The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

/pdf/initial-sequencing-and-analysis-of-the-human-genome-5dapk1rsx1.pdf

Initial sequencing and analysis of the human genome.

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

http://science.sciencemag.org/content/sci/291/5507/1304.full.pdf

The sequence of the human genome.

Probability Distributions.- Linear Models for Regression.- Linear Models for Classification.- Neural Networks.- Kernel Methods.- Sparse Kernel Machines.- Graphical Models.- Mixture Models and EM.- Approximate Inference.- Sampling Methods.- Continuous Latent Variables.- Sequential Data.- Combining Models.

Pattern Recognition and Machine Learning

As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.

/pdf/the-human-genome-browser-at-ucsc-4zclgthyly.pdf

The Human Genome Browser at UCSC

Much of the work directed at the creation of arrays for protein analysis relies on the use of highly selective molecules as receptors[1] In these arrays, the receptors are immobilized and exposed to a mixture of target molecules[1d] or to heterogeneous biological samples such as serum from cancer patients[2] However, such arrays generally only detect the molecules they were programmed to detect, and are ill-suited for the detection of protein variants, such as sequence changes, deletions, and insertions

/pdf/identifying-protein-variants-with-cross-reactive-aptamer-u4gdrc50ba.pdf

Identifying protein variants with cross-reactive aptamer arrays.

Single-stranded DNA (ssDNA) oligonucleotides are useful as aptamers, hybridization probes and for emerging applications in DNA nanotechnology. Current methods to purify ssDNA require both a strand-separation step and a separate size-separation step but may still leave double-stranded DNA (dsDNA) impurities in the sample. Here, we use commercially available acrydite DNA primers to immobilize one strand of a PCR product within a polyacrylamide matrix. Electrophoresis moves the non-crosslinked DNA into the gel where the single-stranded product of desired size can be recovered. Our results show this method produces high yields of pure ssDNA.

Purification of single-stranded DNA by co-polymerization with acrylamide and electrophoresis

During the past decade, aptasensors have largely been designed on the basis of the notion that ligand-modulated equilibration between aptamer conformations could be exploited for sensing. One implementation of this strategy has been to denature the aptamer with an antisense oligonucleotide, wait for dissociation of the antisense oligonucleotide, and stabilize the folded, signaling conformer with a ligand. However, there is a large kinetic barrier associated with releasing the oligonucleotide from the aptamer to again obtain an active, binding conformation. If the length of the antisense oligonucleotide is decreased to make dissociation from the aptamer more favorable, higher background signals are observed. To improve the general methodology for developing aptasensors, we have developed a novel and robust strategy for aptasensor design in which an oligonucleotide kinetically competes with the ligand for binding rather than having to be released from a stable duplex. While the oligonucleotide can induce co...

Engineering Signaling Aptamers That Rely on Kinetic Rather Than Equilibrium Competition

This unit describes the process of gene shuffling, also known as sexual PCR. Gene shuffling is a facile method for the generation of sequence libraries containing the information from a family of related genes. Essentially, related genes are fragmented by DNase I digestion and reassembled by primer-less PCR. The resulting chimeric genes can then be screened or selected for a desired function.

/pdf/library-generation-by-gene-shuffling-zsasuxkv6o.pdf

Library Generation by Gene Shuffling

A series of semi-specific peptides reported in the literature to bind various epitopes on cell surfaces were used in a differential sensing array to pattern cell line identity. The peptides were conjugated to thiazole orange to act as both a fluorescence reporter and a DNA intercalator. Fluorescence data for the peptides exposed to cells, with and without exogenous double stranded DNA (dsDNA), led to chemometric fingerprints for eight cancer cell lines. In contrast to the use of structures meant to act in completely non-specific ways, the use of a limited level of specificity generated linear discriminant score plots with high dimensionality, i.e. several principle components carrying significant variance. The arrays were found to correctly classify the cell lines from 60% to 100% depending upon the cell line. Due to the high dimensionality score plots, the identification of cell lines that were not part of the training set was examined. Support vector machines were used as a novelty detection routine and showed that a cancer line not part of the original training set could be correctly identified as being novel.

Andrew D. Ellington

Papers

Identifying protein variants with cross-reactive aptamer arrays.

Purification of single-stranded DNA by co-polymerization with acrylamide and electrophoresis

Engineering Signaling Aptamers That Rely on Kinetic Rather Than Equilibrium Competition

Library Generation by Gene Shuffling

Differential array sensing for cancer cell classification and novelty detection