Jennifer Asano

A physical map of the mouse genome

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.

Oligonucleotide Microarray Analysis of Genomic Imbalance in Children with Mental Retardation

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.

Alternative expression analysis by RNA sequencing

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/.

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

BackgroundGenomic 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.ResultsWe 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.ConclusionWe 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.

LongSAGE profiling of nine human embryonic stem cell lines

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.

ALEXA: a microarray design platform for alternative expression analysis

To the editor: Eukaryotic genomes are predicted to contain about 7,000–29,000 genes1. Each of these genes may be alternatively processed to produce multiple distinct mRNAs by alternative transcript initiation, splicing and polyadenylation (collectively referred to as alternative expression). Although analysis of available transcript resources indicates that up to ~75% of genes are alternatively processed, most microarray expression platforms cannot detect alternative transcripts2. Proof-of-principle experiments have described the use of oligonucleotide microarrays to profile transcript isoforms generated by alternative expression, but resources to create such arrays are lacking3,4. To address this limitation we created a microarray design platform for alternative expression analysis (ALEXA), which is capable of designing arrays that can detect all of the major categories of alternative expression. The ALEXA platform facilitates selection and annotation of oligonucleotide probes representing alternative expression events for any species in the EnsEMBL database1. For each target gene, probes are selected within every exon, intron, exon junction and exon boundary. This approach allows for the detection of constitutive and alternative exons, canonical exon junctions, junctions of known or new exon-skipping events, alternative exon boundaries and retained introns (Supplementary Fig. 1 online). We designed the platform to be flexible to the user’s experimental interests and preferred array manufacturer. The user may limit probe selection to known alternative expression events or include all possible exon junctions and boundaries to drive the discovery of transcripts. Probes may be designed for an arbitrary subset of genes or for all genes. Most technical parameters of the design can be modified by the user, including: the amount and types of control probes; the use of varying or fixed probe length; and the thresholds for filtering of probe sequences. The probe design process begins with retrieval of genomic sequences from EnsEMBL, removal of pseudogenes, masking of repeat elements and extraction of probe sequences. Random probe sequences are generated to uniformly represent the melting temperature and length of all experimental probes. Extracted and randomly generated probes are scored according to their melting temperature, folding potential, complexity and specificity (Supplementary Methods online). Although several publications have described using microarrays to study alternative expression in model organisms and specific tissues2, to our knowledge ours is the first report of a resource that makes alternative expression microarray designs readily available. Using the ALEXA approach, we precomputed microarray designs representing ~100 million probe sequences for ten EnsEMBL genomes (Supplementary Table 1 online). We assessed the ALEXA approach by using a prototype human array to profile the expression of alternative mRNA isoforms in 5-fluorouracil (5-FU)sensitive and resistant colorectal cancer cell lines5 and compared the results to those from the Affymetrix ‘GeneChip Human Exon 1.0 ST’ array (see Supplementary Results, Supplementary Fig. 2 and Supplementary Table 2 online). Genes and exons differentially expressed between 5-FU–sensitive and resistant cells were identified by both platforms (with significant overlap), but ALEXA arrays provided additional information on the connectivity and boundaries of exons (Table 1). Furthermore, alternative expression events identified by ALEXA were significantly enriched for known alternative expression events represented in publicly available mRNA and expressed sequence tag (EST) databases (Supplementary Results and Supplementary Data 1 online). Finally, we demonstrated the advantage of the ALEXA approach by identifying several differentially expressed known and predicted isoforms with potential relevance to 5-FU resistance (Supplementary Fig. 3 and Supplementary Tables 3 and 4 online). The approach and resources described in this work have considerable potential to advance studies of gene regulation, transcript processing, human disease and evolutionary biology (Supplementary Discussion online). The source code, precomputed array designs and related materials to assist in the creation of custom alternative expression microarrays are available on the ALEXA website (http://www.alexaplatform.org).

Digital Gene Expression by Tag Sequencing on the Illumina Genome Analyzer

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). Curr. Protoc. Hum. Genet. 65:11.11.1‐11.11.36

Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.

A Modified Polymerase Chain Reaction-Long Serial Analysis of Gene Expression Protocol Identifies Novel Transcripts in Human CD34 Bone Marrow Cells

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 (<103 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.

Resuspension of DNA sequencing reaction products in agarose increases sequence quality on an automated sequencer.

We are investigating approaches to increase DNA sequencing quality. Since a majorfactor in sequence generation is the cost of reagents and sample preparations, we have developed and optimized methods to sequence directly plasmid DNA isolated from alkaline lysis preparations. These methods remove the costly PCR and post-sequencing purification steps but can result in low sequence quality when using standard resuspension protocols on some sequencing platforms. This work outlines a simple, robust, and inexpensive resuspension protocol for DNA sequencing to correct this shortcoming. Resuspending the sequenced products in agarose before electrophoresis results in a substantial and reproducible increase in sequence quality and read length over resuspension in deionized water and has allowed us to use the aforementioned sample preparation methods to cut considerably the overall sequencing costs without sacrificing sequence quality. We demonstrate that resuspension of unpurified sequence products generated from template DNA isolated by a modified alkaline lysis technique in low concentrations of agarose yields a 384% improvement in sequence quality compared to resuspension in deionized water. Utilizing this protocol, we have produced more than 74,000 high-quality, long-read-length sequences from plasmid DNA template on the MegaBACET 1000 platform.