Generation and annotation of the DNA sequences of human chromosomes 2 and 4

Human chromosome 2 is unique to the human lineage in being the product of a head-to-head fusion of two intermediate-sized ancestral chromosomes. Chromosome 4 has received attention primarily related to the search for the Huntington's disease gene, but also for genes associated with Wolf-Hirschhorn syndrome, polycystic kidney disease and a form of muscular dystrophy. Here we present approximately 237 million base pairs of sequence for chromosome 2, and 186 million base pairs for chromosome 4, representing more than 99.6% of their euchromatic sequences. Our initial analyses have identified 1,346 protein-coding genes and 1,239 pseudogenes on chromosome 2, and 796 protein-coding genes and 778 pseudogenes on chromosome 4. Extensive analyses confirm the underlying construction of the sequence, and expand our understanding of the structure and evolution of mammalian chromosomes, including gene deserts, segmental duplications and highly variant regions.     

Global variation in copy number in the human genome

Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.     

Accurate whole human genome sequencing using reversible terminator chemistry

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.     

Chromatography of carbohydrates

The coupling of high-performance anion-exchange chromatography with pulsed amperometric detection provides a much needed analytical tool for the analysis of the carbohydrates of glycoconjugates.     

Dissecting the catalytic triad of a serine protease

Serine proteases are present in virtually all organisms and function both inside and outside the cell; they exist as two families, the 'trypsin-like' and the 'subtilisin-like', that have independently evolved a similar catalytic device characterized by the Ser, His, Asp triad, an oxyanion binding site, and possibly other determinants that stabilize the transition state (Fig. 1). For Bacillus amyloliquefaciens subtilisin, these functional elements impart a total rate enhancement of at least 10(9) to 10(10) times the non-enzymatic hydrolysis of amide bonds. We have examined the catalytic importance and interplay between residues within the catalytic triad by individual or multiple replacement with alanine(s), using site-directed mutagenesis of the cloned B. amyloliquefaciens subtilisin gene. Alanine substitutions were chosen to minimize unfavourable steric contacts and to avoid imposing new charge interactions or hydrogen bonds from the substituted side chains. In contrast to the effect of mutations in residues involved in substrate binding, the mutations in the catalytic triad greatly reduce the turnover number and cause only minor effects on the Michaelis constant. Kinetic analyses of the multiple mutants demonstrate that the residues within the triad interact synergistically to accelerate amide bond hydrolysis by a factor of approximately 2 X 10(6).