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.

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.

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.

January’s issue of Cold Spring Harbor Protocols wraps up the second volume of our ongoing Emerging Model Organisms series. The idea behind the series is that technical advances have allowed for great expansion in the range of organisms used for research. Each set of articles is meant to introduce the reader to a new organism, to explain why it’s useful for laboratory research and to provide information on husbandry, genetics and genomics, and a set of basic laboratory protocols. The first set of 23 emerging model systems was collected in a laboratory manual, and the current set of 18 will soon be as well. January’s organisms are:

The Rabbit (Oryctolagus cuniculus): The rabbit is a valuable animal model for a variety of biomedical research areas including in vitro fertilization, early embryology and organogenesis, neurophysiology, ophthalmology, and cardiovascular research. The rabbit is also used as a model for toxicology studies and analyses of drug effects on embryo and fetal development, as well as for research involving the immune system, not to mention its common use in antibody production. Christoph Viebahn and colleagues from the University of Göttingen provide an overview of the rabbit as an experimental system, and protocols for mating and embryo isolation, dissection and fixation of embryos, embryo culture, staining and imaging, immunofluorescence, in situ hybridization, mounting, embedding and sectioning, embryo transfer, artificial insemination and cryopreservation of embryos.

Paramecium tetraurelia: Paramecium makes an interesting unicellular model, as the authors note:

Paramecium tetraurelia is a widely distributed, free-living unicellular organism that feeds on bacteria and can easily be cultured in the laboratory. Its position within the phylum Ciliophora, remote from the most commonly used models, offers an interesting perspective on the basic cellular and molecular processes of eukaryotic life. Its large size and complex cellular organization facilitate morphogenetic studies of conserved structures, such as cilia and basal bodies, as well as electrophysiological studies of swimming behavior. Like all ciliates, P. tetraurelia contains two distinct types of nuclei, the germline micronucleus (MIC) and the somatic macronucleus (MAC), which differentiate from copies of the zygotic nucleus after fertilization. The sexual cycle can be managed by controlling food uptake, allowing the study of a developmentally regulated differentiation program in synchronous cultures. Spectacular genome rearrangements occur during the development of the somatic macronucleus. Their epigenetic control by RNA-mediated homology-dependent mechanisms, which might underlie long-known cases of non-Mendelian inheritance, provides evolutionary insight into the diversity of small RNA pathways involved in genome regulation. Being endowed with two alternative modes of sexual reproduction (conjugation and autogamy), P. tetraurelia is ideally suited for genetic analyses, and the recent sequencing of its macronuclear genome revealed one of the largest numbers of genes in any eukaryote. Together with the development of new molecular techniques, including complementation cloning and an easily implemented technique for reverse genetics based on RNA interference (RNAi), these features make P. tetraurelia a very attractive unicellular model.

Eric Meyer and colleagues from the CNRS have written an overview of P tetraurelia as a model system, and protocols for maintaining cell lines, mass culture, gene silencing, DNA microinjection, immunocytochemistry, and fluorescence in situ hybridization.

We have some new organisms in the works for Volume 3, but would welcome your suggestions.

Metagenomics, the study of DNA isolated from naturally occurring populations and samples, is rapidly growing. Improvements to cloning and sequencing techniques are allowing researchers to study organism in environmental samples, and new knowledge of species interactions and community dynamics is emerging. The identification of microorganisms in these samples is of vital importance to their interpretation. In the January issue of Cold Spring Harbor Protocols, Annelie Wendeberg of the Helmholtz Centre for Environmental Research presents a protocol for Fluorescence In Situ Hybridization for the Identification of Environmental Microbes. The methods described allow the phylogenetic identification of microorganisms in environmental samples (e.g., water and sediments) by means of fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes followed by signal amplification with catalyzed reporter deposition (CARD). The protocol is one of January’s featured articles, and like all featured articles in Cold Spring Harbor Protocols, it is freely accessible to subscribers and non-subscribers alike.

With the recent progress in understanding epigenetic mechanisms, methods for profiling patterns of DNA modification have become important tools for analysis of gene regulation. DNA methylation, in which cytosine is modified to form 5-methylcytosine, is a well-characterized epigenetic modification essential for normal development in plants and mammals. In the December issue of Cold Spring Harbor Protocols, Jon Reinders presents Amplification of Bisulfite-Converted DNA for Genome-Wide DNA Methylation Profiling. This method utilizes the treatment of DNA with sodium bisulfite, which converts unmethylated cytosine to uracil (5-methylcytosine is not converted). This is followed with PCR amplification, where the uracil amplifies as thymine, creating a C-to-T transition. The genome can then be analyzed for these transitions using an array-based platform. Reinders protocol mitigates the major issues with bisulfite conversion (DNA fragmentation and poor reproducibility) and reduces bias during the amplification step. While the protocol is optimized for use in Arabidopsis, it can potentially be adapted for use in other organisms.

We’re getting toward the end of the second volume of our Emerging Model Organisms series in Cold Spring Harbor Protocols, and November’s issue brings us a look at the Hawaiian Bobtail Squid and the genus Dioscorea, or True Yams.

Euprymna scolopes, the Hawaiian Bobtail Squid (our cover model this month, see below) is a cephalopod that’s well-suited for study in the laboratory. E. scolopes is primarily studied in three contexts:
1) as a model for cephalopod development–the embryos and protective chorions are clear, making it amenable for the observations and manipulations common in other studied model systems
2) as a model of animal-bacteria symbioses with the luminous marine bacterium Vibrio fischeri
3) as a system for studying the interaction of tissues with light, as the squid features a specialized light organ.

Heinz Gert de Couet and colleagues supply an overview of the Hawaiian Bobtail Squid as a model system, along with protocols for Preparation of Genomic DNA, Confocal Immunocytochemistry, Whole-Mount In Situ Hybridization (parts 1 and 2), and Culture and Observation.

Dioscorea is a large genus of plants that are monocots but that look like dicots, and are closely related to the phylogenetically derived group containing the grasses. It’s interesting evolutionarily because of the position it occupies, as a link between the eudicots and grasses–groups that contain all the model flowering plant species. The true yam is also important as a food crop. R. Geeta and colleagues provide an overview of the genus, and protocols for husbandry, culturing tissues, management of plantlets, controlled crosses, and DNA extraction.

CSH Protocols November Cover

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.