High-Throughput Analysis


Cap analysis gene expression (CAGE) is a method used to discover new promoters and for quantifying gene activity, providing data essential for studies of regulatory gene networks. But CAGE requires large amounts of RNA, which are often not obtainable from rare specimens. In the January issue of Cold Spring Harbor Protocols Piero Carninci and colleagues from the RIKEN Yokohama Institute’s Omics Science Center present NanoCAGE: A High-Resolution Technique to Discover and Interrogate Cell Transcriptomes, a method that can capture information from as little as 10 nanograms of total RNA. The protocol describes how to rapidly prepare nanoCAGE libraries which can be sequenced with high sensitivity. As one of January’s featured articles, the protocol is freely available to subscribers and non-subscribers alike.

N-terminalomics is a high-throughput strategy for identifying proteins by selectively enriching for and sequencing their N-terminal peptides by mass spectrometry. In the November issue of Cold Spring Harbor Protocols, Samie Jaffrey and colleagues from Cornell University present a newly-developed N-terminalomic approach, N-CLAP (N-terminalomics by Chemical Labeling of the alpha-Amine of Proteins). N-CLAP: Global Profiling of N-Termini by Chemoselective Labeling of the alpha-Amine of Proteins describes the use of Edman chemistry to modify all of the amines in proteins, followed by the generation of a new unmodified amine at the N-terminus after the removal of the first amino acid by peptide bond cleavage. The alpha-amine at the protein N-terminus is labeled with a cleavable biotin affinity tag, which facilitates the downstream purification of the N-terminal peptides. Peptides are eluted by cleaving the biotin affinity tag and identified by tandem mass spectrometry (MS/MS). N-CLAP can be used for the identification of signaling peptides for mature proteins as well as for global profiling of cleavage events that occur during cell signaling, such as apoptosis.

Large segments of DNA can vary in copy number between individuals. Such copy number variations (CNVs) contribute greatly to genetic diversity and are also thought to be associated with susceptibility or resistance to some diseases, including cancer. Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS), featured in the September issue of Cold Spring Harbor Protocols, provides an assay for determining the copy number of any allele in the genome. The method, from Raphael Kopan and colleagues at Washington University, takes advantage of the fact that pyrosequencing can accurately measure the ratio of DNA fragments in a mixture that differ by a single nucleotide. A reference allele with a known copy number and a query allele with an unknown copy number are engineered with single nucleotide variations, and the ratio seen between these probes and genomic DNA reflects the copy number. RQPS can be used to measure copy number of any transgene, differentiate homozygotes from heterozygotes, detect the CNV of endogenous genes, and screen embryonic stem cells targeted with bacterial artificial chromosome (BAC) vectors. RQPS is rapid, inexpensive, sensitive, and adaptable to high-throughput approaches. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

Improvements in automation and acquisition time have made the microscope a viable platform for performing hundreds of concurrent parallel experiments. Using these sorts of tools, it is now possible to run high-throughput screens for protein function and interaction in living cells, examining dynamic cellular processes to distinguish between primary and secondary phenotypes, and to study the phenotype kinetics. In the August issue of Cold Spring Harbor Protocols, Jan Ellenberg and colleagues from the EMBL present High-Throughput Microscopy Using Live Mammalian Cells, an overview of how to screen live cells using imaging technologies. The article examines each aspect of the general screening process and considers specific examples in the processing of time-lapse experiments. The techniques discussed are based on the use of cultured mammalian cells, but the concepts are easily transferred to cultured cells from other species like Drosophila and small organisms such as C. elegans.

While 454-based pyrosequencing has led to great advances, an intrinsic artifact of the process leads to artificial over-representation of more than 10% of the original DNA sequencing templates. This is particularly problematic in metagenomic studies, where the abundance of any sequence in a dataset is often used for comparative community analysis. It’s important to remove these artificial replicates before analysis. This phenomenon can skew data interpretation when making comparisons between datasets. As metagenome datasets become more plentiful, the ability to apply more robust statistical tests becomes increasingly important, and the validity of the input datasets becomes more crucial. Tools such as MG-RAST (covered in the January issue of Cold Spring Harbor Protocols in Using the Metagenomics RAST Server (MG-RAST) for Analyzing Shotgun Metagenomes) have the capability to remove exact duplicates, but this captures only a subset of the artificial replicates. In the April issue of Cold Spring Harbor Protocols, Tracy Teal and Thomas Schmidt from Michigan State University present an instruction set for Identifying and Removing Artificial Replicates from 454 Pyrosequencing Data. Their 454 Replicate Filter is a web-based tool that incorporates the algorithm cd-hit. This protocol provides details on how to use the replicate filter and obtain a file of unique sequences for use in metagenomic or transcriptomic analyses. This allows users to obtain a more accurate quantitative representation of the sequence diversity in a dataset.

The goal of tissue engineering is to recapitulate healthy human organs and tissue structures in culture, and then transplant them into patients, where they are fully integrated. This is a complicated process, and the use of high-throughput imaging systems that allow researchers to directly monitor transplanted tissues in live animals over time is important for improving the culturing and implantation techniques, as well as the design of artificial tissue scaffolds. By using transgenic animals with cell-specific fluorescent reporters, parameters such as tissue perfusion, donor cell survival, and donor-host cell interaction/integration can be observed. In the April issue of Cold Spring Harbor Protocols, Mary Dickinson and colleagues from the Baylor College of Medicine present a protocol for the use of The Mouse Cornea as a Transplantation Site for Live Imaging of Engineered Tissue Constructs. This is a modified version of the classical corneal micropocket angiogenesis assay, which employs it as a live imaging “window” to monitor angiogenic hydrogel tissue constructs. As one of April’s featured articles, it is freely available to subscribers and nonsubscribers alike.

The use of recombinant proteins, antibodies, small molecules, or nucleic acids as affinity reagents is a simple yet powerful strategy to study the protein/bait interactions that drive biological processes. Analysis via mass spectrometry rather than western blotting extends the identification of interactors, often allowing detection of thousands of proteins from complex mixtures. But this increased sensitivity can lead to problems distinguishing specific interactions from background noise. In the March issue of Cold Spring Harbor Protocols, Shao-En Ong from the Broad Institute of MIT and Harvard presents Unbiased Identification of Protein/Bait Interactions Using Biochemical Enrichment and Quantitative Proteomics. This method uses quantitative proteomics approaches to compare enrichment with the bait of interest against samples using control baits to allow sensitive detection and discrimination of specific protein/bait interactions. As one of March’s featured articles, it is freely available to subscribers and non-subscribers alike.

The incorporation of thymidine analogues, such as 5-bromo-2′-deoxyuridine (BrdU), into newly synthesized DNA is a powerful tool for analysis of DNA replication, repair and other aspects of DNA metabolism. In Genome-Wide Analysis of DNA Synthesis by BrdU Immunoprecipitation on Tiling Microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae, Oscar Aparicio and colleagues from the University of Southern California couple BrdU immunoprecipitation with DNA microarrays to enable genome-wide identification of BrdU-labeled chromosomal DNA. BrdU-IP-chip has many potential applications and has already been used to identify replication origins, make quantitative comparisons of origin firing between strains, and examine replication fork progression. As one of February’s featured articles in Cold Spring Harbor Protocols, the protocol is freely available to subscribers and non-subscribers alike.

Mapping DNase I hypersensitive sites has long been the standard method for identifying genetic regulatory elements such as promoters, enhancers, silencers, insulators, and locus control regions. Sequences that are nucleosome-depleted, presumably to provide access for transcription factors, are selectively digested by DNase I. Traditional low-throughput methods use Southern blots to then identify these hypersensitive sites. In the February issue of Cold Spring Harbor Protocols, Gregory Crawford and colleagues from Duke University present DNase-seq: A High-Resolution Technique for Mapping Active Gene Regulatory Elements Across the Genome from Mammalian Cells. DNase-seq is a high-throughput method that identifies DNase I hypersensitive sites across the whole genome by capturing DNase-digested fragments and applying next-generation sequencing techniques. In a single experiment, DNase-seq can identify most active regulatory regions from potentially any cell type, from any species with a sequenced genome. As one of February’s featured articles, it is freely available to subscribers and non-subscribers alike.

The introduction of high-throughput laboratory methods has greatly increased the pace of research into the genetics of complex diseases. Instead of focusing only on one or a few coding variants in a small sample of individuals, the ability to accurately and efficiently genotype many individuals and to cover more of the variation within individual genes has resulted in genetic studies with greater statistical power. Laboratory Methods for High-Throughput Genotyping, from Howard Edenberg and Yunlong Liu at the University of Indiana, presents an overview of the commonly used methods for high-throughput single-nucleotide polymorphism (SNP) genotyping for different stages of genetic studies and briefly reviews some of the high-throughput sequencing methods just coming into use. The authors also discuss recent developments in “next-generation” sequencing that will enable other kinds of studies. The article is excerpted from the recently published Genetics of Complex Human Diseases laboratory manual. It is featured in the November issue of Cold Spring Harbor Protocols, and like all our featured articles, is freely available to subscribers and non-subscribers alike.

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