A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing.

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 approximately 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.

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing.

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.

Analysis of one million base pairs of Neanderthal DNA.

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.

Genome sequencing in microfabricated high-density picolitre reactors.

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.

Illness-related stigma, mood and adjustment to illness in persons with hepatitis C.

We examined stigma in persons with hepatitis C and its relationship with mood and adjustment to illness. We studied 87 persons awaiting interferon treatment for hepatitis C at St James's Hospital, Dublin. Stigma was assessed using Fife's Experience of Illness scale. A structured clinical interview was used to establish DSM-IV diagnosis. The Hospital Anxiety and Depression Scale (HADS) and Beck Depression Inventory (BDI) were also used as measures of mood. Factor analysis and clustering around latent variables analysis were used to assess scale structure and reliability. The stigma scale had an overall reliability of 0.94. A strong dimension of fear of disclosure emerged, from item analysis, together with dimensions of social isolation and social rejection. Stigma was higher in those in manual occupations and the unemployed than in those in non-manual occupation. There were high levels in those with disease associated with injecting drug use and iatrogenic disease caused by transfusion or anti-D blood products, and low levels in those who had been treated for haemophilia with contaminated products or whose hepatitis was of unknown origin. Adjusted for confounders, a 1-decile increase in stigma score had an odds ratio of 1.4 for DSM-IV depression and similar associations with depression on the HADS and BDI. Stigma was also associated with poorer work and social adjustment, lower acceptance of illness, higher subjective levels of symptoms and greater subjective impairment of memory and concentration. These associations were replicated in the non-depressed subsample. The results underline the strong link between stigma and well-being in hepatitis C. However, they also suggest that stigma is a complex construct that will require further research to elucidate.

Calculating effect size for continuous variables: D537--a robust, quantile-based approach.

The calculation of effect size is an important step in measuring the potential real-life significance of the effect of an intervention. In the case of continuous data, Cohen's d is frequently used. This scales the difference between the means of two groups, or the mean difference between pairs of measurements, by dividing by the standard deviation. However, outlying values, especially in small studies, can influence the size of d. This article presents D537, a robust formula for d that is based on rank statistics. The median is used as a measure of difference, while the scaling factor is the range between the 30th and 70th percentiles of the distribution; a range that is equal to one standard deviation when the data are normally distributed. When data are normally distributed, the value of D537 is equal to that of Cohen's d. As D537 is based on the 30th, 50th and 70th percentiles, it is robust to outliers.

Patterns of damage in genomic DNA sequences from a Neandertal.

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.