The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.DNA sequencing has markedly changed the nature of biomedical research and medicine. Reductions in the cost, complexity and time required to sequence large amounts of DNA, including improvements in the ability to sequence bacterial and eukaryotic genomes, will have significant scientific, economic and cultural impact. Largescale sequencing projects, including whole-genome sequencing, have usually required the cloning of DNA fragments into bacterial vectors, amplification and purification of individual templates, followed by Sanger sequencing 1 using fluorescent chain-terminating nucleotide analogues 2 and either slab gel or capillary electrophoresis. Current estimates put the cost of sequencing a human genome between $10 million and $25 million 3 . Alternative sequencing methods have been described 4-8 ; however, no technology has displaced the use of bacterial vectors and Sanger sequencing as the main generators of sequence information.Here we describe an integrated system whose throughput routinely enables applications requiring millions of bases of sequence information, including whole-genome sequencing. Our focus has been on the co-development of an emulsion-based method 9-11 to isolate and amplify DNA fragments in vitro, and of a fabricated substrate and instrument that performs pyrophosphate-based sequencing (pyrosequencing 5,12 ) in picolitre-sized wells.In a typical run we generate over 25 million bases with a Phred quality score of 20 or better (predicted to have an accuracy of 99% or higher). Although this Phred 20 quality throughput is significantly higher than that of Sanger sequencing by capillary electrophoresis, it is currently at the cost of substantially shorter reads and lower average individual read accuracy. Sanger-based capillary electrophoresis sequencing systems produce up to 700 bases of sequence information from each of 96 DNA templates at an average read accuracy of 99.4% in 1 h, or 67,000 bases per hour, with substantially all of the bases having Phred 20 or better quality 23 . We further characterize the performance ...

BackgroundWith the exception of APOE ε4 allele, the common genetic risk factors for sporadic Alzheimer's Disease (AD) are unknown.Methods and FindingsWe completed a genome-wide association study on 381 participants in the ADNI (Alzheimer's Disease Neuroimaging Initiative) study. Samples were genotyped using the Illumina Human610-Quad BeadChip. 516,645 unique Single Nucleotide Polymorphisms (SNPs) were included in the analysis following quality control measures. The genotype data and raw genetic data are freely available for download (LONI, http://www.loni.ucla.edu/ADNI/Data/). Two analyses were completed: a standard case-control analysis, and a novel approach using hippocampal atrophy measured on MRI as an objectively defined, quantitative phenotype. A General Linear Model was applied to identify SNPs for which there was an interaction between the genotype and diagnosis on the quantitative trait. The case-control analysis identified APOE and a new risk gene, TOMM40 (translocase of outer mitochondrial membrane 40), at a genome-wide significance level of≤10−6 (10−11 for a haplotype). TOMM40 risk alleles were approximately twice as frequent in AD subjects as controls. The quantitative trait analysis identified 21 genes or chromosomal areas with at least one SNP with a p-value≤10−6, which can be considered potential “new” candidate loci to explore in the etiology of sporadic AD. These candidates included EFNA5, CAND1, MAGI2, ARSB, and PRUNE2, genes involved in the regulation of protein degradation, apoptosis, neuronal loss and neurodevelopment. Thus, we identified common genetic variants associated with the increased risk of developing AD in the ADNI cohort, and present publicly available genome-wide data. Supportive evidence based on case-control studies and biological plausibility by gene annotation is provided. Currently no available sample with both imaging and genetic data is available for replication.ConclusionsUsing hippocampal atrophy as a quantitative phenotype in a genome-wide scan, we have identified candidate risk genes for sporadic Alzheimer's disease that merit further investigation.

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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.

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