Matthew Hickenbotham

DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome

Acute myeloid leukaemia is a highly malignant haematopoietic tumour that affects about 13,000 adults in the United States each year. The treatment of this disease has changed little in the past two decades, because most of the genetic events that initiate the disease remain undiscovered. Whole-genome sequencing is now possible at a reasonable cost and timeframe to use this approach for the unbiased discovery of tumour-specific somatic mutations that alter the protein-coding genes. Here we present the results obtained from sequencing a typical acute myeloid leukaemia genome, and its matched normal counterpart obtained from the same patient’s skin. We discovered ten genes with acquired mutations; two were previously described mutations that are thought to contribute to tumour progression, and eight were new mutations present in virtually all tumour cells at presentation and relapse, the function of which is not yet known. Our study establishes whole-genome sequencing as an unbiased method for discovering cancer-initiating mutations in previously unidentified genes that may respond to targeted therapies.

Whole-genome sequencing and variant discovery in C. elegans

Massively parallel sequencing instruments enable rapid and inexpensive DNA sequence data production. Because these instruments are new, their data require characterization with respect to accuracy and utility. To address this, we sequenced a Caernohabditis elegans N2 Bristol strain isolate using the Solexa Sequence Analyzer, and compared the reads to the reference genome to characterize the data and to evaluate coverage and representation. Massively parallel sequencing facilitates strain-to-reference comparison for genome-wide sequence variant discovery. Owing to the short-read-length sequences produced, we developed a revised approach to determine the regions of the genome to which short reads could be uniquely mapped. We then aligned Solexa reads from C. elegans strain CB4858 to the reference, and screened for single-nucleotide polymorphisms (SNPs) and small indels. This study demonstrates the utility of massively parallel short read sequencing for whole genome resequencing and for accurate discovery of genome-wide polymorphisms.

Viral Discovery and Sequence Recovery Using DNA Microarrays

Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.

Extending assembly of short DNA sequences to handle error

UNLABELLED Inexpensive de novo genome sequencing, particularly in organisms with small genomes, is now possible using several new sequencing technologies. Some of these technologies such as that from Illuminas Solexa Sequencing, produce high genomic coverage by generating a very large number of small reads ( approximately 30 bp). While prior work shows that partial assembly can be performed by k-mer extension in error-free reads, this algorithm is unsuccessful with the sequencing error rates found in practice. We present VCAKE (Verified Consensus Assembly by K-mer Extension), a modification of simple k-mer extension that overcomes error by using high depth coverage. Though it is a simple modification of a previous approach, we show significant improvements in assembly results on simulated and experimental datasets that include error. AVAILABILITY http://152.2.15.114/~labweb/VCAKE

Analysis of the prostate cancer cell line LNCaP transcriptome using a sequencing-by-synthesis approach

BackgroundHigh throughput sequencing-by-synthesis is an emerging technology that allows the rapid production of millions of bases of data. Although the sequence reads are short, they can readily be used for re-sequencing. By re-sequencing the mRNA products of a cell, one may rapidly discover polymorphisms and splice variants particular to that cell.ResultsWe present the utility of massively parallel sequencing by synthesis for profiling the transcriptome of a human prostate cancer cell-line, LNCaP, that has been treated with the synthetic androgen, R1881. Through the generation of approximately 20 megabases (MB) of EST data, we detect transcription from over 10,000 gene loci, 25 previously undescribed alternative splicing events involving known exons, and over 1,500 high quality single nucleotide discrepancies with the reference human sequence. Further, we map nearly 10,000 ESTs to positions on the genome where no transcription is currently predicted to occur. We also characterize various obstacles with using sequencing by synthesis for transcriptome analysis and propose solutions to these problems.ConclusionThe use of high-throughput sequencing-by-synthesis methods for transcript profiling allows the specific and sensitive detection of many of a cells transcripts, and also allows the discovery of high quality base discrepancies, and alternative splice variants. Thus, this technology may provide an effective means of understanding various disease states, discovering novel targets for disease treatment, and discovery of novel transcripts.

The value of avian genomics to the conservation of wildlife.

BackgroundGenomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution.Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care).In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes.ResultsGenotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current populations genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology.Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m form, originally thought to be the derived condition, might actually be the ancestral one.ConclusionMining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease.In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology.

Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank

IntroductionOur efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined.MethodsUsing normal breast tissue from 20 premenopausal donors to KTB, the changes in the mRNA of the normal breast epithelium as a function of phase of the menstrual cycle and hormonal contraception were assayed using next-generation whole transcriptome sequencing (RNA-Seq).ResultsIn total, 255 genes representing 1.4% of all genes were deemed to have statistically significant differential expression between the two phases of the menstrual cycle. The overwhelming majority (221; 87%) of the genes have higher expression during the luteal phase. These data provide important insights into the processes occurring during each phase of the menstrual cycle. There was only a single gene significantly differentially expressed when comparing the epithelium of women using hormonal contraception to those in the luteal phase.ConclusionsWe have taken advantage of a unique research resource, the KTB, to complete the first-ever next-generation transcriptome sequencing of the epithelial compartment of 20 normal human breast specimens. This work has produced a comprehensive catalog of the differences in the expression of protein-coding genes as a function of the phase of the menstrual cycle. These data constitute the beginning of a reference data set of the normal mammary gland, which can be consulted for comparison with data developed from malignant specimens, or to mine the effects of the hormonal flux that occurs during the menstrual cycle.

Gallus GBrowse: a unified genomic database for the chicken

Gallus GBrowse (http://birdbase.net/cgi-bin/gbrowse/gallus/) provides online access to genomic and other information about the chicken, Gallus gallus. The information provided by this resource includes predicted genes and Gene Ontology (GO) terms, links to Gallus In Situ Hybridization Analysis (GEISHA), Unigene and Reactome, the genomic positions of chicken genetic markers, SNPs and microarray probes, and mappings from turkey, condor and zebra finch DNA and EST sequences to the chicken genome. We also provide a BLAT server (http://birdbase.net/cgi-bin/webBlat) for matching user-provided sequences to the chicken genome. These tools make the Gallus GBrowse server a valuable resource for researchers seeking genomic information regarding the chicken and other avian species.

Flipping NextGen: using biological systems to characterize NextGen sequencing technologies

Finally, de-novo sequencing (i.e. without a reference) would require a minimum of 1/2 of the sequence length to be unique in order to allow sufficient contig extension in the assembly process. For example, 40–50 bp reads are necessary for de-novo characterization of these systems uniquely defined by 20–25 bp reads. As of 2009, short read NextGen sequencing technologies have moved to 50 bp and beyond, ushering in what is expected to be the start of a revolution in genomics. Conclusion These results establish a lower bound on sequence length (x) required to sufficiently conduct re-sequencing, transcriptome, and de-novo sequencing projects. The asymptotic nature of the results also provides a guide for what percentage of the total space (y) we might expect to define in genomes/transcriptomes of similar size and complexity. from UT-ORNL-KBRIN Bioinformatics Summit 2009 Pikeville, TN, USA. 20–22 March 2009

Abstract 2216: Next-generation whole transcriptome sequencing of triple-negative breast tumors and normal tissues

Background: Triple-negative breast cancer (TNBC) disproportionally affects pre-menopausal women and women of African-American descent, and has been plagued by the absence of targeted therapies leading to poor survival. The paucity of therapeutic targets in TNBC impels us to utilize new technologies that can determine novel targets on a global scale. Using next-generation sequencing, we embarked on a study to analyze the whole transcriptomes of TNBC tumors compared to normal breast tissues in order to comprehensively identify novel targets by analyzing all full length transcripts expressed in these tissues. Methods: Normal breast tissues from healthy pre-menopausal volunteers with no history of disease were procured from the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. To eliminate bias from stromal tissue, epithelial cells were laser capture microdissected and RNA extracted from captured cells. cDNA libraries from 10 TNBC tumors and 10 normal breast tissues were subsequently sequenced on an ABI SOLiD3 sequencer using a 50bp fragment run. For gene expression, mapping of reads to the human genome was performed using the ABI Whole Transcriptome Pipeline and outputs were imported into Partek Genomics Suite for analysis. To analyze for gene fusions, reads were mapped to the genome using the SOLiD Analysis Pipeline Tool, followed by an alignment to Refseq to map reads crossing exon-exon junctions. A composite transcriptome was formed from areas of the genome with significant expression (17% of the genome sequence) and served as a concise search space for identifying fusions. Reads not mapping to the genome or to RefSeq (a rich source of fusion reads) were then mapped to the composite transcriptome using BLAT to facilitate a highly accurate split-read alignment. Using a custom developed pipeline, reads that spanned transcribed regions from two different chromosomes, or to loci farther than 200kb apart on the same chromosome, were considered as candidate fusions. Results/Discussion: Sequencing of the 10 TNBC tumors and 10 normal samples produced 1.1 billion reads equaling 58.15GB of data. Mapping of the reads to the genome revealed 1.6 million transcribed regions (exons) of significant expression. A preliminary analysis of gene expression shows 55.2% of the transcribed loci to have significant differential expression between tumor and normal. Network-node, non-coding RNA, and statistical analyses are currently ongoing. In a further interim analysis, we bioinformatically identified several interchromosomal fusions that were present in a majority of the tumors but were absent in the normal samples. RT-PCR validation of these candidate fusions in a larger validation cohort of TNBC tumors and normal breast tissues is ongoing. A multitude of additional analyses including but not limited to: novel transcripts, alternative splicing, and presence of viral genes are also planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2216.