59] and SeqClean [60] (http://compbio. dfci.harvard.edu/tgi/software/) were used in order to remove remaining PCR oligos introduced during the cDNA normalization protocol.”

“Indeed, studies capitalizing on the benefits of NGS technologies in (previously) genetically poorly known species are becoming more common (Vera et al. 2008; Hohenlohe et al. 2010; Bruneaux et al. 2013) as are reviews outlining the benefits of NGS-based approaches in ecological and evolutionary research (e.”

“It is particularly useful as a shotgun method for generating EST data, and its costs are relatively low [33,38].”

“…for nonmodel species such as whitefish by making genome sequencing, transcriptome sequencing and single nucleotide polymorphism (SNP) discovery efficient and accessible, even in the absence of a reference genome (Quinn et al. 2008; Vera et al. 2008; Kristiansson et al. 2009; Renaut et al. 2010).”

“The potential of this approach has been demonstrated in animals lacking reference genome sequences such as the coral Acropora millepora [18], the whitefly Bemisia tabaci [19], the butterfly Melitaea cinxia [20], the mosquito Anopheles funestus [21] and the planarian Schmidtea mediterranea [22,23].”

“Next Generation Sequencing technologies (NGS) are rapidly invading many evolutionary and ecological fields, such as phylogenomics, molecular evolution, population genomics and molecular ecology.”

“Needless to say, the most important current step in the application of genomics to studies of ecology and evolution is the introduction of next-generation (massive parallel) sequencing technology (Ellegren 2008; Vera et al. 2008).”

“These NGS platforms can detect millions of transcripts and can be used for new gene discovery and expression profiling independent of a reference genome [48-50].”

“This redundancy is often observed with the MIRA program [83], and could reflect biological variation among transcripts originating from a single gene, for example alternative splicing or allelic variation.”

“similar approaches (Swiss-prot and NR = average 33%, range 12 - 73%, GO database = average 20%, range 18 33% [32-44]).”

“Transcriptome sequencing through 454 pyrosequencing is regarded as a prime choice for novel gene discovery in non-model organisms and many studies have used de novo assembly of transcriptome data to produce and characterize genome-level resources for non-model organisms [32], [33], [34].”

“sets of transcriptome contigs assembled with different methods [17], a second run of assembly was conducted using the previously obtained contigs and singlets as input.”

“The rapid development of next-generation sequencing technologies opened the possibility to sequence de novo the transcriptome of this non-model species (Vera et al., 2008; Ekblom and Galindo, 2011; Ozsolak and Milos, 2011).”

“The 454 GS FLX sequencing technology has made EST-based resources more readily accessible for non-model organism transcriptomes [2,3].”

“New technologies such as RNA-seq can be exploited to develop expressed sequence tags (ESTs), which can be used to construct a microarray (Vera et al. 2008), or can be used to directly estimate transcript abundance and sequence variation.”

“The non-model species community is rapidly harnessing the transcriptome, with an explosion of RNA-Seq studies published over the past 5 years, predominantly using the longer sequencing reads of the 454 FLX technology for the generation of EST databases containing sequence and SNP information [8-12].”

“Although there have been just a few 454 transcriptome sequencing efforts to date [27-31], our results suggest that for studies of this type, 454 sequencing alone could largely replace traditional Sanger Sequencing.”

“, [13]], SNP detection [14,15], or transcriptome sequencing [16].”

“This is higher than those reported in other de novo transcriptome analyses [40-45], including that of an arctic crustacean [23], but much lower than the 43.”

“Compared with the gene chip technique, which is based on the premise of a large number of genes or molecular database information, RNA-Seq can be studied in non-model organisms without corresponding genome sequences as a reference [10-12].”

“9%) were assembled into contigs with a high N50 and an average contig length of 1,216 bp, which is longer than those reported before, which range from 197 bp [46] to 500 bp [47].”

“High-throughput sequencing of RNA (RNA-Seq) has been widely applied to many organisms including non-model species (e.g., Vera et al. 2008; Hale et al. 2009; Meyer et al. 2009; Liu et al. 2011; Hou et al. 2011; Zhao et al. 2012), of which 454-based RNA-Seq was and is still being widely used (e.g.,…”

“…genomewide studies in non-model organisms are now much more achievable (Tautz et al. 2010; Ekblom & Galindo 2011), and several studies have demonstrated that novel functional genomic insights can be gained for species without fully sequenced genomes (e.g. Vera et al. 2008; Papakostas et al. 2012).”

“The transcriptome coverage obtained here is significantly greater than that from another recent survey of a butterfly, Melitaea cinxia, in which around 600 K 454 sequence reads showed similarity to 6289 B. mori proteins (Vera et al. 2008).”

“Most of the EST processing protocols were adapted from those described by Vera and collaborators.(30) Based on preliminary data using different computational assembly pipelines, including the 454 GS Table 1.”

“For example, Vera et al. (2008) also found microbial ª 2 0 1 0 T H E A U T H O R S .”

“These provide powerful tools for the understanding of genome variations in crop species at the DNA, RNA and protein levels, and particularly for nonmodel plant species (Vera et al. 2008).”

“[55], lake sturgeon [56] and coral [57].”

“Pyrosequencing of ESTs provides an efficient way to generate sequence data for non-model organisms in the form of transcriptome sequencing, and can be used to characterize gene expression and identify novel genes [21,22,23].”

“NGS is not limited to detecting transcripts from known genomic sequences, and therefore is particularly attractive for studies in non-model organisms (Vera et al. 2008; Kristiansson et al. 2009; Meyer et al. 2009).”

“58% in a bat, Artibeus jamaicensis [77],, 18% in a butterfly, Melitaea cinxia [59],, and Rapid Evolution of Urban White-Footed Mice”

“Consequently, it has become a powerful platform for profiling the transcriptomes of both model and non-model organisms [32,33,34].”

“Data generated by Roche-454 sequencing produces longer fragments and may be more suited for species without significant sequence data already available [49], but overall sequencing output and coverage is lower than Illumina [48].”

“Recently, deep-sequencing technologies that deliver millions of short reads from platforms such as Roche/454, AB SOLiD and Illumina were used for de novo assemblies and transcriptome analysis of the organisms where reference sequence is absent (Collins et al. 2008; Vera et al. 2008; Morozova et al. 2009; Wang et al. 2…”

“As a consequence, these technologies have been used in several studies of non-model species, such as the Granville butterfly [22], the Propertius duskywing and the Anise swallowtail butterflies [23], the migratory locust Locusta migratoria [24] and the primitively eusocial wasp, Polistes metricus [25].”

“Considerable sequencing depth can be obtained, making it possible to identify transcriptome expression variation [29].”

“, 2009) and Melitaea cinxia (Vera et al., 2008).”

“The obtained contigs and singletons were further reassembled by performing a second round to decrease the redundancy caused by the heuristic nature of the assembly process [34].”

“In recent years this has lead to the establishment of myriad data sets ranging from increased coverage of variation in the human genome at the individual level [1-5] to the sequencing of non-model prokaryotic and eukaryotic genomes and transcriptomes [6-11].”

“The application of new sequencing technologies, such as pyrosequencing, to traditional ecological models expands the horizon for ecological genomic studies [24-29].”

“In this approach, population of total or fractionated RNA is converted to library of cDNA with adaptors attached to both the ends. cDNA molecules are then sequenced in high throughput manner and short sequences are obtained (Holt and Jones 2008; Vera et al. 2008).”

“It has been reported that next generation sequencing methods are an excellent tool for the discovery of novel genes [19-21,57-59].”

“Most of the reads were similar in length to the results from those studies [28,29] and longer than those used in the previous 454EST studies [30-32].”

“…length is considerably higher than the average isotig length achieved from de novo assemblies of transcriptomes of other species (Melitaea cinxia, Vera et al. 2008; Bathymodiolus azoricus, Bettencourt et al. 2010; Laternula elliptica, Clark et al. 2010; Concholepas concholepas, Cárdenas et al.…”

“…data even in the absence of a reference genome sequence and have spurred a recent flurry of transcriptome projects in non-model organisms (e.g. Vera et al. 2008; Meyer et al. 2009; Schwartz et al. 2010), including numerous angiosperms (Novaes et al. 2008; Alagna et al. 2009; Barakat et al.…”

“Bacterial diversity was characterized by sequencing 16S rRNA gene sequences amplified by PCR from genomic DNA extractions, using the Roche 454 Titanium platform [33].”

“This method not only detects transcripts that correspond to existing genomic sequences, but it can also be used for de novo assembly of short reads for gene discovery and expression profiling in organisms for which there is no reference genome [17,20-26].”

“ESTs have been useful for development of molecular markers [4,5,6,7,8], comparative genomics [9,10] and for genetic analysis of adaptive traits [11,12].”

“0 (Vera et al. 2008)] and singletons were annotated using Blast homology searches against predicted gene sets of silkmoth (Bombyx mori, Lepidoptera: Bombycidae) and fruit fly (Drosophila melanogaster, Diptera: Drosophilidae) genomes, and the UniProt database (Vera et al.”

“In this study, we use 454 FLX pyrosequencing to characterize a portion of the chum salmon transcriptome; we conducted three sequencing runs on cDNA from testes from each of two chum salmon from different populations (cf., Ellegren 2008; Vera et al. 2008) where we detected thousands of putative SNPs.”

“Since RNA-seq is not restricted to detecting transcripts of known genes, it is particularly useful for non-model organisms with no available genomic data [22].”

“discovery in many studies [9,10], and has significantly accelerated and improved the sensitivity of gene expression profiling.”

“The 947,623 resulting sequences and quality scores were analysed using PipeMeta, a menu based, desktop interface pipeline toolset for Windows [59,60].”

“Note that the 50% of genes identified after our de novo sequencing of reptilian transcriptomes is larger than the proportion (about 25%) of transcript identification in previous efforts [27,51-53].”

“…capable of producing millions of DNA sequence reads in a single run allows de novo transcriptome assembly and a fast, cost-effective, and reliable method for development of functional genomic tools for non-model species tractable for ecological and studies (Mardis, 2008; Vera et al., 2008).”

“The GS20 Melitaea cinxia dataset from whole larvae is published by Vera et al [40] and was down-loaded from NCBI's Short Read Archive after communication with Howard Fescemyer (Pennsylvania State University).”

“Meanwhile, the data collections are also valuable for gene annotation and discovery [23,24], comparative genomics [25], development of molecular markers [26,27], and population genomics studies on genetic variation associated with adaptive traits [28].”

“Xenobiotic sequences from intracellular parasites are commonly found in transcriptome data of wild species [10].”

“It has enormous potential for studies of non-model organisms because transcript abundance can be quantified in the absence of genomic sequence data (e.g., Vera et al., 2008; Ekblom et al., 2010; O’Neil et al., 2010).”