Combining next-generation pyrosequencing with microarray for large scale expression analysis in non-model species

<p>Abstract</p> <p>Background</p> <p>The next generation sequencing technologies provide new options to characterize the transcriptome and to develop affordable tools for functional genomics. We describe here an innovative approach for this purpose and demonstrate its potential also for non-model species.</p> <p>Results</p> <p>The method we developed is based on 454 sequencing of 3' cDNA fragments from a normalized library constructed from pooled RNAs to generate, through <it>de novo </it>reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence, is not available. This "virtual transcriptome" provides extensive coverage depth, and can be used for the setting up of a comprehensive microarray based expression analysis. We evaluated the potential of this approach by monitoring gene expression during berry maturation in <it>Vitis vinifera </it>as if no other sequence information was available for this species. The microarray designed on the berries' transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proved to be more informative than one of the most comprehensive grape microarrays available to date, the GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization, which could detect the expression of 15,556 genes in the same samples.</p> <p>Conclusion</p> <p>This approach provides a powerful method to rapidly build up an extensive catalog of unique transcripts that can be successfully used to develop a microarray for large scale analysis of gene expression in any species, without the need for prior sequence knowledge.</p

Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome

<p>Abstract</p> <p>Background</p> <p>With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (<it>Salmo salar</it>) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering ~1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library.</p> <p>Results</p> <p>An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with ~30× coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (~0.09× coverage) were incorporated. The addition of paired end sequencing reads (additional ~26× coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (~10.5× coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly.</p> <p>Conclusion</p> <p>These results represent the first use of GS FLX paired end reads for <it>de novo </it>sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to <it>de novo </it>sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.</p

Evaluation of the capacities of mouse TCR profiling from short read RNA-seq data.

Profiling T cell receptor (TCR) repertoire via short read transcriptome sequencing (RNA-Seq) has a unique advantage of probing simultaneously TCRs and the genome-wide RNA expression of other genes. However, compared to targeted amplicon approaches, the shorter read length is more prone to mapping error. In addition, only a small percentage of the genome-wide reads may cover the TCR loci and thus the repertoire could be significantly under-sampled. Although this approach has been applied in a few studies, the utility of transcriptome sequencing in probing TCR repertoires has not been evaluated extensively. Here we present a systematic assessment of RNA-Seq in TCR profiling. We evaluate the power of both Fluidigm C1 full-length single cell RNA-Seq and bulk RNA-Seq in characterizing the repertoires of different diversities under either naïve conditions or after immunogenic challenges. Standard read length and sequencing coverage were employed so that the evaluation was conducted in accord with the current RNA-Seq practices. Despite high sequencing depth in bulk RNA-Seq, we encountered difficulty quantifying TCRs with low transcript abundance (<1%). Nevertheless, top enriched TCRs with an abundance of 1-3% or higher can be faithfully detected and quantified. When top TCR sequences are of interest and transcriptome sequencing is available, it is worthwhile to conduct a TCR profiling using the RNA-Seq data