ABSTRACT
The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.
Subject(s)
Chromosome Mapping/methods , DNA, Neoplasm/genetics , Mutation , Neoplasms/genetics , Base Sequence , Humans , Molecular Sequence Data , Neoplasms/diagnosis , Sensitivity and SpecificityABSTRACT
Neanderthals are the extinct hominid group most closely related to contemporary humans, so their genome offers a unique opportunity to identify genetic changes specific to anatomically fully modern humans. We have identified a 38,000-year-old Neanderthal fossil that is exceptionally free of contamination from modern human DNA. Direct high-throughput sequencing of a DNA extract from this fossil has thus far yielded over one million base pairs of hominoid nuclear DNA sequences. Comparison with the human and chimpanzee genomes reveals that modern human and Neanderthal DNA sequences diverged on average about 500,000 years ago. Existing technology and fossil resources are now sufficient to initiate a Neanderthal genome-sequencing effort.
Subject(s)
DNA/analysis , DNA/genetics , Fossils , Hominidae/genetics , Animals , DNA, Mitochondrial/analysis , DNA, Mitochondrial/genetics , Humans , Phylogeny , Polymorphism, Genetic/genetics , Population Density , Sequence Analysis, DNAABSTRACT
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.
Subject(s)
Genome, Bacterial , Genomics/instrumentation , Microchemistry/instrumentation , Mycoplasma genitalium/genetics , Sequence Analysis, DNA/instrumentation , Electrophoresis, Capillary , Emulsions , Fiber Optic Technology , Genomics/economics , Microchemistry/economics , Polymerase Chain Reaction , Reproducibility of Results , Sensitivity and Specificity , Sequence Analysis, DNA/economics , Time FactorsABSTRACT
High-throughput direct sequencing techniques have recently opened the possibility to sequence genomes from Pleistocene organisms. Here we analyze DNA sequences determined from a Neandertal, a mammoth, and a cave bear. We show that purines are overrepresented at positions adjacent to the breaks in the ancient DNA, suggesting that depurination has contributed to its degradation. We furthermore show that substitutions resulting from miscoding cytosine residues are vastly overrepresented in the DNA sequences and drastically clustered in the ends of the molecules, whereas other substitutions are rare. We present a model where the observed substitution patterns are used to estimate the rate of deamination of cytosine residues in single- and double-stranded portions of the DNA, the length of single-stranded ends, and the frequency of nicks. The results suggest that reliable genome sequences can be obtained from Pleistocene organisms.