Abstract:
:The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Harakalova M,Mokry M,Hrdlickova B,Renkens I,Duran K,van Roekel H,Lansu N,van Roosmalen M,de Bruijn E,Nijman IJ,Kloosterman WP,Cuppen Edoi
10.1038/nprot.2011.396subject
Has Abstractpub_date
2011-11-03 00:00:00pages
1870-86issue
12eissn
1754-2189issn
1750-2799pii
nprot.2011.396journal_volume
6pub_type
杂志文章相关文献
Nature Protocols文献大全abstract::RNA sequencing (RNA-seq) has been rapidly adopted for the profiling of transcriptomes in many areas of biology, including studies into gene regulation, development and disease. Of particular interest is the discovery of differentially expressed genes across different conditions (e.g., tissues, perturbations) while opt...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.099
更新日期:2013-09-01 00:00:00
abstract::The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the ɛ-amino group of lysine residues. This 'SUMOylation' alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific conseque...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.095
更新日期:2015-09-01 00:00:00
abstract::We describe a simple and versatile scheme to prepare a series of poly(ethylene glycol)-based bidentate ligands that permit strong interactions with colloidal semiconductor nanocrystals (quantum dots, QDs) and gold nanoparticles (AuNPs) alike and promote their dispersion in aqueous solutions. These ligands are synthesi...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.243
更新日期:2009-01-01 00:00:00
abstract::This protocol explains how to discover functional signals in genomic sequences by detecting over- or under-represented oligonucleotides (words) or spaced pairs thereof (dyads) with the Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/). Two typical applications are presented: (i) predicting transcription...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.98
更新日期:2008-01-01 00:00:00
abstract::The analysis of large microbiome data sets holds great promise for the delineation of the biological and metabolic functioning of living organisms and their role in the environment. In the midst of this genomic puzzle, viruses, especially those that infect microbial communities, represent a major reservoir of genetic ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2017.063
更新日期:2017-08-01 00:00:00
abstract::RNase H-dependent PCR-enabled T-cell receptor sequencing (rhTCRseq) can be used to determine paired alpha/beta T-cell receptor (TCR) clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples. With the enhanced specificity of RNase H-dependent PCR (rhPCR), it achieves TCR-specific...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-019-0195-x
更新日期:2019-08-01 00:00:00
abstract::The telomeric repeat amplification protocol (TRAP) is a two-step process for analyzing telomerase activity in cell or tissue extracts. Recent modifications of this sensitive assay include elimination of radioactivity by using a fluorescently labeled primer instead of a radiolabeled primer. In addition, the TRAP assay ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.239
更新日期:2006-01-01 00:00:00
abstract::Glycoproteins are involved in diverse biological processes ranging from extracellular contact and recognition to intracellular signaling. Crystal structures of glycoproteins would yield tremendous insight into these processes. But glycoprotein structural analysis has been hindered by difficulties in expressing milligr...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.29
更新日期:2009-01-01 00:00:00
abstract::Traumatic brain injury (TBI) is a major cause of mortality and morbidity. Various attempts have been made to replicate clinical TBI using animal models. The fluid-percussion model (FP) is one of the oldest and most commonly used models of experimentally induced TBI. Both central (CFP) and lateral (LFP) variations of t...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.112
更新日期:2010-09-01 00:00:00
abstract::Influenza A viruses (IAVs) cause epidemics and pandemics that result in considerable financial burden and loss of human life. To manage annual IAV epidemics and prepare for future pandemics, an improved understanding of how IAVs emerge, transmit, cause disease and acquire pandemic potential is urgently needed. Fundame...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.180
更新日期:2014-11-01 00:00:00
abstract::Over the past 20 years, the starlet sea anemone, Nematostella vectensis, a small estuarine animal, has emerged as a powerful model system for field and laboratory studies of development, evolution, genomics, molecular biology and toxicology. Here we describe how to collect Nematostella, culture it through its entire s...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.044
更新日期:2013-05-01 00:00:00
abstract::An important goal of the analysis of sequenced genomes of microbial pathogens is to improve the therapy of infectious diseases. In this context, a major challenge is to detect genomic-level evolutionary changes that increase microbial virulence. TimeZone, a genome analysis software package, is designed to detect footp...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.031
更新日期:2013-04-01 00:00:00
abstract::Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0105-7
更新日期:2019-02-01 00:00:00
abstract::As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-stat...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.254
更新日期:2006-01-01 00:00:00
abstract::The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.167
更新日期:2010-12-01 00:00:00
abstract::Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.315
更新日期:2007-01-01 00:00:00
abstract::Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time,...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.116
更新日期:2013-10-01 00:00:00
abstract::Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site an...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.010
更新日期:2013-03-01 00:00:00
abstract::This protocol describes an optimized method for direct in vitro monitoring of homo- and heterotypic axon-axon interactions involved in the developmental assembly of neural circuits. The assay exploits a classical example of heterotypic axonal interactions by modeling the sequential extension of spinal motor and somato...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.442
更新日期:2012-01-26 00:00:00
abstract::Mass spectrometry imaging (MSI) enables label-free spatial mapping of hundreds of biomolecules in tissue sections. This capability provides valuable information on tissue heterogeneity that is difficult to obtain using population-averaged assays. Despite substantial developments in both instrumentation and methodology...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-019-0237-4
更新日期:2019-12-01 00:00:00
abstract::Biominerals serve as critical structures of living systems and play important roles in biochemical processes. Understanding their crystallization mechanisms is therefore central to many areas of biology, biogeoscience, and biochemistry. Some biominerals, such as bone and dentin, are hierarchical nanocomposite structur...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0027-4
更新日期:2018-09-01 00:00:00
abstract::With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, ter...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.126
更新日期:2016-11-01 00:00:00
abstract::In most organisms, one crossover (CO) event inhibits the chances of another nearby event. The term used to describe this phenomenon is 'CO interference'. Here, we describe a protocol for quickly generating large data sets that are amenable to CO interference analysis in the flowering plant, Arabidopsis thaliana. We em...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.491
更新日期:2008-01-01 00:00:00
abstract::To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC an...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.115
更新日期:2013-10-01 00:00:00
abstract::This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.055
更新日期:2012-05-31 00:00:00
abstract::The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-00428-7
更新日期:2020-12-16 00:00:00
abstract::Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and comb...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.186
更新日期:2006-01-01 00:00:00
abstract::Many techniques have been developed for studying inducible gene expression, but all of them are multicomponent systems consisting of cis-acting elements at the DNA or RNA level, trans-acting regulator proteins and/or small molecules as inducers. RNA thermometers are the only known single-component regulators of gene e...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.112
更新日期:2009-01-01 00:00:00
abstract::Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein o...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.7
更新日期:2006-01-01 00:00:00
abstract::We describe a protocol for the insertion of ultrashort single-walled carbon nanotubes (SWCNTs) to form nanopores in a Montal-Mueller lipid bilayer. The SWCNTs are designed to bind to a specific analyte of interest; binding will result in the reduction of current in single-channel recording experiments. The first stage...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.112
更新日期:2015-11-01 00:00:00