Publications by authors named "Jennifer Asano"

14 Publications

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Alternative expression analysis by RNA sequencing.

Nat Methods 2010 Oct 12;7(10):843-7. Epub 2010 Sep 12.

Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada.

In alternative expression analysis by sequencing (ALEXA-seq), we 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, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We 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. We 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/.
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http://dx.doi.org/10.1038/nmeth.1503DOI Listing
October 2010

Digital gene expression by tag sequencing on the illumina genome analyzer.

Curr Protoc Hum Genet 2010 Apr;Chapter 11:Unit 11.11.1-36

BCCA Genome Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada.

This unit provides a protocol for performing digital gene expression profiling on the Illumina Genome Analyzer sequencing platform. Tag sequencing (Tag-seq) is an implementation of the LongSAGE protocol on the Illumina sequencing platform that increases utility while reducing both the cost and time required to generate gene expression profiles. The ultra-high-throughput sequencing capability of the Illumina platform allows the cost-effective generation of libraries containing an average of 20 million tags, a 200-fold improvement over classical LongSAGE. Tag-seq has less sequence composition bias, leading to a better representation of AT-rich tag sequences, and allows a more accurate profiling of a subset of the transcriptome characterized by AT-rich genes expressed at levels below the threshold of detection of LongSAGE (Morrissy et al., 2009).
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http://dx.doi.org/10.1002/0471142905.hg1111s65DOI Listing
April 2010

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data.

BMC Bioinformatics 2007 Oct 2;8:368. Epub 2007 Oct 2.

Genome Sciences Centre, BC Cancer Agency, British Columbia Cancer Agency, Suite 100, 570 West 7th Avenue, Vancouver, BC, V5Z 4S6, Canada.

Background: Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results: We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion: We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.
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http://dx.doi.org/10.1186/1471-2105-8-368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2148068PMC
October 2007

LongSAGE profiling of nine human embryonic stem cell lines.

Genome Biol 2007 ;8(6):R113

Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada, V5Z 1L3.

To facilitate discovery of novel human embryonic stem cell (ESC) transcripts, we generated 2.5 million LongSAGE tags from 9 human ESC lines. Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells, and identified novel ESC transcripts, at least one of which may represent a marker of the pluripotent state.
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http://dx.doi.org/10.1186/gb-2007-8-6-r113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2394759PMC
February 2008

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation.

Plant J 2007 Jun 3;50(6):1063-78. Epub 2007 May 3.

Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 +/- 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa, version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.
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http://dx.doi.org/10.1111/j.1365-313X.2007.03112.xDOI Listing
June 2007

A modified polymerase chain reaction-long serial analysis of gene expression protocol identifies novel transcripts in human CD34+ bone marrow cells.

Stem Cells 2007 Jul 5;25(7):1681-9. Epub 2007 Apr 5.

Terry Fox Laboratory, Vancouver, BC, Canada V5Z 1L3.

Transcriptome profiling offers a powerful approach to investigating developmental processes. Long serial analysis of gene expression (LongSAGE) is particularly attractive for this purpose because of its inherent quantitative features and independence of both hybridization variables and prior knowledge of transcript identity. Here, we describe the validation and initial application of a modified protocol for amplifying cDNA preparations from <10 ng of RNA (<10(3) cells) to allow representative LongSAGE libraries to be constructed from rare stem cell-enriched populations. Quantitative real-time polymerase chain reaction (Q-RT-PCR) analyses and comparison of tag frequencies in replicate LongSAGE libraries produced from amplified and nonamplified cDNA preparations demonstrated preservation of the relative levels of different transcripts originally present at widely varying levels. This PCR-LongSAGE protocol was then used to obtain a 200,000-tag library from the CD34+ subset of normal adult human bone marrow cells. Analysis of this library revealed many anticipated transcripts, as well as transcripts not previously known to be present in CD34+ hematopoietic cells. The latter included numerous novel tags that mapped to unique and conserved sites in the human genome but not previously identified as transcribed elements in human cells. Q-RT-PCR was used to demonstrate that 10 of these novel tags were expressed in cDNA pools and present in extracts of other sources of normal human CD34+ hematopoietic cells. These findings illustrate the power of LongSAGE to identify new transcripts in stem cell-enriched populations and indicate the potential of this approach to be extended to other sources of rare cells. Disclosure of potential conflicts of interest is found at the end of this article.
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http://dx.doi.org/10.1634/stemcells.2006-0794DOI Listing
July 2007

Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines.

Genome Res 2007 Jan 29;17(1):108-16. Epub 2006 Nov 29.

Canada's Michael Smith Genome Sciences Centre, BC Cancer Research Centre, BC Cancer Agency, Vancouver, British Columbia V5Z 4S6, Canada.

We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of approximately 5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.
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http://dx.doi.org/10.1101/gr.5488207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1716260PMC
January 2007

Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation.

Am J Hum Genet 2006 Sep 25;79(3):500-13. Epub 2006 Jul 25.

Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.
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http://dx.doi.org/10.1086/507471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1559542PMC
September 2006

A mouse atlas of gene expression: large-scale digital gene-expression profiles from precisely defined developing C57BL/6J mouse tissues and cells.

Proc Natl Acad Sci U S A 2005 Dec 13;102(51):18485-90. Epub 2005 Dec 13.

Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Research Centre, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4S6.

We analyzed 8.55 million LongSAGE tags generated from 72 libraries. Each LongSAGE library was prepared from a different mouse tissue. Analysis of the data revealed extensive overlap with existing gene data sets and evidence for the existence of approximately 24,000 previously undescribed genomic loci. The visual cortex, pancreas, mammary gland, preimplantation embryo, and placenta contain the largest number of differentially expressed transcripts, 25% of which are previously undescribed loci.
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http://dx.doi.org/10.1073/pnas.0509455102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1311911PMC
December 2005

Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome.

Genome Res 2004 Apr;14(4):766-79

Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada V5Z 4E6.

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.
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http://dx.doi.org/10.1101/gr.2336604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC383324PMC
April 2004

The Genome sequence of the SARS-associated coronavirus.

Science 2003 May 1;300(5624):1399-404. Epub 2003 May 1.

British Columbia Cancer Agency (BCCA) Genome Sciences Centre, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada.

We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.
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http://dx.doi.org/10.1126/science.1085953DOI Listing
May 2003

A physical map of the mouse genome.

Nature 2002 Aug 4;418(6899):743-50. Epub 2002 Aug 4.

The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.
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http://dx.doi.org/10.1038/nature00957DOI Listing
August 2002

An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones.

Nucleic Acids Res 2002 Jun;30(11):2460-8

Genome Sciences Centre, BC Cancer Agency, 600 West 10th Avenue, Vancouver, BC V5Z 4E6, Canada.

We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC117194PMC
http://dx.doi.org/10.1093/nar/30.11.2460DOI Listing
June 2002