Jarret Glasscock

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.

Evolutionary toggling of the MAPT 17q21.31 inversion region

Using comparative sequencing approaches, we investigated the evolutionary history of the European-enriched 17q21.31 MAPT inversion polymorphism. We present a detailed, BAC-based sequence assembly of the inverted human H2 haplotype and compare it to the sequence structure and genetic variation of the corresponding 1.5-Mb region for the noninverted H1 human haplotype and that of chimpanzee and orangutan. We found that inversion of the MAPT region is similarly polymorphic in other great ape species, and we present evidence that the inversions occurred independently in chimpanzees and humans. In humans, the inversion breakpoints correspond to core duplications with the LRRC37 gene family. Our analysis favors the H2 configuration and sequence haplotype as the likely great ape and human ancestral state, with inversion recurrences during primate evolution. We show that the H2 architecture has evolved more extensive sequence homology, perhaps explaining its tendency to undergo microdeletion associated with mental retardation in European populations.

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.

A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas.

Background:Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing.Methods:The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used.Results:Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability.Conclusions:A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (http://clinicaltrials.gov/ct2/show/NCT02220855).

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 4858: Next-generation whole transcriptome sequencing of thymic malignancies

Background: Thymomas & Thymic Carcinomas are rare malignancies with approximately 500 cases in the US per year. Apart from standard chemotherapy, treatment options are limited for those patients who become refractory to therapy or present with distant metastasis. Further, histological subtyping of these tumors has proven challenging, resulting in substantial discordance between pathologists and hindering the development of targeted therapy. A major impediment to therapeutic advancement is an inadequate understanding of the transcriptional biology of these cancers. Using next-generation sequencing, we embarked on a study to survey the transcriptomes of thymic malignancies to comprehensively identify novel biology by analyzing all full length transcripts expressed in these tissues. Methods: Frozen thymomas, thymic carcinoma, and normal tissues were obtained from the Indiana University Simon Cancer Center. The WHO (2004 classification) subtypes represented in our sample set include: (4) type A, (2) A/B, (1) B2, (5) B3, (1) C, and (3) normal tissues. Tissues were reviewed and classified by one pathologist (S.B.) who was blinded to subsequent analyses. cDNA libraries were prepared and sequenced on an Applied Biosystems (AB) SOLiD3+ sequencer using a 50bp fragment run. For gene expression, mapping of reads to the genome was performed using the AB BioScope 1.0 Pipeline and outputs imported into Partek Genomics Suite for analysis. In Partek, mapped reads were cross-referenced against known genes from the UCSC database followed by statistical comparison of RPKM values for each gene between subtypes. Dimensionality reduction analyses (PCA & hierarchical clustering) were also performed in Partek. Results: RNA sequencing of the 16 tissues produced 736 million reads equaling 37GB of data of which 24.4GB (66%) mapped to the human genome. These initial sequencing outputs represent only a portion, as additional paired-end sequencing of these samples is ongoing. In our preliminary data analysis, unsupervised hierarchical clustering of RPKM values from 20,600 RefSeq genes revealed 100% concordance between gene expression clusters and WHO subtype. A subsequent unsupervised clustering of 705 pre-miRNAs also showed substantial concordance between clusters and subtype. When analyzing differential gene expression between the three subtypes most represented in our data set (A, A/B, B3), we report 318 genes to be differentially expressed between A vs. A/B, 799 genes for A/B vs. B3, and 1524 genes for A vs. B3. Discussion: We report preliminary data from RNA-sequencing of thymic malignancies. Initial analyses reveal that this technology could be used to accurately subtype these tumors. Further paired-end sequencing of these samples and additional tumors is ongoing. Subsequent analyses of these data include identifying gene fusions, mutations, alternative splicing, noncoding RNAs, novel transcribed regions, and potential viral genomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4858. doi:10.1158/1538-7445.AM2011-4858

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.