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1.
BMC Genomics ; 18(1): 515, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28679365

RESUMO

BACKGROUND: RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. METHODS: Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. RESULTS: This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. CONCLUSIONS: These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.


Assuntos
Biblioteca Gênica , RNA Mensageiro , Análise de Sequência de RNA/métodos , Manejo de Espécimes/normas , Células HL-60 , Humanos
3.
Oncotarget ; 7(18): 25930-48, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27036018

RESUMO

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.


Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Biomarcadores Tumorais/análise , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Fosfoproteínas/biossíntese , Esferoides Celulares/patologia , Fatores de Transcrição , Proteínas de Sinalização YAP
4.
Cancer Cell ; 29(3): 394-406, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26977886

RESUMO

Malignant rhabdoid tumors (MRTs) are rare lethal tumors of childhood that most commonly occur in the kidney and brain. MRTs are driven by SMARCB1 loss, but the molecular consequences of SMARCB1 loss in extra-cranial tumors have not been comprehensively described and genomic resources for analyses of extra-cranial MRT are limited. To provide such data, we used whole-genome sequencing, whole-genome bisulfite sequencing, whole transcriptome (RNA-seq) and microRNA sequencing (miRNA-seq), and histone modification profiling to characterize extra-cranial MRTs. Our analyses revealed gene expression and methylation subgroups and focused on dysregulated pathways, including those involved in neural crest development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Tumor Rabdoide/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica/genética , Histonas/genética , Humanos , MicroRNAs/genética , Proteína SMARCB1 , Fatores de Transcrição/genética , Transcriptoma/genética
5.
J Pathol ; 230(3): 249-60, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23616356

RESUMO

Parathyroid carcinoma is a rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level, and the deleterious effects of hypercalcaemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long-term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested potential roles for the tumour suppressor MEN1 and proto-oncogene RET in benign parathyroid tumourigenesis, while the tumour suppressor HRPT2 and proto-oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumour specimens were analysed using high-throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well-characterized cancer genes such as mTOR, MLL2, CDKN2C, and PIK3CA. Comparison of acquired mutations in patient-matched primary and recurrent tumours revealed loss of PIK3CA activating mutation during the evolution of the tumour from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single-base resolution. Loss of the short arm of chromosome 1, along with somatic missense and truncating mutations in CDKN2C and THRAP3, respectively, provides new evidence for the potential role of these genes as tumour suppressors in parathyroid cancer. The key somatic mutations identified in this study can serve as novel diagnostic markers as well as therapeutic targets.


Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Genômica , Recidiva Local de Neoplasia/genética , Neoplasias das Paratireoides/genética , Adulto , Sequência de Bases , Cálcio/sangue , Transformação Celular Neoplásica , Classe I de Fosfatidilinositol 3-Quinases , Inibidor de Quinase Dependente de Ciclina p18/genética , DNA de Neoplasias/química , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Dosagem de Genes , Fusão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Hormônio Paratireóideo/metabolismo , Neoplasias das Paratireoides/patologia , Neoplasias das Paratireoides/cirurgia , Fosfatidilinositol 3-Quinases/genética , Polimorfismo de Nucleotídeo Único , Proto-Oncogene Mas , RNA Neoplásico/genética , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética
6.
J Biomed Biotechnol ; 2011: 560124, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20981149

RESUMO

We have developed a new approach to screen bacterial artificial chromosome (BAC) libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380) with temperature inducible homologous recombination (HR) capability. We amplified one library segment, induced HR at 42°C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular/métodos , Biblioteca Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Recombinação Genética , Ágar , Animais , Impressões Digitais de DNA , Escherichia coli/genética , Marcação de Genes , Genoma/genética , Resistência a Canamicina/genética , Reação em Cadeia da Polimerase , Pongo/genética
7.
Genes Chromosomes Cancer ; 49(8): 669-81, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20544841

RESUMO

A multiplatform approach, including conventional cytogenetic techniques, BAC array comparative genomic hybridization, and Affymetrix 500K SNP arrays, was applied to the study of the tumor genomes of 25 follicular lymphoma biopsy samples with paired normal DNA samples to characterize balanced translocations, copy number imbalances, and copy-neutral loss of heterozygosity (cnLOH). In addition to the t(14;18), eight unique balanced translocations were found. Commonly reported FL-associated copy number regions were revealed including losses of 1p32-36, 6q, and 10q, and gains of 1q, 6p, 7, 12, 18, and X. The most frequent regions affected by copy-neutral loss of heterozygosity were 1p36.33 (28%), 6p21.3 (20%), 12q21.2-q24.33 (16%), and 16p13.3 (24%). We also identified by SNP analysis, 45 aberrant regions that each affected one gene, including CDKN2A, CDKN2B, FHIT, KIT, PEX14, and PTPRD, which were associated with canonical pathways involved in tumor development. This study illustrates the power of using complementary high-resolution platforms on paired tumor/normal specimens and computational analysis to provide potential insights into the significance of single-gene somatic aberrations in FL tumorigenesis.


Assuntos
Dosagem de Genes , Genoma Humano , Perda de Heterozigosidade , Linfoma Folicular/genética , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genética , Cromossomos Artificiais Bacterianos , Cromossomos Humanos Par 12/genética , Biologia Computacional , Análise Citogenética , Feminino , Perfilação da Expressão Gênica , Humanos , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Estudos Prospectivos
8.
Genome Biol ; 8(10): R224, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17953769

RESUMO

We present a method, called fingerprint profiling (FPP), that uses restriction digest fingerprints of bacterial artificial chromosome clones to detect and classify rearrangements in the human genome. The approach uses alignment of experimental fingerprint patterns to in silico digests of the sequence assembly and is capable of detecting micro-deletions (1-5 kb) and balanced rearrangements. Our method has compelling potential for use as a whole-genome method for the identification and characterization of human genome rearrangements.


Assuntos
Cromossomos Artificiais Bacterianos , Impressões Digitais de DNA/métodos , Rearranjo Gênico/genética , Genoma Humano/genética , Simulação por Computador , Primers do DNA , Enzimas de Restrição do DNA , Humanos
9.
Nature ; 432(7018): 761-4, 2004 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-15592415

RESUMO

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones. In an accompanying paper we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes.


Assuntos
Galinhas/genética , Genoma , Genômica , Mapeamento Físico do Cromossomo , Animais , Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular , Mapeamento de Sequências Contíguas , Impressões Digitais de DNA , Ligação Genética/genética , Sitios de Sequências Rotuladas
10.
J Bacteriol ; 186(22): 7783-95, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15516593

RESUMO

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.


Assuntos
Plasmídeos/genética , Bifenilos Policlorados/metabolismo , Rhodococcus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Biodegradação Ambiental , Sistema Enzimático do Citocromo P-450/genética , Transferência Genética Horizontal , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Origem de Replicação , Rhodococcus/metabolismo , Análise de Sequência de DNA , Telômero
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