Assembly, annotation and polymorphism analysis of a draft transcriptome sequence for a fast-growing Eucalyptus plantation tree

Background: De novo assembly of transcript sequences produced by short-read DNA sequencing technologies offers a rapid approach to obtain expressed gene catalogs for non-model organisms. A draft genome sequence will be produced in 2010 for a Eucalyptus tree species (E. grandis) representing the most important hardwood fibre crop in the world. Genome annotation of this valuable woody plant and genetic dissection of its superior growth and productivity will be greatly facilitated by the availability of a comprehensive collection of expressed gene sequences from multiple tissues and organs. Results: We present an extensive expressed gene catalog for a commercially grown E. grandis × E. urophylla hybrid clone constructed using only Illumina mRNA-Seq technology and de novo assembly. A total of 18,894 transcriptderived contigs, a large proportion of which represent full-length protein coding genes were assembled and annotated. Analysis of assembly quality, length and diversity show that this dataset represent the most comprehensive expressed gene catalog for any Eucalyptus tree. mRNA-Seq analysis furthermore allowed digital expression profiling of all of the assembled transcripts across diverse xylogenic and non-xylogenic tissues, which is invaluable for ascribing putative gene functions. Conclusions: De novo assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in Eucalyptus and other non-model organisms. The transcriptome resource (Eucspresso, http:// eucspresso.bi.up.ac.za/) generated by this study will be of value for genomic analysis of woody biomass production in Eucalyptus and for comparative genomic analysis of growth and development in woody and herbaceous plants. Background Ultra-high-throughput second-generation DNA sequencing technologies from companies such as Roche (454 pyrosequencing), Illumina (sequencing by synthesis, Solexa GA) and Applied Biosystems (sequencing by ligation, SOLiD), are increasingly being used for novel exploratory genomics in small to medium-sized laboratories. “Short-read” (36 72 nt) technologies such as those of Illumina and Applied Biosystems have proven to be exceptionally successful in a wide variety of whole-transcriptome investigations [1-5], but most of these studies have relied on prior sequence knowledge such as an annotated genome for qualitative and quantitative transcriptome analyses. Genome assembly of short sequences without any auxiliary knowledge has primarily utilized 454 sequencing data, due to the longer individual read lengths of 150-400 base pairs (bp). However, short-read sequencing (Illumina GA and SOLiD) has been successfully used for de novo assembly of small bacterial genomes (2-5 Mbp), where 36 bp reads have been assembled [6-8] and hybrid approaches, where genomes are de novo assembled using a combination of reads from multiple sequencing platforms to overcome the inherent limitations of each technology, have been used to successfully assemble genomes of up to 40 Mbp [9,10]. More recently, the sequencing of the giant panda genome was demonstrated [11] using de novo assembly of sequence derived from a single platform (Illumina), but utilizing a * Correspondence: zander.myburg@fabi.up.ac.za † Contributed equally Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa Full list of author information is available at the end of the article Mizrachi et al. BMC Genomics 2010, 11:681 http://www.biomedcentral. com/1471-2164/11/681 © 2010 Mizrachi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. combination of different insert sizes, allowing assembly of an estimated 94% of the genome (2.25 Gbp). De novo assembly of large, highly repetitive and highly heterozygous eukaryotic genomes from short-read data remains a challenge. In transcriptome studies, 454 pyrosequencing has proven very useful for generating ESTs representing the majority of expressed genes. This has enabled gene discovery in a variety of previously uncharacterized eukaryotic organisms with no or little a priori DNA sequence information [12-16]. However, relatively few published studies have attempted de novo assembly of whole-transcriptome sequences from short-read data such as that generated by Illumina GA or SOLiD technologies. Assembly of short (36-72 bp) read data into accurate, contiguous transcript sequences has only recently been reported [17-19] demonstrating that assembly of long, potentially full-length, transcript assemblies is indeed possible. Eucalyptus tree species and hybrids presently constitute the most widely planted (≈ 20 Mha) and commercially important hardwood fibre crop in the world. They are mainly utilized for timber, pulp and paper production [20]. Their fast growth rates and wide adaptability may in future allow sustainable and cost efficient production of woody biomass for bioenergy generation [21,22]. Eucalyptus will soon be only the second forest plantation genus (after Populus) for which a reference genome sequence will be completed by end 2010 [23]. To support the genome annotation effort, there is much value in having a dataset of genes with strong transcriptional evidence across a range of tissues and developmental stages. Until recently, limited amounts of Eucalyptus EST/unigene data were available in public databases, mainly due to the fact that commercial interests have necessitated private EST collections [24]. As of March 2010, aside from a mixed-species collection of ≈56,000 nucleotide sequences on NCBI (≈ 37,000 of which are Sanger EST sequences) and which contain extensive redundancy, the largest effort to date to generate a comprehensive catalogue of expressed genes in a single Eucalyptus species was based on 454 sequencing of cDNA fragments from E. grandis trees [15]. While this study provided an excellent representation of expressed genes and gene ontology classes in E. grandis, the relatively short lengths of the assembled contigs (mean length of 389 bp for all contigs longer than 200 bp) meant that very few complete gene models were represented. There remains therefore a fundamental need for a high-quality expressed gene catalog for Eucalyptus, to support genome annotation efforts and discern authentically expressed genes from predicted gene models, as well as for future genomics research, which will include transcriptome, proteome and metabolome profiling. In the process of producing such a high-quality expressed gene catalog for Eucalyptus, we addressed three main questions: First, is it feasible to de novo assemble Illumina mRNA-Seq data into contiguous, near full-length gene model sequences for Eucalyptus? Second, what genes make up the expressed gene catalog for a fast-growing Eucalyptus plantation tree? Finally, can we re-use the mRNA-Seq data to create a tissue and organ-specific digital expression profile for each assembled contig? We addressed these questions by generating a comprehensive set of expressed gene sequences from a commercially grown Eucalyptus hybrid (E. grandis × E. urophylla) clone using Illumina mRNA-Seq technology and de novo short-read assembly. We report herein the complete annotation of the expressed gene catalog based on comparative analysis with the published Arabidopsis thaliana [25], Populus trichocarpa [26] and Vitis vinifera [27] protein-coding datasets. We describe an interactive database of annotated transcript sequences, coding sequences (CDSs) and derived protein sequences (Eucspresso, http:// eucspresso.bi.up.ac.za/, CA Hefer, E Mizrachi, AA Myburg, F Joubert, unpublished), which will be continuously updated and curated in association with the Eucalyptus Genome Network (EUCAGEN, http://www. eucagen.org) as part of an effort to initiate a publicly accessible database for Eucalyptus transcriptomics research similar to that produced for Populus [28]. Results De novo assembly, validation and annotation of contigs In total, 62 million paired-end reads of raw mRNA-Seq data (6.90 Gbp) representing poly(A)-selected RNA from six Eucalyptus tissues and varying in lengths from 36 bp to 60 bp, were generated in 14 lanes on Illumina GA and GAII instruments. Following a sequence filtering process to exclude low quality and ribosomal RNAderived reads, we assembled 36 million paired-end reads (3.93 Gbp, Additional file 1 Table S1 and Figure S1, NCBI Sequence Read Archive accession SRA012408) of non-normalized mRNA sequence, using the Velvet short-read assembler (version 0.7.30, [29]). In total, 18,894 RNA-derived contigs were assembled (comprising 22.1 Mbp of transcriptome sequence) that were greater than 200 bp in length (mean = 1170 bp, Figure 1 and Additional file 2), with a median coverage per base (CPB) per contig of 37×, ranging from 8× (minimum coverage cut-off for assembly) to 5,262× (Additional file 1 Figure S2). We performed ab inito CDS prediction using GENSCAN [30] and found that 15,713 contigs (83.2%) contained a predicted CDS (Additional file 1 Table S3). Analysis of the predicted coding sequences using Anaconda [31] identified 6,208 contigs that contained Mizrachi et al. 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