The complete genome of an individual by massively parallel DNA sequencing.

The association of genetic variation with disease and drug response, and improvements in nucleic acid technologies, have given great optimism for the impact of 'genomic medicine'. However, the formidable size of the diploid human genome, approximately 6 gigabases, has prevented the routine application of sequencing methods to deciphering complete individual human genomes. To realize the full potential of genomics for human health, this limitation must be overcome. Here we report the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. This sequence was completed in two months at approximately one-hundredth of the cost of traditional capillary electrophoresis methods. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence. In addition, we accurately identified small-scale (2-40,000 base pair (bp)) insertion and deletion polymorphism as well as copy number variation resulting in the large-scale gain and loss of chromosomal segments ranging from 26,000 to 1.5 million base pairs. Overall, these results agree well with recent results of sequencing of a single individual by traditional methods. However, in addition to being faster and significantly less expensive, this sequencing technology avoids the arbitrary loss of genomic sequences inherent in random shotgun sequencing by bacterial cloning because it amplifies DNA in a cell-free system. As a result, we further demonstrate the acquisition of novel human sequence, including novel genes not previously identified by traditional genomic sequencing. This is the first genome sequenced by next-generation technologies. Therefore it is a pilot for the future challenges of 'personalized genome sequencing'.

The (un)shared responsibility in the reverse logistics of portable batteries: A Brazilian case.

The Brazilian National Solid Waste Policy (BNSWP) determines that reverse logistics (RL) of waste batteries is mandatory and adopts the principle of shared responsibility among stakeholders. In this work, we sought to diagnose the current state of the RL chain for batteries in Vale do Aço, Brazil, and identify challenges and potentialities related to its improvement. Data were obtained by means of questionnaires sent to the main stakeholders involved in the batteries RL chain and by consultation of official Brazilian databases. The results showed that the main obstacles to the implementation of the RL of portable batteries in the region are: the lack of information by consumers and retailers regarding the BNSWP and management of waste batteries; the existence of few collection and consolidation points in the region; lack of municipal waste management plans; and lack of sustainability of waste picker organizations. The identified potentialities were: the good receptivity of environmental education actions by the population; the possibility of taking advantage of direct logistics of local retail chains to collect waste; the existence of a special waste management and transportation company in the region; and the existence of waste picker organizations interested in integrating the RL system. Finally, by a SWOT analysis, strategies for the implementation of a more effective RL system were drawn up. This work can serve as a basis for the structuring and implementation of the batteries RL system at the Metropolitan Region of Vale do Aço (MRVA) and can be a reference for other settings.

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.

Stimulation of 3'-->5' exonuclease and 3'-phosphodiesterase activities of yeast apn2 by proliferating cell nuclear antigen.

The Apn2 protein of Saccharomyces cerevisiae contains 3'-->5' exonuclease and 3'-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3'-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3'-->5' exonuclease and 3'-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.

Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2.

In the presence of proliferating cell nuclear antigen, yeast DNA polymerase delta (Pol delta) replicated DNA at a rate of 40-60 nt/s. When downstream double-stranded DNA was encountered, Pol delta paused, but most replication complexes proceeded to carry out strand-displacement synthesis at a rate of 1.5 nt/s. In the presence of the flap endonuclease FEN1 (Rad27), the complex carried out nick translation (1.7 nt/s). The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1. Maturation in the presence of DNA ligase was studied with various downstream primers. Downstream DNA primers, RNA primers, and small 5'-flaps were efficiently matured by Pol delta and FEN1, and Dna2 did not stimulate maturation. However, maturation of long 5'-flaps to which replication protein A can bind required both DNA2 and FEN1. The maturation kinetics were optimal with a slight molar excess over DNA of Pol delta, FEN1, and proliferating cell nuclear antigen. A large molar excess of DNA ligase substantially enhanced the rate of maturation and shortened the nick-translation patch (nucleotides excised past the RNA/DNA junction before ligation) to 4-6 nt from 8-12 nt with equimolar ligase. These results suggest that FEN1, but not DNA ligase, is a stable component of the maturation complex.