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  • 1
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 19, No. 9 ( 2009-09), p. 1606-1615
    Abstract: Utilizing the full power of next-generation sequencing often requires the ability to perform large-scale multiplex enrichment of many specific genomic loci in multiple samples. Several technologies have been recently developed but await substantial improvements. We report the 10,000-fold improvement of a previously developed padlock-based approach, and apply the assay to identifying genetic variations in hypermutable CpG regions across human chromosome 21. From ∼3 million reads derived from a single Illumina Genome Analyzer lane, ∼94% (∼50,500) target sites can be observed with at least one read. The uniformity of coverage was also greatly improved; up to 93% and 57% of all targets fell within a 100- and 10-fold coverage range, respectively. Alleles at 〉 400,000 target base positions were determined across six subjects and examined for single nucleotide polymorphisms (SNPs), and the concordance with independently obtained genotypes was 98.4%–100%. We detected 〉 500 SNPs not currently in dbSNP, 362 of which were in targeted CpG locations. Transitions in CpG sites were at least 13.7 times more abundant than non-CpG transitions. Fractions of polymorphic CpG sites are lower in CpG-rich regions and show higher correlation with human–chimpanzee divergence within CpG versus non-CpG sites. This is consistent with the hypothesis that methylation rate heterogeneity along chromosomes contributes to mutation rate variation in humans. Our success suggests that targeted CpG resequencing is an efficient way to identify common and rare genetic variations. In addition, the significantly improved padlock capture technology can be readily applied to other projects that require multiplex sample preparation.
    Type of Medium: Online Resource
    ISSN: 1088-9051
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2009
    detail.hit.zdb_id: 1483456-X
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  • 2
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    Cold Spring Harbor Laboratory ; 2016
    In:  Cold Spring Harbor Protocols Vol. 2016, No. 5 ( 2016-05), p. pdb.top086793-
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2016, No. 5 ( 2016-05), p. pdb.top086793-
    Abstract: In light of the multitude of new Cas9-mediated functionalities, the ability to carry out multiple Cas9-enabled processes simultaneously and in a single cell is becoming increasingly valuable. Accomplishing this aim requires a set of Cas9–guide RNA (gRNA) pairings that are functionally independent and insulated from one another. For instance, two such protein–gRNA complexes would allow for concurrent activation and editing at independent target sites in the same cell. The problem of establishing orthogonal CRISPR systems can be decomposed into three stages. First, putatively orthogonal systems must be identified with an emphasis on minimizing sequence similarity of the Cas9 protein and its associated RNAs. Second, the systems must be characterized well enough to effectively express and target the systems using gRNAs. Third, the systems should be established as orthogonal to one another by testing for activity and cross talk. Here, we describe the value of these orthogonal CRISPR systems, outline steps for selecting and characterizing potentially orthogonal Cas9–gRNA pairs, and discuss considerations for the desired specificity in Cas9-coupled functions.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2016
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  • 3
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    Online Resource
    Cold Spring Harbor Laboratory ; 2002
    In:  Genome Research Vol. 12, No. 7 ( 2002-07-01), p. 1019-1028
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 12, No. 7 ( 2002-07-01), p. 1019-1028
    Abstract: Gene expression is regulated by transcription factors that interact with cis -regulatory elements. Predicting these elements from sequence data has proven difficult. We describe here a successful computational search for elements that direct expression in a particular temporal-spatial pattern in the Drosophila embryo, based on a single well characterized enhancer model. The fly genome was searched to identify sequence elements containing the same combination of transcription factors as those found in the model. Experimental evaluation of the search results demonstrates that our method can correctly predict regulatory elements and highlights the importance of functional testing as a means of identifying false-positive results. We also show that the search results enable the identification of additional relevant sequence motifs whose functions can be empirically validated. This approach, combined with gene expression and phylogenetic sequence data, allows for genome-wide identification of related regulatory elements, an important step toward understanding the genetic regulatory networks involved in development. [Sequence data reported in this paper have been deposited in GenBank with accession nos. AF513981 ( Eve MHE) and AF513982 ( Hbr DME). Supplementary material is available online at http://www.genome.org . The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: R. Blackman]
    Type of Medium: Online Resource
    ISSN: 1088-9051 , 1549-5469
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2002
    detail.hit.zdb_id: 1483456-X
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  • 4
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    Online Resource
    Cold Spring Harbor Laboratory ; 2004
    In:  Genome Research Vol. 14, No. 2 ( 2004-02), p. 201-208
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 14, No. 2 ( 2004-02), p. 201-208
    Abstract: Various computational approaches have been developed for predicting cis -regulatory DNA elements in prokaryotic genomes. We describe a novel method for predicting transcription-factor-binding sites in Escherichia coli . Our method takes advantage of the principle that transcription factors frequently coregulate gene expression, but without requiring prior knowledge of which groups of genes are coregulated. Using position weight matrices for 49 known transcription factors, we examined spacings between pairs of matrix hits. These pairs were assigned probabilities according to the overrepresentation of their separation distance. The functions of many open reading frames (ORFs) downstream from predicted binding sites are unknown, and may correspond to novel regulon members. For five predictions, knockouts with mutated replacements of the predicted binding sites were created in E. coli MG1655. Quantitative real-time PCR (RT-PCR) indicates that for each of the knockouts, at least one gene immediately downstream exhibits a statistically significant change in mRNA expression. This approach may be useful in analyzing binding sites in a variety of organisms.
    Type of Medium: Online Resource
    ISSN: 1088-9051
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2004
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  • 5
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    Online Resource
    Cold Spring Harbor Laboratory ; 2016
    In:  Cold Spring Harbor Protocols Vol. 2016, No. 5 ( 2016-05), p. pdb.prot090183-
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2016, No. 5 ( 2016-05), p. pdb.prot090183-
    Abstract: This protocol outlines a general approach for characterizing the protospacer-adjacent motifs (PAMs) of Cas9 orthologs. It uses a three-plasmid system: One plasmid carries Cas9 and its tracrRNA, a second targeting vector contains the spacer and repeat, and the third plasmid encodes the targeted sequence (as the protospacer) with varying PAM sequences. It leverages the Cas9 nuclease activity to cleave and destroy plasmids that bear a compatible PAM. The level of depletion of a library of targeted plasmids after Cas9-mediated selection can then be assessed by deep sequencing to reveal candidate PAMs for downstream validation.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2016
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  • 6
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    Online Resource
    Cold Spring Harbor Laboratory ; 2011
    In:  Genome Research Vol. 21, No. 9 ( 2011-09), p. 1506-1511
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 21, No. 9 ( 2011-09), p. 1506-1511
    Abstract: Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number.
    Type of Medium: Online Resource
    ISSN: 1088-9051
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2011
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  • 7
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    Online Resource
    Cold Spring Harbor Laboratory ; 2005
    In:  Genome Research Vol. 15, No. 6 ( 2005-06), p. 848-855
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 15, No. 6 ( 2005-06), p. 848-855
    Abstract: With the completion of full genome sequences and advancement in high-throughput technologies, in silico methods have been successfully used to integrate diverse data sources toward unraveling the combinatorial nature of transcriptional regulation. So far, almost all of these studies are restricted to lower eukaryotes such as budding yeast. We describe here a computational search for functional transcription-factor (TF) combinations using phylogenetically conserved sequences and microarray-based expression data. Taking into account both orientational and positional constraints, we investigated the overrepresentation of binding sites in the vicinity of one another and whether these combinations result in more coherent expression profiles. Without any prior biological knowledge, the search led to the discovery of several experimentally established TF associations, as well as some novel ones. In particular, we identified a regulatory module controlling cell cycle-dependent transcription of G 2 -M genes and expanded its functional generality. We also detected many homotypic combinations, supporting the importance of binding-site density in transcriptional regulation of higher eukaryotes.
    Type of Medium: Online Resource
    ISSN: 1088-9051
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2005
    detail.hit.zdb_id: 1483456-X
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  • 8
    Online Resource
    Online Resource
    Cold Spring Harbor Laboratory ; 2000
    In:  Genome Research Vol. 10, No. 4 ( 2000-04-01), p. 431-445
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 10, No. 4 ( 2000-04-01), p. 431-445
    Abstract: We report steps toward the systematic management, standardization, and analysis of functional genomics data. We developed the ExpressDB database for yeast RNA expression data and loaded it with ∼17.5 million pieces of data reported by 11 studies with three different kinds of high-throughput RNA assays. A web-based tool supports queries across the data from these studies. We examined comparability of data by converting data from 9 studies (217 conditions) into mRNA relative abundance estimates (ERAs) and by clustering of conditions by ERAs. We report on generation of ERAs and condition clustering for non-microarray data (5 studies, 63 conditions) and describe initial attempts to generate microarray-based ERAs (4 studies, 154 conditions), which exhibit increased error, on our web site http://arep.med.harvard.edu/ExpressDB . We recommend standards for data reporting, suggest research into improving comparability of microarray data through quantifying and standardizing control condition RNA populations, and also suggest research into the calibration of different RNA assays. We introduce a model for a database that integrates different kinds of functional genomics data, Biomolecule Interaction, Growth and Expression Database (BIGED).
    Type of Medium: Online Resource
    ISSN: 1088-9051 , 1549-5469
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2000
    detail.hit.zdb_id: 1483456-X
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  • 9
    Online Resource
    Online Resource
    Cold Spring Harbor Laboratory ; 2000
    In:  Genome Research Vol. 10, No. 6 ( 2000-06-01), p. 744-757
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 10, No. 6 ( 2000-06-01), p. 744-757
    Abstract: Regulatory motifs can be found by local multiple alignment of upstream regions from coregulated sets of genes, or regulons. We searched for regulatory motifs using the program AlignACE together with a set of filters that helped us choose the motifs most likely to be biologically relevant in 17 complete microbial genomes. We searched the upstream regions of potentially coregulated genes grouped by three methods: (1) genes that make up functional pathways; (2) genes homologous to regulons from a well-studied species ( Escherichia coli ); and (3) groups of genes derived from conserved operons. This last group is based on the observation that genes making up homologous regulons in different species are often assorted into coregulated operons in different combinations. This allows partial reconstruction of regulons by looking at operon structure across several species. Unlike other methods for predicting regulons, this method does not depend on the availability of experimental data other than the genome sequence and the locations of genes. New, statistically significant motifs were found in the genome sequence of each organism using each grouping method. The most significant new motif was found upstream of genes in the methane-metabolism functional group in Methanobacterium thermoautotrophicum . We found that at least 27% of the known E. coli DNA-regulatory motifs are conserved in one or more distantly related eubacteria. We also observed significant motifs that differed from the E. coli motif in other organisms upstream of sets of genes homologous to known E. coli regulons, including Crp, LexA, and ArcA in Bacillus subtilis ; four anaerobic regulons in Archaeoglobus fulgidus (NarL, NarP, Fnr, and ModE); and the PhoB, PurR, RpoH, and FhlA regulons in other archaebacterial species. We also used motif conservation to aid in finding new motifs by grouping upstream regions from closely related bacteria, thus increasing the number of instances of the motif in the sequence to be aligned. For example, by grouping upstream sequences from three archaebacterial species, we found a conserved motif that may regulate ferrous ion transport that was not found in individual genomes. Discovery of conserved motifs becomes easier as the number of closely related genome sequences increases.
    Type of Medium: Online Resource
    ISSN: 1088-9051 , 1549-5469
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2000
    detail.hit.zdb_id: 1483456-X
    SSG: 12
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  • 10
    Online Resource
    Online Resource
    Cold Spring Harbor Laboratory ; 2002
    In:  Genome Research Vol. 12, No. 11 ( 2002-11-01), p. 1723-1731
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 12, No. 11 ( 2002-11-01), p. 1723-1731
    Abstract: Combinatorial regulation is an important feature of eukaryotic transcription. However, only a limited number of studies have characterized this aspect on a whole-genome level. We have conducted a genome-wide computational survey to identify cis-regulatory motif pairs that co-occur in a significantly high number of promoters in the S. cerevisiae genome. A pair of novel motifs, mRRPE and PAC, co-occur most highly in the genome, primarily in the promoters of genes involved in rRNA transcription and processing. The two motifs show significant positional and orientational bias with mRRPE being closer to the ATG than PAC in most promoters. Two additional rRNA-related motifs, mRRSE3 and mRRSE10, also co-occur with mRRPE and PAC. mRRPE and PAC are the primary determinants of expression profiles while mRRSE3 and mRRSE10 modulate these patterns. We describe a new computational approach for studying the functional significance of the physical locations of promoter elements that combine analyses of genome sequence and microarray data. Applying this methodology to the regulatory cassette containing the four rRNA motifs demonstrates that the relative promoter locations of these elements have a profound effect on the expression patterns of the downstream genes. These findings provide a function for these novel motifs and insight into the mechanism by which they regulate gene expression. The methodology introduced here should prove particularly useful for analyzing transcriptional regulation in more complex genomes.
    Type of Medium: Online Resource
    ISSN: 1088-9051 , 1549-5469
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2002
    detail.hit.zdb_id: 1483456-X
    SSG: 12
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