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The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome. [ABSTRACT FROM AUTHOR]

The 454 Sequencer has dramatically increased the volume of sequencing conducted by the scientific community and expanded the range of problems that can be addressed by the direct readouts of DNA sequence. Key breakthroughs in the development of the 454 sequencing platform included higher throughput, simplified all in vitro sample preparation and the miniaturization of sequencing chemistries, enabling massively parallel sequencing reactions to be carried out at a scale and cost not previously possible. Together with other recently released next-generation technologies, the 454 platform has started to democratize sequencing, providing individual laboratories with access to capacities that rival those previously found only at a handful of large sequencing centers. Over the past 18 months, 454 sequencing has led to a better understanding of the structure of the human genome, allowed the first non-Sanger sequence of an individual human and opened up new approaches to identify small RNAs. To make next-generation technologies more widely accessible, they must become easier to use and less costly. In the longer term, the principles established by 454 sequencing might reduce cost further, potentially enabling personalized genomics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls. Results: All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR. Conclusion: Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA. [ABSTRACT FROM AUTHOR]

Life beyond Earth may be based on RNA or DNA if such life is related to life on Earth through shared ancestry due to meteoritic exchange, such as may be the case for Mars, or if delivery of similar building blocks to habitable environments has biased the evolution of life toward utilizing nucleic acids. In this case, in situ sequencing is a powerful approach to identify and characterize such life without the limitations or expense of returning samples to Earth, and can monitor forward contamination. A new semiconductor sequencing technology based on sensing hydrogen ions released during nucleotide incorporation can enable massively parallel sequencing in a small, robust, optics-free CMOS chip format. We demonstrate that these sequencing chips survive several analogues of space radiation at doses consistent with a 2-year Mars mission, including protons with solar particle event-distributed energy levels and 1 GeV oxygen and iron ions. We find no measurable impact of irradiation at 1 and 5 Gy doses on sequencing quality nor on low-level hardware characteristics. Further testing is required to study the impacts of soft errors as well as to characterize performance under neutron and gamma irradiation and at higher doses, which would be expected during operation in environments with significant trapped energetic particles such as during a mission to Europa. Our results support future efforts to use in situ sequencing to test theories of panspermia and/or whether life has a common chemical basis. Key Words: Laboratory simulation experiments-Life-detection instruments-Nucleic acids-Mars-Panspermia. Astrobiology 13, 560-569. [ABSTRACT FROM AUTHOR]

Summary: A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from ∼0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 ± 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small. [Copyright &y& Elsevier]