Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer

Tyrosine kinase (TK) fusions are attractive drug targets in cancers. However, rapid identification of these lesions has been hampered by experimental limitations. Our in silico analysis of known cancer-derived TK fusions revealed that most breakpoints occur within a defined region upstream of a conserved GXGXXG kinase motif. We therefore designed a novel DNA-based targeted sequencing approach to screen systematically for fusions within the 90 human TKs; it should detect 92% of known TK fusions. We deliberately paired ‘in-solution’ DNA capture with 454 sequencing to minimize starting material requirements, take advantage of long sequence reads, and facilitate mapping of fusions. To validate this platform, we analyzed genomic DNA from thyroid cancer cells (TPC-1) and leukemia cells (KG-1) with fusions known only at the mRNA level. We readily identified for the first time the genomic fusion sequences of CCDC6-RET in TPC-1 cells and FGFR1OP2-FGFR1 in KG-1 cells. These data demonstrate the feasibility of this approach to identify TK fusions across multiple human cancers in a high-throughput, unbiased manner. This method is distinct from other similar efforts, because it focuses specifically on targets with therapeutic potential, uses only 1.5 µg of DNA, and circumvents the need for complex computational sequence analysis.     

Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans

The rapid accumulation of genomic sequences from a large number of eukaryotes, including numerous filamentous fungi, has created a tremendous scientific potential, which can only be realized if precise site-directed genome modifications, like gene deletions, promoter replacements, in-frame GFP fusions and specific point mutations can be made rapidly and reliably. The development of gene-targeting techniques in filamentous fungi and other higher eukaryotes has been hampered because foreign DNA is predominantly integrated randomly into the genome. For Aspergillus nidulans, we have developed a flexible method for gene-targeting employing a bipartite gene-targeting substrate. This substrate is made solely by PCR, which obviates the need for bacterial subcloning steps. The method reduces the number of false positives and can be used to produce virtually any genome alteration. A major advance of the method is that it allows multiple subsequent genome manipulations to be performed as the selectable marker is recycled.     

Prediction of genomic islands in seven human pathogens using the Z-Island method

We adopted the method of Zhang and Zhang (the Z-Island method) to identify genomic islands in seven human pathogens, analyzing their chromosomal DNA sequences. The Z-Island method is a theoretical method for predicting genomic islands in bacterial genomes; it consists of determination of the cumulative GC profile and computation of codon usage bias. Thirty-one genomic islands were found in seven pathogens using this method. Further analysis demonstrated that most have the known conserved features; this increases the probability that they are real genomic islands. Eleven genomic islands were found to code for products involved in causing disease (virulence factors) or in resistance to antibiotics (resistance factors). This finding could be useful for research on the pathogenicity of these bacteria and helpful in the treatment of the diseases that they cause. In a comparison of the distribution of mobility elements in genomic islands predicted by different methods, the Z-Island method gave lower false-positive rates. The Z-Island method was found to detect more known genomic islands than the two methods that we compared it with, SIGI-HMM and IslandPick. Furthermore, it maintained a better balance between specificity and sensitivity. The only inconvenience is that the steps for finding genomic islands by the Z-Island method are semi-automatic.     

Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.     

Creating precise GFP fusions in plasmids using yeast homologous recombination

Using a combination of primer amplification, homologous recombination, and yeast genetics, we established a method for creating precise promoter and protein fusions in genes originating from organisms other than yeast. One major advantage of this new method is its versatility. Fusions can be produced within a target gene without constraints regarding the site of insertion. Thus, fusions can be generated within a target sequence exactly at the site desired, and all sequences upstream and downstream of the insertion site were preserved. To illustrate the general applicability of this technique, we fused the gene encoding GFP to a Caenorhabditis elegans homologue of the dishevelled gene, dsh-2. This approach is not restricted to GFP fusions but can be utilized to create fusions between almost any two sequences regardless of the source. Therefore, this method provides a flexible alternative to other PCR-mediated techniques.     

Comprehensive DNA signature discovery and validation

DNA signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species. Here we describe Insignia, a new, comprehensive system for the rapid identification of signatures in the genomes of bacteria and viruses. With the availability of hundreds of complete bacterial and viral genome sequences, it is now possible to use computational methods to identify signature sequences in all of these species, and to use these signatures as the basis for diagnostic assays to detect and genotype microbes in both environmental and clinical samples. The success of such assays critically depends on the methods used to identify signatures that properly differentiate between the target genomes and the sample background. We have used Insignia to compute accurate signatures for most bacterial genomes and made them available through our Web site. A sample of these signatures has been successfully tested on a set of 46 Vibrio cholerae strains, and the results indicate that the signatures are highly sensitive for detection as well as specific for discrimination between these strains and their near relatives. Our approach, whereby the entire genomic complement of organisms are compared to identify probe targets, is a promising method for diagnostic assay development, and it provides assay designers with the flexibility to choose probes from the most relevant genes or genomic regions. The Insignia system is freely accessible via a Web interface and has been released as open source software at: http://insignia.cbcb.umd.edu.     

Targeted assembly of short sequence reads

As next-generation sequence (NGS) production continues to increase, analysis is becoming a significant bottleneck. However, in situations where information is required only for specific sequence variants, it is not necessary to assemble or align whole genome data sets in their entirety. Rather, NGS data sets can be mined for the presence of sequence variants of interest by localized assembly, which is a faster, easier, and more accurate approach. We present TASR, a streamlined assembler that interrogates very large NGS data sets for the presence of specific variants by only considering reads within the sequence space of input target sequences provided by the user. The NGS data set is searched for reads with an exact match to all possible short words within the target sequence, and these reads are then assembled stringently to generate a consensus of the target and flanking sequence. Typically, variants of a particular locus are provided as different target sequences, and the presence of the variant in the data set being interrogated is revealed by a successful assembly outcome. However, TASR can also be used to find unknown sequences that flank a given target. We demonstrate that TASR has utility in finding or confirming genomic mutations, polymorphisms, fusions and integration events. Targeted assembly is a powerful method for interrogating large data sets for the presence of sequence variants of interest. TASR is a fast, flexible and easy to use tool for targeted assembly.     

Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles

Breakage-fusion-bridge (BFB) cycle is a series of chromosome breaks and duplications that could lead to the increased copy number of a genomic segment (gene amplification). A critical step of BFB cycles leading to gene amplification is a palindromic fusion of sister chromatids following the rupture of a dicentric chromosome during mitosis. It is currently unknown how sister chromatid fusion is produced from a mitotic break. To delineate the process, we took an integrated genomic, cytogenetic and molecular approach for the recurrent MCL1 amplicon at chromosome 1 in human tumor cells. A newly developed next-generation sequencing-based approach identified a cluster of palindromic fusions within the amplicon at ∼50-kb intervals, indicating a series of breaks and fusions by BFB cycles. The physical location of the amplicon (at the end of a broken chromosome) further indicated BFB cycles as underlying processes. Three palindromic fusions were mediated by the homologies between two nearby inverted Alu repeats, whereas the other two fusions exhibited microhomology-mediated events. Such breakpoint sequences indicate that homology-mediated fold-back capping of broken ends followed by DNA replication is an underlying mechanism of sister chromatid fusion. Our results elucidate nucleotide-level events during BFB cycles and end processing for naturally occurring mitotic breaks.     

Detection of non-coding RNAs on the basis of predicted secondary structure formation free energy change

Background  

Non-coding RNAs (ncRNAs) have a multitude of roles in the cell, many of which remain to be discovered. However, it is difficult to detect novel ncRNAs in biochemical screens. To advance biological knowledge, computational methods that can accurately detect ncRNAs in sequenced genomes are therefore desirable. The increasing number of genomic sequences provides a rich dataset for computational comparative sequence analysis and detection of novel ncRNAs.     

Large multiple organism gene finding by collapsed Gibbs sampling.

The Gibbs sampling method has been widely used for sequence analysis after it was successfully applied to the problem of identifying regulatory motif sequences upstream of genes. Since then, numerous variants of the original idea have emerged: however, in all cases the application has been to finding short motifs in collections of short sequences (typically less than 100 nucleotides long). In this paper, we introduce a Gibbs sampling approach for identifying genes in multiple large genomic sequences up to hundreds of kilobases long. This approach leverages the evolutionary relationships between the sequences to improve the gene predictions, without explicitly aligning the sequences. We have applied our method to the analysis of genomic sequence from 14 genomic regions, totaling roughly 1.8 Mb of sequence in each organism. We show that our approach compares favorably with existing ab initio approaches to gene finding, including pairwise comparison based gene prediction methods which make explicit use of alignments. Furthermore, excellent performance can be obtained with as little as four organisms, and the method overcomes a number of difficulties of previous comparison based gene finding approaches: it is robust with respect to genomic rearrangements, can work with draft sequence, and is fast (linear in the number and length of the sequences). It can also be seamlessly integrated with Gibbs sampling motif detection methods.     

The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia

The Arabidopsis thaliana MERISTEM LAYER 1 (ATML1) gene is expressed in the epidermis of developing embryos and shoot meristems. To identify regulatory sequences necessary for epidermis-specific expression, three fusions of overlapping ATML1 genomic sequences to the GUS reporter gene were introduced into Arabidopsis plants. All fusion genes conferred epidermis-specific expression of both GUS mRNA and protein activity but varied in both the timing and relative levels of expression, suggesting partial redundancy of ATML1 regulatory elements. This study defines L1-specific regulatory sequences that are sufficient to direct foreign gene expression in a layer-specific manner.     

End joining at Caenorhabditis elegans telomeres

Critically shortened telomeres can be subjected to DNA repair events that generate end-to-end chromosome fusions. The resulting dicentric chromosomes can enter breakage–fusion–bridge cycles, thereby impeding elucidation of the structures of the initial fusion events and a mechanistic understanding of their genesis. Current models for the molecular basis of fusion of critically shortened, uncapped telomeres rely on PCR assays that typically capture fusion breakpoints created by direct ligation of chromosome ends. Here we use independent approaches that rely on distinctive features of Caenorhabditis elegans to study the frequency of direct end-to-end chromosome fusion in telomerase mutants: (1) holocentric chromosomes that allow for genetic isolation of stable end-to-end fusions and (2) unique subtelomeric sequences that allow for thorough PCR analysis of samples of genomic DNA harboring multiple end-to-end fusions. Surprisingly, only a minority of end-to-end fusion events resulted from direct end joining with no additional genome rearrangements. We also demonstrate that deficiency for the C. elegans Ku DNA repair heterodimer does not affect telomere length or cause synthetic effects in the absence of telomerase.     

“Word” Preference in the Genomic Text and Genome Evolution: Different Modes of n-tuplet Usage in Coding and Noncoding Sequences

Extensive work on n-tuplet occurrence in genomic sequences has revealed the correlation of their usage with sequence origin. Parallel to that, there exist different restrictions in the nucleotide composition of coding and noncoding sequences that may result in distinct modes of usage of n-tuplets. The relatively simple approaches described herein focus on such differences. They are based on simple summation measures of n-tuplet frequencies, computed after filtering the background nucleotide composition. Among the main targets of this work is to draw some conclusions on the qualitative differences in the composition of genomic sequences depending on their functionality. Moreover, an evolutionary model is formulated, including simple forms of ubiquitous events of genome dynamics: genomic fusions, genome shuffling due to transpositions, replication slippage, and point mutations. This model is shown to be able to reproduce all the statistical features of genomic sequences discussed herein. [Reviewing Editor: Dr. Brian Morton]     

Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA–probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA–probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader–Willy syndrome, Angelman syndrome or acute myeloid leukemia.