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1.  A New Whole Genome Amplification Method for Studying Clonal Evolution Patterns in Malignant Colorectal Polyps 
Genes, chromosomes & cancer  2012;51(5):490-500.
To identify the genetic drivers of colorectal tumorigenesis, we applied array comparative genomic hybridization (aCGH) to 13 formalin-fixed paraffin-embedded (FFPE) samples of early, localized human colon adenocarcinomas arising in high-grade adenomas (so called “malignant polyps”). These lesions are small and hence the amount of DNA is limited. Additionally, the quality of DNA is compromised due to the fragmentation as a consequence of formalin fixation. To overcome these problems, we optimized a newly developed isothermal whole genome amplification system (NuGEN Ovation® WGA FFPE System). Starting with 100 ng of FFPE DNA, the amplification system produced 4.01 ± 0.29 μg (mean ± standard deviation) of DNA. The excellent quality of amplified DNA was further indicated by a high signal-to-noise ratio and a low derivative log2 ratio spread. Both, the amount of amplified DNA and aCGH performance were independent of the age of the FFPE blocks and the associated degradation of the extracted DNA. We observed losses of chromosome arms 5q and 18q in the adenoma components of the malignant polyp samples, while the embedded early carcinomas revealed losses of 8p, 17p, and 18, and gains of 7, 13, and 20. Aberrations detected in the adenoma components were invariably maintained in the embedded carcinomas. This approach demonstrates that using isothermally whole genome amplified FFPE DNA is technically suitable for aCGH. In addition to demonstrating the clonal origin of the adenoma and carcinoma part within a malignant polyp, the gain of chromosome arm 20q was an indicator for progression from adenoma to carcinoma.
PMCID: PMC3535186  PMID: 22334367
2.  Deep Clonal Profiling of Formalin Fixed Paraffin Embedded Clinical Samples 
PLoS ONE  2012;7(11):e50586.
Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.
PMCID: PMC3511535  PMID: 23226320
3.  Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions 
Genome Biology  2009;10(7):R80.
Distinct developmental fates in Drosophila melanogaster are specified by quantitative differences in transcription factor occupancy on a common set of bound regions.
We previously established that six sequence-specific transcription factors that initiate anterior/posterior patterning in Drosophila bind to overlapping sets of thousands of genomic regions in blastoderm embryos. While regions bound at high levels include known and probable functional targets, more poorly bound regions are preferentially associated with housekeeping genes and/or genes not transcribed in the blastoderm, and are frequently found in protein coding sequences or in less conserved non-coding DNA, suggesting that many are likely non-functional.
Here we show that an additional 15 transcription factors that regulate other aspects of embryo patterning show a similar quantitative continuum of function and binding to thousands of genomic regions in vivo. Collectively, the 21 regulators show a surprisingly high overlap in the regions they bind given that they belong to 11 DNA binding domain families, specify distinct developmental fates, and can act via different cis-regulatory modules. We demonstrate, however, that quantitative differences in relative levels of binding to shared targets correlate with the known biological and transcriptional regulatory specificities of these factors.
It is likely that the overlap in binding of biochemically and functionally unrelated transcription factors arises from the high concentrations of these proteins in nuclei, which, coupled with their broad DNA binding specificities, directs them to regions of open chromatin. We suggest that most animal transcription factors will be found to show a similar broad overlapping pattern of binding in vivo, with specificity achieved by modulating the amount, rather than the identity, of bound factor.
PMCID: PMC2728534  PMID: 19627575
5.  Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm  
PLoS Biology  2008;6(2):e27.
Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior–posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal–ventral patterning genes, whose expression we show to be quantitatively modulated by anterior–posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets.
Author Summary
One of the largest classes of regulatory proteins in animals, sequence-specific DNA binding transcription factors determine in which cells genes will be expressed and so control the development of an animal from a single cell to a morphologically complex adult. Understanding how this process is coordinated depends on knowing the number and types of genes that each transcription factor binds and regulates. Using immunoprecipitation of in vivo crosslinked chromatin coupled with DNA microarray hybridization (ChIP/chip), we have determined the genomic binding sites in early embryos of six transcription factors that play a crucial role in early development of the fruit fly Drosophila melanogaster. We find that these proteins bind to several thousand genomic regions that lie close to approximately half the protein coding genes. Although this is a much larger number of genes than these factors are generally thought to regulate, we go on to show that whereas the more highly bound genes generally look to be functional targets, many of the genes bound at lower levels do not appear to be regulated by these factors. Our conclusions differ from those of other groups who have not distinguished between different levels of DNA binding in vivo using similar assays and who have generally assumed that all detected binding is functional.
ChIP/chip analysis indicates that sequence-specific transcription factors bind to overlapping sets of thousands of genomic regions in Drosophila embryos, but most regions are bound at low levels and many may not be functional targets of these factors.
PMCID: PMC2235902  PMID: 18271625
6.  Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP) 
Nucleic Acids Research  2005;33(21):e185.
cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5′- and 3′-untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. Expressed Sequence Tag (EST) sequencing is the method of choice for recovering cDNAs representing many of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently. We describe a PCR-based method for directed screening of plasmid cDNA libraries. We demonstrate its utility in a screen of libraries used in our Drosophila EST projects for 153 transcription factor genes that were not represented by full-length cDNA clones in our Drosophila Gene Collection. We recovered high-quality, full-length cDNAs for 72 genes and variously compromised clones for an additional 32 genes. The method can be used at any scale, from the isolation of cDNA clones for a particular gene of interest, to the improvement of large gene collections in model organisms and the human. Finally, we discuss the relative merits of directed cDNA library screening and RT–PCR approaches.
PMCID: PMC1301602  PMID: 16326860
7.  The lords of the genomes 
Genome Biology  2004;5(10):349.
A report on The Biology of Genomes meeting, Cold Spring Harbor, USA, 12-16 May 2004.
A report on The Biology of Genomes meeting, Cold Spring Harbor, USA, 12-16 May 2004.
PMCID: PMC545592  PMID: 15461811
8.  Annotation of the Drosophila melanogaster euchromatic genome: a systematic review 
Genome Biology  2002;3(12):research0083.1-83.22.
The recent completion of the Drosophila melanogaster genomic sequence to high quality, and the availability of a greatly expanded set of Drosophila cDNA sequences, afforded FlyBase the opportunity to significantly improve genomic annotations.
The recent completion of the Drosophila melanogaster genomic sequence to high quality and the availability of a greatly expanded set of Drosophila cDNA sequences, aligning to 78% of the predicted euchromatic genes, afforded FlyBase the opportunity to significantly improve genomic annotations. We made the annotation process more rigorous by inspecting each gene visually, utilizing a comprehensive set of curation rules, requiring traceable evidence for each gene model, and comparing each predicted peptide to SWISS-PROT and TrEMBL sequences.
Although the number of predicted protein-coding genes in Drosophila remains essentially unchanged, the revised annotation significantly improves gene models, resulting in structural changes to 85% of the transcripts and 45% of the predicted proteins. We annotated transposable elements and non-protein-coding RNAs as new features, and extended the annotation of untranslated (UTR) sequences and alternative transcripts to include more than 70% and 20% of genes, respectively. Finally, cDNA sequence provided evidence for dicistronic transcripts, neighboring genes with overlapping UTRs on the same DNA sequence strand, alternatively spliced genes that encode distinct, non-overlapping peptides, and numerous nested genes.
Identification of so many unusual gene models not only suggests that some mechanisms for gene regulation are more prevalent than previously believed, but also underscores the complex challenges of eukaryotic gene prediction. At present, experimental data and human curation remain essential to generate high-quality genome annotations.
PMCID: PMC151185  PMID: 12537572
9.  Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome 
Genome Biology  2002;3(12):research0086.1-86.2.
Analysis of conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons.
It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.
We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences.
Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.
PMCID: PMC151188  PMID: 12537575
10.  A Drosophila full-length cDNA resource 
Genome Biology  2002;3(12):research0080.1-80.8.
High-quality full-insert sequence for 8,921 putative full-length cDNA clones in the Drosophila Gene Collection has been generated and compared to the annotated Release 3 genomic sequence. More than 5,300 cDNAs have been identifieed that contain a complete and accurate protein-coding sequence, corresponding to at least one splice form for 40% of the predicted D. melanogaster genes.
A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages.
We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40% of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing.
We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.
PMCID: PMC151182  PMID: 12537569
11.  Finishing a whole-genome shotgun: Release 3 of the Drosophila melanogaster euchromatic genome sequence 
Genome Biology  2002;3(12):research0079.1-79.14.
The Drosophila melanogaster genome was the first metazoan genome to be sequenced by whole-genome shotgun. Now, the sequence has been finished in a process designed to close gaps, improve sequence quality and validate the assembly.
The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions.
Our finishing process was designed to close gaps, improve sequence quality and validate the assembly. Sequence traces derived from the WGS and draft sequencing of individual bacterial artificial chromosomes (BACs) were assembled into BAC-sized segments. These segments were brought to high quality, and then joined to constitute the sequence of each chromosome arm. Overall assembly was verified by comparison to a physical map of fingerprinted BAC clones. In the current version of the 116.9 Mb euchromatic genome, called Release 3, the six euchromatic chromosome arms are represented by 13 scaffolds with a total of 37 sequence gaps. We compared Release 3 to Release 2; in autosomal regions of unique sequence, the error rate of Release 2 was one in 20,000 bp.
The WGS strategy can efficiently produce a high-quality sequence of a metazoan genome while generating the reagents required for sequence finishing. However, the initial method of repeat assembly was flawed. The sequence we report here, Release 3, is a reliable resource for molecular genetic experimentation and computational analysis.
PMCID: PMC151181  PMID: 12537568
12.  TAK1 Participates in c-Jun N-Terminal Kinase Signaling during Drosophila Development 
Molecular and Cellular Biology  2000;20(9):3015-3026.
Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-β/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated a Drosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH2-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouse TAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.
PMCID: PMC85571  PMID: 10757786

Results 1-12 (12)