RESUMEN
Results of massive parallel sequencing-by-synthesis vary depending on the sequencing approach. CoolMPS™ is a new sequencing chemistry that incorporates bases by labeled antibodies. To evaluate the performance, we sequenced 240 human non-coding RNA samples (dementia patients and controls) with and without CoolMPS. The Q30 value as indicator of the per base sequencing quality increased from 91.8 to 94%. The higher quality was reached across the whole read length. Likewise, the percentage of reads mapping to the human genome increased from 84.9 to 86.2%. For both technologies, we computed similar distributions between different RNA classes (miRNA, piRNA, tRNA, snoRNA and yRNA) and within the classes. While standard sequencing-by-synthesis allowed to recover more annotated miRNAs, CoolMPS yielded more novel miRNAs. The correlation between the two methods was 0.97. Evaluating the diagnostic performance, we observed lower minimal P-values for CoolMPS (adjusted P-value of 0.0006 versus 0.0004) and larger effect sizes (Cohen's d of 0.878 versus 0.9). Validating 19 miRNAs resulted in a correlation of 0.852 between CoolMPS and reverse transcriptase-quantitative polymerase chain reaction. Comparison to data generated with Illumina technology confirmed a known shift in the overall RNA composition. With CoolMPS we evaluated a novel sequencing-by-synthesis technology showing high performance for the analysis of non-coding RNAs.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN no Traducido/química , Análisis de Secuencia de ARN/métodos , Especificidad de Anticuerpos , Biomarcadores , Biología Computacional , ADN Complementario/genética , Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Demencia/sangre , Demencia/genética , Técnica del Anticuerpo Fluorescente Directa , Biblioteca de Genes , Humanos , Biopsia Líquida , MicroARNs/química , MicroARNs/genética , Nucleótidos/inmunología , ARN no Traducido/síntesis química , ARN no Traducido/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaAsunto(s)
Cirugía Colorrectal , Infecciones por Coronavirus/epidemiología , Enfermedades Intestinales/cirugía , Pandemias/estadística & datos numéricos , Neumonía Viral/epidemiología , Actitud del Personal de Salud , Actitud Frente a la Salud , Betacoronavirus , COVID-19 , Procedimientos Quirúrgicos Electivos , Humanos , Entrevistas como Asunto , Práctica Profesional , SARS-CoV-2 , Telemedicina , Estados Unidos/epidemiologíaRESUMEN
Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review board- approved "open consent" process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10). We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain-we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.
Asunto(s)
Bases de Datos Genéticas , Variación Genética , Genoma Humano/genética , Fenotipo , Medicina de Precisión/métodos , Programas Informáticos , Línea Celular , Recolección de Datos , Humanos , Medicina de Precisión/tendencias , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Surgical coaching interventions have been recommended as a method of technological skills improvement for individual surgeons and lifelong occupational learning. Patient outcomes for laparoscopic colectomy vary significantly based on surgeon experience and case volume. As surgical coaching is an emerging area, little is known about how surgeons view coaching interventions. METHODS: Semistructured interviews with 68 colorectal surgeons from across the country who were e-mail recruited from the American Society of Colon and Rectal Surgeons focused on exploring the attitudes surrounding surgical coaching programs among colorectal surgeons. Interviews were performed via telephone, audio-recorded, and transcribed verbatim with redaction of identifying information. Interviews were analyzed by iterative steps informed by thematic analysis. RESULTS: Surgeons reported the desire to participate in coaching programs to improve patient outcomes through technical skill advancement, to keep pace with surgical innovation, and to fulfill a desire for lifelong learning. However, surgeons varied in their beliefs over who should be coached, who should coach, the format of coaching, and the topics addressed in coaching. Obstacles identified included time, financial and medicolegal concerns, balance with resident education, and vulnerability. CONCLUSION: Widespread enthusiasm for surgical coaching programs exists among colorectal surgeons. However, there is variability in what surgeons believe an ideal surgical coaching program would look like. Therefore, in alignment with adult learning theory, we recommend the creation of several different models of surgical coaching to allow each surgeon to benefit from this advancement in continuous professional development.
Asunto(s)
Neoplasias Colorrectales , Tutoría , Cirujanos , Adulto , Humanos , Tutoría/métodos , Investigación Cualitativa , Cirujanos/educaciónRESUMEN
There is a rapidly developing need for new technologies to amplify millions of different targets from genomic DNA for high throughput genotyping and population gene-sequencing from diverse species. Here we describe a novel approach for the specific selection and amplification of genomic DNA fragments of interest that eliminates the need for costly and time consuming synthesis and testing of potentially millions of amplicon-specific primers. This technique relies upon Type IIs restriction enzyme digestion of genomic DNA and ligation of the fragments to double-sided adapters to form closed-circular DNA molecules. The novel use of double-sided adapters, assembled through the combinatorial use of two small universal sets of oligonucleotide building blocks, provides greater selection capacity by utilizing both sides of the adapter in a sequence-specific ligation event. As demonstrated, formation of circular structures results in protection of the desired molecules from nuclease treatment and enables a level of selectivity high enough to isolate single, or multiple, pre-defined fragments from the human genome when digested at over five million sites. Priming sites incorporated into the adapter allows the utilization of a common pair of primers for the amplification of any adapter-captured DNA fragment of interest.
Asunto(s)
ADN/genética , Genoma , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/aislamiento & purificación , ADN/metabolismo , ADN Circular/genética , ADN Circular/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Escherichia coli/genética , Genoma Bacteriano , Genoma Humano , Humanos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Sensibilidad y EspecificidadRESUMEN
Genome sequencing of large numbers of individuals promises to advance the understanding, treatment, and prevention of human diseases, among other applications. We describe a genome sequencing platform that achieves efficient imaging and low reagent consumption with combinatorial probe anchor ligation chemistry to independently assay each base from patterned nanoarrays of self-assembling DNA nanoballs. We sequenced three human genomes with this platform, generating an average of 45- to 87-fold coverage per genome and identifying 3.2 to 4.5 million sequence variants per genome. Validation of one genome data set demonstrates a sequence accuracy of about 1 false variant per 100 kilobases. The high accuracy, affordable cost of $4400 for sequencing consumables, and scalability of this platform enable complete human genome sequencing for the detection of rare variants in large-scale genetic studies.
Asunto(s)
ADN/química , Genoma Humano , Análisis por Micromatrices , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Biología Computacional , Costos y Análisis de Costo , ADN/genética , Bases de Datos de Ácidos Nucleicos , Biblioteca Genómica , Genotipo , Haplotipos , Proyecto Genoma Humano , Humanos , Masculino , Nanoestructuras , Nanotecnología , Técnicas de Amplificación de Ácido Nucleico , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/economía , Análisis de Secuencia de ADN/instrumentación , Análisis de Secuencia de ADN/normas , Programas InformáticosRESUMEN
The TLX/HOX11 subfamily of divergent homeobox genes are involved in various aspects of embryogenesis and, in the case of TLX1/HOX11 and TLX3/HOX11L2, feature prominently as oncogenes in human T-cell acute lymphoblastic leukaemia. TLX1 possesses immortalising activity in a wide variety of blood cell lineages, however, the effect of this oncogene on haemopoietic cell differentiation has not been fully investigated. We therefore constitutively expressed TLX1 in murine bone marrow or fetal liver cells using retroviral transfer followed by transplantation and/or in vitro culture. TLX1 was found to dramatically alter haemopoiesis, promoting the emergence of a non-haemopoietic CD45(-) CD31(+) cell population while markedly inhibiting erythroid and granulocytic cell differentiation. To identify genetic programs perturbed by TLX1, a comparison of transcript profiles from J2E erythroid cells with and without enforced TLX1 expression was undertaken. This revealed a pattern of gene expression indicative of enhanced proliferation coupled to differentiation arrest. Of the genes identified, two, KIT and VEGFC, were found to be potential TLX1 targets based on transcriptional assays. These results demonstrate that TLX1 can act broadly to impair haemopoiesis and divert differentiation to an alternative fate. This may account for its ability to promote the pre-leukaemic state via perturbation of specific gene expression programs.
Asunto(s)
Células de la Médula Ósea/patología , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/genética , Células 3T3 , Animales , Diferenciación Celular/genética , Femenino , Citometría de Flujo , Expresión Génica , Perfilación de la Expresión Génica , Hematopoyesis/genética , Hígado/embriología , Luciferasas/genética , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-kit/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/patología , Transducción Genética , Factor C de Crecimiento Endotelial Vascular/genética , beta-Galactosidasa/genéticaRESUMEN
Expression profiling and genomic DNA sequence comparisons are increasingly being applied to the identification and analysis of the genes that are involved in lipid metabolism. Not only has genome-wide expression profiling aided in the identification of novel genes that are involved in important processes in lipid metabolism such as sterol efflux, but also the utilization of information from these studies has added to our understanding of the regulation of pathways that participate in the process. Coupled with these gene expression studies, cross-species comparison (a technique used to search for sequences that are conserved through evolution) has proven to be a powerful tool to identify important noncoding regulatory sequences and novel genes that are relevant to lipid biology. An example of the value of this approach was the recent chance discovery of a new apolipoprotein gene (that which encodes apolipoprotein AV) that has dramatic effects on triglyceride metabolism in mice and humans.