Research Interests

Genomics and Cancer Medicine

My PhD research focused on genome-wide analysis of alternative mRNA isoforms (arising by alternative splicing, initiation and poly-adenylation) [ref]. The principle goal of this work was to develop computational and genomic methods to identify those mRNA isoforms that represent potential therapeutic, diagnostic, and prognostic biomarkers in cancer. This goal was initially pursued by creating a computational platform for the design and analysis of splicing sensitive microarrays for alternative expression analysis ('ALEXA-arrays') [ref]. Subsequently, the advent of massively parallel RNA sequencing technology allowed me to develop 'ALEXA-Seq' [ref] and apply this approach to almost 20 models of cancer progression and tissue development. Both the splicing microarray and RNA sequencing approaches were published and associated software and databases were made available at our website: www.AlexaPlatform.org. The utility of these approaches was demonstrated by discovery and validation of a novel isoform associated with fluorouracil resistance in a panel of pre- and post-treatment colorectal cancer tumors. Although my PhD thesis work focused on development and application of bioinformatic methods for alternative expression analysis, I am interested in many genomic and informatics approaches to studying cancer biology and medicine. I have participated in analysis and identification of: alternative mRNA isoforms [ref, ref2], miRNAs [ref, ref], copy number variations [ref], point mutations [ref, ref], insertions/deletions, structural rearrangements [ref], and fusion genes [ref] associated with cancer. During the course of these collaborations I gained experience analyzing data from: Sanger sequencing of full-length cDNA clones, Sanger sequencing of genomic PCR amplicons, Affymetrix U133 gene expression arrays, Affymetrix exon expression arrays, custom NimbleGen splicing microarrays (designed by me), Affymetrix SNP 6.0 arrays (for copy number assessment), Roche 454 RNA sequencing, Illumina GAII RNA sequencing, Illumina GAII exon capture sequencing, and Illumina HiSeq RNA sequencing.

Bioinformatics

Many genomics and informatics tasks in cancer research involve using and adapting a combination of existing algorithms, tools, databases, and parallel computing. I have also participated in the development and application of novel bioinformatic methods including creating software and databases where existing approaches have fallen short. I have experience programming in Unix, Perl, SQL, Bash, BASIC, C, C++, HTML, CFML, and the statistical programming language R. I have recent experience with: ALEXA-seq, Cufflinks, Scripture, Tophat, Bowtie, Picard, HMMSplicer, Maq, BWA, NovoAlign, BEDtools, Velvet, Galaxy, the EnsEMBL Perl API, Xapian/Omega, Vector NTI, Consed, PhredPhrap, BLAST, BLAT, clustalW, Bioconductor, Affymetrix’s Expression Console and ExACT, Agilent’s eArray, mdust, RNAfold, pairFold, mySQL, custom UCSC tracks, VMware virtual machines, and countless other commonly used bioinformatics tools.

Alternative mRNA expression

Initial studies following from the Human Genome Project have revealed that the apparent number of genes present in a human is much less than expected for such a complex organism. Recent studies suggest that in fact it is not just the number of genes that gives rise to our complexity but rather the number of functionally distinct versions of a gene that can be encoded from a single gene region. Human genes are comprised of DNA sequences called exons separated by long stretches of DNA sequence called introns which must be removed so that the exons can be assembled into a working copy of the gene. My PhD research focused on the phenomenon of alternative splicing in which one gene is assembled from its component pieces in many different ways to produce a multitude of different functional products. In particular, changes in the forms of certain genes may be important in the progression of cancer and account for the differences in the severity of cancers and response to treatment observed among individuals. By studying the alternative splicing of genes in models of cancer the hope is to identify promising candidates for vaccine and drug development.

Custom splicing microarrays for alternative expression analysis

The use of microarray technology to profile mRNA transcripts generated by alternative splicing is an area of rapid development. To facilitate the use of microarrays to study alternative splicing I created an open source array design platform for alternative expression analysis called ‘ALEXA' (www.AlexaPlatform.org). This platform allows the design and analysis of microarrays of arbitrary density and complexity for alternative expression analysis of most EnsEMBL annotated species. Creation of ALEXA arrays involves the extraction, scoring, filtering and annotation of oligonucleotide probes corresponding to exons, introns, exon boundaries and exon-exon junctions. I used this platform to pre-compute designs for ten EnsEMBL annotated species. To evaluate the ALEXA-array approach I generated a design for the human genome and measured differential expression of alternate isoforms between 5-fluorouracil sensitive and resistant colorectal cancer cell lines. Results generated from ALEXA arrays were compared to those from Affymetrix exon arrays. ALEXA array data was comparable or superior to Affymetrix exon arrays in terms of reproducibility, sensitivity and specificity and provided additional information on the connectivity and boundaries of exons. Analysis results, microarray designs and source code for ALEXA-arrays are available at http://www.alexaplatform.org/alexa_arrays/.

Alternative expression analysis by massively parallel RNA sequencing

In alternative expression analysis by sequencing (ALEXA-seq), I developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, I used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. I assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription-PCR, quantitative PCR and Sanger sequencing. I observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.

Mechanisms of chemotherapy resistance in colorectal cancer

Colorectal cancer (CRC) is the 4th most commonly diagnosed type of cancer and has the 2nd highest number of fatalities in Canada (2007 Canadian Cancer Statistics). Treatment commonly involves surgery and adjuvant or neo-adjuvant systemic chemotherapy. One of the most commonly used chemotherapy drugs in the treatment of CRC is the uracil analog, 5-fluorouracil (5-FU). The high mortality rate associated with CRC is due in part to resistance to 5-FU treatment. The factors that determine 5-FU efficacy are complex and it is likely that any clinical test of 5-FU response that is based on the characterization of only one or two of the many genes involved will fall short of being applicable to the general population. Despite its common usage, the response rate to 5-FU is low (as low as 20% when used as a single agent). While for some patients these drugs are highly effective, for others they either have no favourable response or an adverse reaction. Developing an accurate means to identify responders and determine appropriate dosage is critical to improving this situation. If we can understand what causes resistance to 5-FU, we can use this information to avoid giving the drug to patients that are unlikely to benefit from it. Furthermore, if we can understand what causes resistance to drugs, we can use this information to design new drugs to combat or avoid this resistance. To pursue this goal we developed and applied genomic methods to profile the expression, RNA processing and sequence polymorphisms of human transcripts and to identify those events associated with resistance to 5-FU in colorectal cancer. We selected a series of colon cancer cell lines representing a cancer that is sensitive to chemotherapy or resistant to one of four commonly used chemotherapy drugs. By studying the differences in gene structure correlated with this change in drug response we gained insight into why chemotherapy initially seems to work well in some patients but becomes less effective over time. This study represents one contribution to the ultimate goal of one day tailoring cancer treatments to the needs of individual patients and lessening the incidence, morbidity and mortality from cancer.

Identifying, characterizing, and processing tumors for clinical validation studies

During the course of participating in genomic and bioinformatic analysis of cancer, I became interested (out of necessity) in many of the technical and logistical challenges faced in attempting clinical validation of our predictions. This has lead me to a greater appreciation of the complexity and importance of integrated teams in cancer genomics research. My own experience has involved working with students, scientists, surgeons, oncologists, pathologists and hospital and biobank administrators. I have gained experience in the principles and writing of ethics applications. I have found that exposure to patient records, such as oncology, surgical, and pathology reports is invaluable in gaining perspective on the treatment and outcomes of a particular cancer. I also gained an appreciation for the art of isolating nucleic acid material from patient samples and how this contrasts with isolation from tissue culture sources. This appreciation was gained by extensive experimentation with RNA and DNA isolation from cultured cells, fresh frozen archival samples from tumour banks, and formalin fixed paraffin embedded (FFPE) tumor blocks from several anatomical pathology departments across the province. Ultimately I used my experience to train and supervise students to assist in these activities, without whom obtaining the hundreds of samples needed for our studies would not have been possible.