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

What's so great about RNA?

As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of synthetic biology. However, synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment. Curr. Protoc. Mol. Biol. 99:3.24.1-3.24.10. © 2012 by John Wiley & Sons, Inc.


Keywords:

CEL I;
error correction;
synthetic DNA;
Surveyor nuclease;
synthetic biology;
oligonucleotide;
oligonucleotide synthesis

Enrichment of Error-Free Synthetic DNA Sequences by CEL I Nuclease

Publisher Summary
Reagentless biosensors are sensors that can detect a target analyte in a homogenous format, that is, without the addition of reagents other than the sample. A reagentless biosensor has many advantages over conventional diagnostic assays. The most obvious advantage is its simplicity and ease of use. Functional nucleic acids can be engineered, based largely on an appreciation of their secondary structures and chemically synthesized in bulk. It has proven remarkably easy to incorporate aptamers and ribozymes into reagentless biosensors that directly transduce ligand recognition to optical signals. By simply cleaving apart or adjoining flurophores and quenchers on oligonucleotide substrates, one can potentially construct reagentless rizozyme or aptazyme chips that could simultaneously report the concentration of multiple different analytes. Prototypes of such chips have now been made. While reagentless nucleic acid biosensors may ultimately prove less sensitive or robust than the reagentless protein biosensors, it is nonetheless likely that nucleic acid biosensors are much more amenable to generation by high-throughput selection method.

Chapter 12 – Nucleic Acids for Reagentless Biosensors

The availability of custom synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing “in-house” gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides. Curr. Protoc. Mol. Biol. 99:3.23.1-3.23.18. © 2012 by John Wiley & Sons, Inc.


Keywords:

gene synthesis;
synthetic genes;
synthetic DNA;
synthetic biology;
protein expression;
gene assembly;
oligonucleotides;
oligonucleotide assembly;
oligonucleotide synthesis;
inside-out nucleation;
PCR

Design and assembly of large synthetic DNA constructs.

Publisher Summary Reagentless biosensors are sensors that can detect a target analyte in a homogenous format, that is, without the addition of reagents other than the sample. Reagentless biosensors can be constructed by integrating a signaling or reporter component with a biological macromolecule, allowing the biomolecule to directly transduce the molecular recognition event into a detectable signal. Similarly, reagentless electrochemical biosensors have been made by electrostatic self-deposition of redox polyelectrolyte mediators and enzymes. Aptazymes have proven remarkably plastic and can be activated by metals, small organic molecules, peptides, and proteins. By simply cleaving apart or adjoining fluorophores and quenchers on oligonucleotide substrates, one can potentially construct reagentless ribozyme or aptazyme chips that could simultaneously report the concentration of multiple different analytes. Prototypes of such chips have now been made. While reagentless nucleic acid biosensors may ultimately prove less sensitive or robust than reagentless protein biosensors, it is nonetheless likely that nucleic acid biosensors will prove much more amenable to generation by high throughput selection methods, and thus may be the best vehicle for developing chips that can acquire organismal proteomes and metabolomes.

Andrew D. Ellington

Papers

What's so great about RNA?

Enrichment of Error-Free Synthetic DNA Sequences by CEL I Nuclease

Chapter 12 – Nucleic Acids for Reagentless Biosensors

Design and assembly of large synthetic DNA constructs.

Nucleic acids for reagentless biosensors