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.

Our long-running series of articles highlighting emerging model organisms continues in September with three entries, The Starlet Sea Anemone (Nematostella vectensis), Cephalochordates (Amphioxus or Lancelets) and The Western Clawed Frog (Xenopus tropicalis).

The slow rate of sequence evolution, the presumed high degree of preservation of ancestral traits, the ease of culturing, and the availability and experimental tractability of the early embryos have made Nematostella a prime cnidarian model for a number of biological studies. It serves not only as a model system for cnidarians, but also as an important representative of its phylum in comparisons with other lower Metazoa or Bilateria. Ulrich Technau and colleagues provide an overview of Nematostella, and protocols for spawning, in situ hybridization, antibody and phalloidin staining and BrdU labeling.

Cephalochordates, commonly called amphioxus or lancelets, are marine invertebrate chordates. Studies on cephalochordates have answered some long-standing questions concerning the evolution of vertebrates from their invertebrate ancestors and have also generated interesting avenues for further investigation of the evolutionary origin of developmental mechanisms that led to the emergence of the vertebrate body plan. Linda Holland and colleagues provide background on Cephalochordates, along with detailed methods for Amphioxus embryo collection, in situ hybridization, DNA extraction, and RNA extraction and extracting RNA from small amounts of tissue for RT-PCR.

Xenopus tropicalis is a small, wholly aquatic frog that is a diploid relative of Xenopus laevis. It shares many of the advantages of X. laevis as a model organism for studying aspects of vertebrate biology, particularly the genetic, biochemical, and environmental factors that influence vertebrate development from embryonic stages through adulthood. X. tropicalis is also finding uses as an important test species for assessing the impact of environmental toxins and disease on amphibians, which are in decline in many areas of the world due to water-borne pollutants and infectious agents such as the chytrid fungus. Frank Conlon and colleagues have contributed an overview of X. tropicalis, along with protocols for natural mating, in vitro fertilization, and tissue sampling and genomic DNA preparation.

No, this posting isn’t an Aesopian fable, it’s a note on our Emerging Model Organisms featured in July’s issue of Cold Spring Harbor Protocols. This month we’re covering Ants (Formicidae) and the The Painted Turtle, Chrysemys picta.

Painted Turtles have been the subject of study in many areas, including their buoyancy system, the trade-offs between offspring size and number, the ability to “overwinter”, the reptilian lymphatic system, and as an example of temperature dependent sex determination. Nicole Valenzuela from Iowa State University provides The Painted Turtle, Chrysemys picta: A Model System for Vertebrate Evolution, Ecology, and Human Health, along with a protocol for Egg Incubation and Collection of Painted Turtle Embryos.

Like many other organisms included in this series, it’s probably a misnomer to refer to ants as an “emerging model organism” as they’ve long been a key species for studying ecology, evolution, behavior, and development. Chris Smith and colleagues provide Ants (Formicidae): Models for Social Complexity, which gives an overview of ants as a model system. Protocols are available for colony sampling, marking individual ants, ecological sampling, stable isotope and elemental analysis, fat extraction, dissection, DNA isolation, hormone extraction, ecdysteroid extraction and radioimmunoassay, assay of hormone biosynthesis, GC-MS for characterization of semiochemicals, in situ hybridization, and phase-unknown linkage mapping. They’ve also supplied this month’s cover, and we made an extra effort to make sure we used an appropriate species.

Nested Patch PCR is a method designed to identify SNPs and mutations across many targeted loci for many samples in parallel. In the July issue of Cold Spring Harbor Protocols, Robi Mitra and colleagues from Washington University present Nested Patch PCR for Highly Multiplexed Amplification of Genomic Loci, a method where a large number (greater than 90) of targeted loci from genomic DNA are simultaneously amplified in the same reaction. These amplified loci can then be sequenced on a second-generation sequencing machine to detect single nucleotide polymorphisms (SNPs) and mutations.

Methods that employ mulitplexing during PCR reactions are often hampered by increased interprimer interactions that inhibit uniform amplification and increased formation of mispriming products. The protocol presented here was designed to reduce these two problems and results in a high specificity, with 90% of sequencing reads mapping to targeted loci. Nested Patch PCR is well-suited for the amplification of an intermediate number (100-1000) of targeted regions across a large number of samples and it offers a simple workflow that is compatible with 96-well plates and sample-specific DNA barcodes.

Microbial populations have traditionally been studied in carefully controlled, laboratory-grown cultures. New metagenomic approaches are being developed to study these organisms in environmental or medical samples. The July issue of Cold Spring Harbor Protocols presents a method developed by Holger Daims from the University of Vienna for quantifying populations of microorganisms in a variety of naturally occurring conditions such as plankton samples or biofilms. Use of Fluorescence In Situ Hybridization and the daime Image Analysis Program for the Cultivation-Independent Quantification of Microorganisms in Environmental and Medical Samples combines fluorescent in situ hybridization using rRNA-targeted probes with digital image analysis. The results show an organism’s “biovolume fraction” in a given sample; this indicates the share of biochemical reaction space occupied by the quantified population and can be more relevant ecologically than absolute cell numbers. Like all of our featured articles, this protocol is freely available to subscribers and non-subscribers alike.

High-throughput whole-genome analysis is becoming a standard laboratory approach for investigating cellular processes. Next-generation sequencing is replacing microarrays as the technique of choice for genome-scale analysis, because it offers advantages in both sensitivity and scale. The June issue of Cold Spring Harbor Protocols features Native Chromatin Preparation and Illumina/Solexa Library Construction from Keji Zhao and colleagues at the National Heart, Lung and Blood Institute. The article describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Solexa/Illumina Genome Analyzer. Step-by-step instructions are given for purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.

Biofilms are the natural state of an estimated 99% of prokaryotes in the environment and are defined as an aggregation of microorganisms in a self-created matrix on a surface. Examples are plaque on teeth, or the slime on rocks at the bottom of a river. Because these biofilms can’t be effectively cultured in the laboratory, new techniques are being developed to isolate material from the environment, allowing for a better understanding of the microbes responsible for many diseases and infections. The field of metagenomics, “the culture-independent analysis of a mixture of microbial genomes (termed the metagenome) using an approach based either on expression or on sequencing” is rapidly growing. October’s issue of CSH Protocols presents two useful methods for the study of biofilms from the laboratory of Michael J. Franklin, of Montana State University’s Center for Biofilm Engineering.

Isolation of RNA and DNA from Biofilm Samples Obtained by Laser Capture Microdissection Microscopy describes techniques for embedding biofilms in cryoembedding resin, producing thin sections and isolating discrete sections through laser capture microdissection microscopy. RNA or DNA is then extracted from these discrete populations of cells.

qRT-PCR of Microbial Biofilms takes the RNA isolated in the first method and allows analysis of the number of RNA transcripts of specific genes from bacteria growing in biofilms through quantitative reverse transcriptase real time PCR.

The October issue of CSH Protocols presents a new focus on Emerging Model Organisms.

Much of twentieth century biological research has focused on a limited number of model organisms, such as Arabidopsis, C. elegans, mouse, Drosophila, and E. coli. These classical model species, chosen because they are amenable to laboratory research and suitable for studying a range of biological problems, have served to elucidate many biological processes that can be generalized across a wider array of organisms. It is only a slight exaggeration to say that the basic workings of the cell were elucidated mostly from experiments on a few single-celled organisms — primarily E.coli and yeast. Our understanding of animal development was largely based on the genetics of fruit fly and worm and on the manipulation of a handful of amphibians and mouse; most of what we learned about the molecular and developmental biology of plants came from examining Arabidopsis and just a few other species. But biology wasn’t always done this way.
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To aid in the study of genetic diseases, the International Haplotype Map Project has developed a haplotype map of the human genome, a tool that displays common patterns of genetic variation. While data from the project are available for unrestricted public use from the project’s website, the new tools needed to browse the map can be difficult to master for the beginner. This month’s issue of Cold Spring Harbor Protocols features a set of articles with clear, step-by-step instructions for the analysis of HapMap data.

Browsing HapMap Data Using the Genome Browser provides details on how to navigate to and explore HapMap data for a gene or region of interest. Written by Albert Vernon Smith, this protocol shows how to analyze a candidate gene to find out whether there are any common single nucleotide polymorphisms (SNPs) in the immediate vicinity, what those SNPs’ alleles are, and the relative frequencies of the alleles in the population. As one of our featured articles for the month, it’s freely available to subscribers and non-subscribers.

The other articles in the set (subscribers only) are Generating HapMap Data Text Reports Using the Genome Browser, Manipulating HapMap Data Using HaploView, Retrieving HapMap Data Using HapMart, and Retrieving HapMap Data via Bulk Download. If your institution does not yet subscribe and you’d like to see these articles, you can sign up here for a free three month trial.

With the sequencing of the human genome came the startling revelation that the number of copies of a given gene can vary widely between individuals. This Copy Number Variation (or CNV), contributes to our species’ genetic diversity but it has also been linked to genetic diseases. This month’s issue of Cold Spring Harbor Protocols features a new method for detecting copy number variation. Like all of our monthly featured protocols, it’s freely accessible for subscribers and non-subscribers alike.

Copy Number Variation Detection Via High-Density SNP Genotyping
describes the use of PennCNV, a new computational tool for CNV detection in data from genomic arrays. Developed in the laboratory of Maja Bucan at the University of Pennsylvania, the software is freely available for download. Analysis with PennCNV will provide a more comprehensive understanding of genome variation and will aid in studies seeking the causes of genetic diseases. More information on PennCNV can be found in this Genome Research article, PennCNV: An integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data.

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