Targeted sequencing of specific loci of the human genome is a promising approach for maximizing the efficiency of second-generation sequencing technologies for population-based studies of genetic variation. Here we describe microdroplet PCR, which performs 1.5 million separate amplifications in parallel, as an approach for enriching targeted sequences in the human genome. We initially designed primers to 435 exons of 47 genes that were selected for having a broad spectrum of sequence characteristics. Using this primer set we amplified the same six samples by both microdroplet and traditional singleplex PCR and sequenced the products using the Illumina GAII demonstrating that both methods generate similarly high quality data; 84% of the uniquely mapping reads fell within the targeted sequences, uniform coverage of ~90% of the targeted bases, greater than 99% accuracy in sequence variant calls, and high reproducibility between different samples (r2=0.9). We next scaled the microdroplet PCR to 3976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample on both the Illumina GAII and Roche 454 platforms, and obtained data with equally high specificity and sensitivity quality. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel amplification of specific subsets of the human genome for targeted sequencing.

Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55 second cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the microfluidic chip. We show that amplification of a 245 bp Adenovirus product can be detected and quantified in 35 minutes at starting template concentrations as low as one template molecule per 167 droplets (0.003 pg/μL). The frequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.

Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain. We studied a mutant strain of Pichia stipitis, a yeast capable of converting xylose to ethanol. This unusually efficient mutant strain was developed through repeated rounds of chemical mutagenesis, strain selection, transformation, and genetic manipulation over a period of seven years. We resequenced this strain on three different sequencing platforms. Surprisingly, we found fewer than a dozen mutations in open reading frames. All three sequencing technologies were able to identify each single nucleotide mutation given at least 10-15-fold nominal sequence coverage. Our results show that detecting mutations in evolved and engineered organisms is rapid and cost-effective at the whole-genome level using new sequencing technologies. Identification of specific mutations in strains with altered phenotypes will add insight into specific gene functions and guide further metabolic engineering efforts.[Supplemental material is available online at www.genome.org. Complete data sets are available at the NCBI Short Read Archive under accession no. SRA 001158 (ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead).]Pichia stipitis (Pignal) is a haploid yeast related to endosymbionts of beetles that degrade rotting wood (Suh et al. 2003). It is an important organism for bioenergy production from lignocellulosic materials because of its high capacity to ferment xylose and cellobiose to ethanol (Parekh et al. 1988). We previously sequenced the reference strain, Pichia stipitis CBS-6054, resulting in a completely characterized genome of eight chromosomes totaling 15.4 Mb of sequence (Jeffries et al. 2007). This strain has been subjected to chemical mutagenesis, phenotypic selection, genetic engineering, and adaptive evolution in order to develop strains improved for ethanol production. Chemical mutagenesis and selection resulted in small improvements in ethanol production attributable in part to carbon catabolite derepression (Supplemental Fig. 1; Methods). Disruption of CYC1 (cyctochrome c, isoform 1) to create strain Shi21 increased the specific ethanol production rate by 50% and the ethanol yield by 10%; however, the nature of additional mutational events leading to this phenotype was uncharacterized.Traditional methods for identifying mutations are laborand time-intensive, so we tested the ability of next-generation sequencing technologies to determine the differences in this improved strain's entire genome relative to the reference strain. We generated high-coverage, whole-genome data sets using single fragment end reads from three next-generation sequencing platforms: 454 Life Sc...

We have developed an algorithm ("Lever") that systematically maps metazoan DNA regulatory motifs or motif combinations to the sets of genes that they likely regulate. Lever accomplishes this by assessing whether the motifs are enriched within cis regulatory modules (CRMs), predicted by our "PhylCRM" algorithm, in the noncoding sequences surrounding genes in a collection of gene sets. When these gene sets correspond to Gene Ontology (GO) categories, the results of Lever analysis allow the unbiased assignment of functional annotations to the regulatory motifs and also to the candidate CRMs that comprise the genomic motif occurrences. We demonstrate these methods using human myogenic differentiation as a model system, for which we statistically assessed greater than 25,000 pairings of gene sets and motifs / motif combinations. These results allowed us to assign functional annotations to candidate regulatory motifs predicted previously, and to identify gene sets that are likely to be co-regulated via shared regulatory motifs. Lever allows moving beyond the identification of putative regulatory motifs in mammalian genomes, towards understanding their biological roles. This approach is general and can be applied readily to any cell type, gene expression pattern, or organism of interest.

BackgroundCalorie restriction (CR) is the only intervention known to extend lifespan in a wide range of organisms, including mammals. However, the mechanisms by which it regulates mammalian aging remain largely unknown, and the involvement of the TOR and sirtuin pathways (which regulate aging in simpler organisms) remain controversial. Additionally, females of most mammals appear to live longer than males within species; and, although it remains unclear whether this holds true for mice, the relationship between sex-biased and CR-induced gene expression remains largely unexplored.Methodology/Principal FindingsWe generated microarray gene expression data from livers of male mice fed high calorie or CR diets, and we find that CR significantly changes the expression of over 3,000 genes, many between 10- and 50-fold. We compare our data to the GenAge database of known aging-related genes and to prior microarray expression data of genes expressed differently between male and female mice. CR generally feminizes gene expression and many of the most significantly changed individual genes are involved in aging, hormone signaling, and p53-associated regulation of the cell cycle and apoptosis. Among the genes showing the largest and most statistically significant CR-induced expression differences are Ddit4, a key regulator of the TOR pathway, and Nnmt, a regulator of lifespan linked to the sirtuin pathway. Using western analysis we confirmed post-translational inhibition of the TOR pathway.ConclusionsOur data show that CR induces widespread gene expression changes and acts through highly evolutionarily conserved pathways, from microorganisms to mammals, and that its life-extension effects might arise partly from a shift toward a gene expression profile more typical of females.