Next is now: new technologies for sequencing of genomes, transcriptomes, and beyond

Abstract

The sudden availability of DNA sequencing technologies that rapidly produce vast amounts of sequence information has triggered a paradigm shift in genomics, enabling massively parallel surveying of complex nucleic acid populations. The diversity of applications to which these technologies have already been applied demonstrates the immense range of cellular processes and properties that can now be studied at the single-base resolution. These include genome resequencing and polymorphism discovery, mutation mapping, DNA methylation, histone modifications, transcriptome sequencing, gene discovery, alternative splicing identification, small RNA profiling, DNA-protein, and possibly even protein-protein interactions. Thus, these deep sequencing technologies offer plant biologists unprecedented opportunities to increase the understanding of the functions and dynamics of plant cells and populations.

Figure 1. Advanced DNA sequencing technologies underly diverse approaches to unravel plant cellular activities

Massively parallel DNA sequencing of complex nucleic acid populations now enables numerous subsets of genomic and cellular information to be rapidly characterized at unprecedented resolution and breadth. The AnnoJ genome browser (www.annoj.org) excerpt shown above represents approximately 100 kilobases of Arabidopsis thaliana chromosome 1. Single nucleotide polymorphisms between Col-0 and Ler-1 ecotypes (Lister, O’Malley, Ecker, unpublished), single-base DNA methylation maps, strand-specific smRNA and mRNA components of the transcriptome, and RNA-degradation products from Arabidopsis thaliana flower buds, all generated by ultra high-throughput DNA sequencing, have been integrated to illustrate the holistic views of genomic and transcriptional regulation and variation that can now be routinely captured [41,48].

Figure 2. Identification of mutations by deep sequencing

A plant with Col-0 background that harbors a recessive mutation leading to a mutant phenotype is crossed to a wild-type Ler-1 plant. The heterozygous F1 hybrid plant is allowed to self-fertilize to produce a large pool of F2 plants that are segregating for the mutation. A large number of F2 plants that display the mutant phenotype are pooled and their gDNA subjected to deep sequencing. The density of single nucleotide polymorphisms (SNPs) inherent in the Ler-1 strain is subtracted from the density of SNPs indicative of the Col-0 background, identifying a discrete region on the chromosome in which only Col-0 marker SNPs are present. The deep sequencing data in this interval is then scoured for the potential causative mutation.

Figure 3. Integration of multiple deep-sequencing datasets for identification of DNA methylation-repressed transcripts

Shotgun sequencing was used to generate single-base resolution maps of DNA methylation and the smRNA and mRNA components of the transcriptome in wild-type (Col-0) and DNA methyltransferase-deficient mutant (met1-3) plants. Integration of these diverse datasets and visualization in the Anno-J deep-sequencing browser (www.annoj.org) revealed hundreds of intergenic transcribed regions that were normally suppressed in wild-type plants, where loss of DNA methylation in the met1-3 mutant was accompanied by a decrease in smRNA abundance and an increase in transcriptional activity [41].

Figure 4. Massively parallel interrogtion of all pairwise protein interactions for all proteins encoded by a genome by bait-prey recombination and deep-sequencing

Interaction of bait and prey constructs results in the activation of the CRE recombination system and expression of a selective marker gene. Recombination at loxP sites located at the end of each gene forms a chimeric DNA molecule containing the two genes that encode the interacting proteins. Digestion to release the chimeric ORFs followed by paired-end sequencing of its two ends will produces one sequence tag from each of the genes, thus identifying the two proteins that directly interacted. Two complex pools of yeast cells, each one containing the full complement of an organism’s genes fused to either the bait or the prey domain, would be mixed and allowed to mate. Sequencing of the complex pool of chimeric ORFs would reveal all pairwise interaction that occurred, interrogating the hundreds of millions of possible interactions between any two proteins encoded in a eukaryotic genome.