Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery

Abstract

BackgroundMassively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines (Pinus spp.) have hindered the development of genomic resources, despite the ecological and economical importance of the group. While most genomic studies have focused on a single species (P. taeda), genomic level resources for other pines are insufficiently developed to facilitate ecological genomic research. Lodgepole pine (P. contorta) is an ecologically important foundation species of montane forest ecosystems and exhibits substantial adaptive variation across its range in western North America. Here we describe a sequencing study of expressed genes from P. contorta, including their assembly and annotation, and their potential for molecular marker development to support population and association genetic studies.ResultsWe obtained 586,732 sequencing reads from a 454 GS XLR70 Titanium pyrosequencer (mean length: 306 base pairs). A combination of reference-based and de novo assemblies yielded 63,657 contigs, with 239,793 reads remaining as singletons. Based on sequence similarity with known proteins, these sequences represent approximately 17,000 unique genes, many of which are well covered by contig sequences. This sequence collection also included a surprisingly large number of retrotransposon sequences, suggesting that they are highly transcriptionally active in the tissues we sampled. We located and characterized thousands of simple sequence repeats and single nucleotide polymorphisms as potential molecular markers in our assembled and annotated sequences. High quality PCR primers were designed for a substantial number of the SSR loci, and a large number of these were amplified successfully in initial screening.ConclusionsThis sequence collection represents a major genomic resource for P. contorta, and the large number of genetic markers characterized should contribute to future research in this and other pines. Our results illustrate the utility of next generation sequencing as a basis for marker development and population genomics in non-model species.

Key metabolic pathways involved in xenobiotic biotransformation and stress responses revealed by transcriptomics of the mangrove oyster Crassostrea brasiliana

“…This final dataset was then subjected to putative functional annotation using a more rigorous blastx search (E value < 10 -10 ) against the NCBI non-redundant (nr) protein database. The length of contigs was significantly correlated with the number of sequences assembled into them (Pearson ρ = 0.53; n= 20,938; p < 0.0001), as expected for 454 reads (Parchman et al, 2010) and the size distribution of contigs resembled that of previous 454 studies (Bettencourt et al, 2010;Fraser et al, 2011;Hou et al, 2011) (Supplementary Figures S1A and S1B). Files containing the reads have been submitted to the National Center for Biotechnology Information Short Read Archive [GenBank: SRX790900].…”

De-novo characterization of the soft-shelled turtle Pelodiscus sinensis transcriptome using Illumina RNA-Seq technology

“…Compared with Wang et al (2010b), in which only 16.2% had BLAST hits in Nr database, the higher percentage in the present study was partially ascribed to the higher frequency of long sequences (Wang et al, 2010b). As reported by Parchman et al (2010), longer contigs were more likely to have BLAST matches in the protein database.…”

Screening potential SSR markers of the anadromous fish Coilia nasus by de novo transcriptome analysis using Illumina sequencing

“…These unigenes had a mean length of 580 bp (Table 1). Our data indicated that the C. nasus unigenes were roughly shorter than the average lengths of unigenes that were previously reported in other species such as Eriocheir sinensis (382 bp) (He et al, 2012), Acropora millepora (440 bp) (Meyer et al, 2009), Pinus contorta (500 bp) (Parchman et al, 2010), Chlamydomonas spp (665 bp) (Kim et al, 2013), and Camellia sinensis (733 bp) (Wu et al, 2013). These results provide sequence information for future gene cloning and transgenic engineering stud ies.…”

“…Next-generation sequencing (NGS) technologies facilitate generation of a large number of sequences and have been used recently to isolate simple-sequence repeat (SSR) markers in studies of non-model animals, plants, and fish (Parchman et al, 2010). Transcriptome sequencing can yield a subset of genes from the genome that are functionally active in a selected tissue and species of interest (Venter et al, 2001;Martinez-Alcantara et al, 2009;Metzker, 2010;Schatz et al, 2010).…”