RESUMEN
Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Técnicas de Sustitución del Gen , Transgenes/genéticaRESUMEN
Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.
Asunto(s)
Islas de CpG/genética , Enzimas de Restricción del ADN/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T Reguladores/inmunología , Células Cultivadas , Metilación de ADN , Desmetilación , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Especificidad de Órganos , Cultivo Primario de CélulasRESUMEN
A complex microbiota inhabits various microenvironments of the gut, with some symbiotic bacteria having evolved traits to invade the epithelial mucus layer and reside deep within the intestinal tissue of animals. Whether these distinct bacterial communities across gut biogeographies exhibit divergent behaviours is largely unknown. Global transcriptomic analysis to investigate microbial physiology in specific mucosal niches has been hampered technically by an overabundance of host RNA. Here, we employed hybrid selection RNA sequencing (hsRNA-Seq) to enable detailed spatial transcriptomic profiling of a prominent human commensal as it colonizes the colonic lumen, mucus or epithelial tissue of mice. Compared to conventional RNA-Seq, hsRNA-Seq increased reads mapping to the Bacteroides fragilis genome by 48- and 154-fold in mucus and tissue, respectively, allowing for high-fidelity comparisons across biogeographic sites. Near the epithelium, B. fragilis upregulated numerous genes involved in protein synthesis, indicating that bacteria inhabiting the mucosal niche are metabolically active. Further, a specific sulfatase (BF3086) and glycosyl hydrolase (BF3134) were highly induced in mucus and tissue compared to bacteria in the lumen. In-frame deletion of these genes impaired in vitro growth on mucus as a carbon source, as well as mucosal colonization of mice. Mutants in either B. fragilis gene displayed a fitness defect in competing for colonization against bacterial challenge, revealing the importance of site-specific gene expression for robust host-microbial symbiosis. As a versatile tool, hsRNA-Seq can be deployed to explore the in vivo spatial physiology of numerous bacterial pathogens or commensals.
Asunto(s)
Bacteroides fragilis/genética , Bacteroides fragilis/fisiología , Colon/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteroides fragilis/crecimiento & desarrollo , Colitis/microbiología , Femenino , Regulación Bacteriana de la Expresión Génica , Vida Libre de Gérmenes , Humanos , Mucosa Intestinal/microbiología , Masculino , Ratones , Ácidos Sulfónicos , Simbiosis , TranscriptomaRESUMEN
Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.
Asunto(s)
Edición Génica , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/fisiopatología , Animales , Línea Celular , Técnicas de Sustitución del Gen , Humanos , Ratones , Primates , Reproducibilidad de los Resultados , Visión OcularRESUMEN
BACKGROUND: Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations. RESULTS: To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290, and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes. CONCLUSIONS: UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Reordenamiento Génico , Genoma Humano , Mutación INDEL , Osteosarcoma/diagnóstico , Antígenos de Neoplasias/genética , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/genética , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas del Citoesqueleto , Genómica/métodos , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Osteosarcoma/genética , Eliminación de Secuencia , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Although RNA-seq is a powerful tool, the considerable time and cost associated with library construction has limited its utilization for various applications. RNAtag-Seq, an approach to generate multiple RNA-seq libraries in a single reaction, lowers time and cost per sample, and it produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches.
Asunto(s)
Secuencia de Bases , Biblioteca de Genes , Análisis de Secuencia de ARN/métodos , Bacterias/genética , Perfilación de la Expresión Génica/normas , Análisis de Secuencia de ARN/economía , Análisis de Secuencia de ARN/normas , Factores de TiempoRESUMEN
Large microbial gene clusters encode useful functions, including energy utilization and natural product biosynthesis, but genetic manipulation of such systems is slow, difficult and complicated by complex regulation. We exploit the modularity of a refactored Klebsiella oxytoca nitrogen fixation (nif) gene cluster (16 genes, 103 parts) to build genetic permutations that could not be achieved by starting from the wild-type cluster. Constraint-based combinatorial design and DNA assembly are used to build libraries of radically different cluster architectures by varying part choice, gene order, gene orientation and operon occupancy. We construct 84 variants of the nifUSVWZM operon, 145 variants of the nifHDKY operon, 155 variants of the nifHDKYENJ operon and 122 variants of the complete 16-gene pathway. The performance and behavior of these variants are characterized by nitrogenase assay and strand-specific RNA sequencing (RNA-seq), and the results are incorporated into subsequent design cycles. We have produced a fully synthetic cluster that recovers 57% of wild-type activity. Our approach allows the performance of genetic parts to be quantified simultaneously in hundreds of genetic contexts. This parallelized design-build-test-learn cycle, which can access previously unattainable regions of genetic space, should provide a useful, fast tool for genetic optimization and hypothesis testing.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Klebsiella oxytoca/genética , Familia de Multigenes , Fijación del Nitrógeno/genética , Klebsiella oxytoca/fisiología , Nitrogenasa/genética , Operón/genética , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Although the composition of the human microbiome is now well-studied, the microbiota's >8 million genes and their regulation remain largely uncharacterized. This knowledge gap is in part because of the difficulty of acquiring large numbers of samples amenable to functional studies of the microbiota. We conducted what is, to our knowledge, one of the first human microbiome studies in a well-phenotyped prospective cohort incorporating taxonomic, metagenomic, and metatranscriptomic profiling at multiple body sites using self-collected samples. Stool and saliva were provided by eight healthy subjects, with the former preserved by three different methods (freezing, ethanol, and RNAlater) to validate self-collection. Within-subject microbial species, gene, and transcript abundances were highly concordant across sampling methods, with only a small fraction of transcripts (<5%) displaying between-method variation. Next, we investigated relationships between the oral and gut microbial communities, identifying a subset of abundant oral microbes that routinely survive transit to the gut, but with minimal transcriptional activity there. Finally, systematic comparison of the gut metagenome and metatranscriptome revealed that a substantial fraction (41%) of microbial transcripts were not differentially regulated relative to their genomic abundances. Of the remainder, consistently underexpressed pathways included sporulation and amino acid biosynthesis, whereas up-regulated pathways included ribosome biogenesis and methanogenesis. Across subjects, metatranscriptional profiles were significantly more individualized than DNA-level functional profiles, but less variable than microbial composition, indicative of subject-specific whole-community regulation. The results thus detail relationships between community genomic potential and gene expression in the gut, and establish the feasibility of metatranscriptomic investigations in subject-collected and shipped samples.
Asunto(s)
Tracto Gastrointestinal/microbiología , Genómica/métodos , Metagenoma/genética , Microbiota/genética , Transcriptoma/genética , ADN Bacteriano/análisis , Heces/microbiología , Tracto Gastrointestinal/fisiología , Regulación Bacteriana de la Expresión Génica , Humanos , Boca/microbiología , ARN Bacteriano/análisis , Saliva/microbiología , Manejo de Especímenes/métodosRESUMEN
BACKGROUND: Pediatric inflammatory bowel disease (IBD) is challenging to diagnose because of the non-specificity of symptoms; an unequivocal diagnosis can only be made using colonoscopy, which clinicians are reluctant to recommend for children. Diagnosis of pediatric IBD is therefore frequently delayed, leading to inappropriate treatment plans and poor outcomes. We investigated the use of 16S rRNA sequencing of fecal samples and new analytical methods to assess differences in the microbiota of children with IBD and other gastrointestinal disorders. METHODOLOGY/PRINCIPAL FINDINGS: We applied synthetic learning in microbial ecology (SLiME) analysis to 16S sequencing data obtained from i) published surveys of microbiota diversity in IBD and ii) fecal samples from 91 children and young adults who were treated in the gastroenterology program of Children's Hospital (Boston, USA). The developed method accurately distinguished control samples from those of patients with IBD; the area under the receiver-operating-characteristic curve (AUC) value was 0.83 (corresponding to 80.3% sensitivity and 69.7% specificity at a set threshold). The accuracy was maintained among data sets collected by different sampling and sequencing methods. The method identified taxa associated with disease states and distinguished patients with Crohn's disease from those with ulcerative colitis with reasonable accuracy. The findings were validated using samples from an additional group of 68 patients; the validation test identified patients with IBD with an AUC value of 0.84 (e.g. 92% sensitivity, 58.5% specificity). CONCLUSIONS/SIGNIFICANCE: Microbiome-based diagnostics can distinguish pediatric patients with IBD from patients with similar symptoms. Although this test can not replace endoscopy and histological examination as diagnostic tools, classification based on microbial diversity is an effective complementary technique for IBD detection in pediatric patients.
Asunto(s)
Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/patología , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/microbiología , Metagenoma , Adolescente , Adulto , Antibacterianos/uso terapéutico , Biodiversidad , Niño , Preescolar , Estudios de Cohortes , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/microbiología , Enfermedad de Crohn/patología , Demografía , Diagnóstico Diferencial , Heces/microbiología , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/clasificación , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Complejo de Antígeno L1 de Leucocito/metabolismo , Masculino , Metagenoma/genética , Inducción de Remisión , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Programas Informáticos , Adulto JovenRESUMEN
We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA.
Asunto(s)
Escherichia coli/genética , Consorcios Microbianos/genética , Prochlorococcus/genética , ARN Bacteriano/genética , Rhodobacter sphaeroides/genética , Fraccionamiento Químico , ADN Complementario/análisis , ADN Complementario/biosíntesis , Heces/microbiología , Regulación Bacteriana de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Mensajero/análisis , ARN Mensajero/biosíntesisRESUMEN
Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.
Asunto(s)
Artefactos , Bacterias/genética , ARN Ribosómico 16S/análisis , Bacterias/clasificación , Secuencia de Bases , Quimera/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , ADN Ribosómico/genética , Genómica , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , ARN Bacteriano/genética , Análisis de Secuencia de ADN/métodosRESUMEN
We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.
Asunto(s)
Procesamiento Automatizado de Datos , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Análisis de Secuencia de ADN/métodos , Algoritmos , Humanos , MicroesferasRESUMEN
BACKGROUND: Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.
Asunto(s)
Bacteriófagos/genética , ADN Viral/genética , Genoma Viral/genética , Análisis de Secuencia de ADN/métodos , Biología Computacional/métodos , ADN Viral/química , ADN Viral/metabolismo , Desoxirribonucleasas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Prochlorococcus/virologíaRESUMEN
Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.
Asunto(s)
Genes Bacterianos/genética , Genoma Bacteriano/genética , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/genética , Pulmón/microbiología , Animales , Mapeo Cromosómico , Cromosomas Bacterianos/genética , Elementos Transponibles de ADN/genética , Estudio de Asociación del Genoma Completo , Biblioteca Genómica , Haemophilus influenzae/crecimiento & desarrollo , Ratones , Mutagénesis Insercional/métodos , MutaciónRESUMEN
Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that approximately 60% of target bases in the exonic 'catch', and approximately 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.
Asunto(s)
Genómica/métodos , Oligonucleótidos/metabolismo , Análisis de Secuencia de ADN/métodos , Composición de Base/genética , Teorema de Bayes , Biotinilación , Exones/genética , Humanos , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Promising new sequencing technologies, based on sequencing-by-synthesis (SBS), are starting to deliver large amounts of DNA sequence at very low cost. Polymorphism detection is a key application. We describe general methods for improved quality scores and accurate automated polymorphism detection, and apply them to data from the Roche (454) Genome Sequencer 20. We assess our methods using known-truth data sets, which is critical to the validity of the assessments. We developed informative, base-by-base error predictors for this sequencer and used a variant of the phred binning algorithm to combine them into a single empirically derived quality score. These quality scores are more useful than those produced by the system software: They both better predict actual error rates and identify many more high-quality bases. We developed a SNP detection method, with variants for low coverage, high coverage, and PCR amplicon applications, and evaluated it on known-truth data sets. We demonstrate good specificity in single reads, and excellent specificity (no false positives in 215 kb of genome) in high-coverage data.
Asunto(s)
Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas , Diploidia , Genoma Humano , Haploidia , Humanos , Reacción en Cadena de la Polimerasa , Control de Calidad , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.
Asunto(s)
Linaje de la Célula/genética , Cromatina/genética , Cromatina/metabolismo , Genoma/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Alelos , Animales , Islas de CpG/genética , Fibroblastos , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica , Histonas/metabolismo , Masculino , Metilación , Ratones , Regiones Promotoras Genéticas/genética , Transcripción Genética/genéticaRESUMEN
We have designed resequencing microarrays to test the performance of this platform when interrogating a large number of exons (164 total) from genes associated with cancer. To evaluate false positive and negative rates, dideoxy sequencing was done for 335,420 bases interrogated by the arrays. From the array data, calls could be made for approximately 97.5% of the bases, and false positive rates were very low with only a single mutation reported from the array dataset for which the corresponding dideoxy trace had a clean wildtype sequence. For the nucleotide positions where array calls were made, false negative rates were 1.41% for heterozygous mutations. All the homozygous mutations were detected, but 8.11% were erroneously reported as heterozygous changes from the reference sequence by the array analysis software. In addition, 20 non-small cell lung cancer (NSCLC) samples were analyzed using the arrays, and both somatic and germline mutations were found. The most interesting findings were two MET mutations that have recently been implemented in NSCLC. Large scale MALDI-TOF genotyping indicated that one of these mutations (T1010I) might represent a true cancer-causing genotype, whereas the other (N375S) appears to be a common germline polymorphism.