RESUMEN
Whole-genome sequencing (WGS) is becoming widely used in clinical medicine in diagnostic contexts and to inform treatment choice. Here we evaluate the potential of the Oxford Nanopore Technologies (ONT) MinION long-read sequencer for routine WGS by sequencing the reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome and severe immune dysregulation. We develop and apply a novel reference panel-free analytical method to infer and then exploit phase information which improves single-nucleotide variant (SNV) calling performance from otherwise modest levels. In the clinical sample, we identify and directly phase two non-synonymous de novo variants in SAMD9L, (OMIM #159550) inferring that they lie on the same paternal haplotype. Whilst consensus SNV-calling error rates from ONT data remain substantially higher than those from short-read methods, we demonstrate the substantial benefits of analytical innovation. Ongoing improvements to base-calling and SNV-calling methodology must continue for nanopore sequencing to establish itself as a primary method for clinical WGS.
Asunto(s)
Pruebas Genéticas/métodos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nanoporos , Secuenciación Completa del Genoma/métodos , Adulto , Ataxia Cerebelosa/diagnóstico , Ataxia Cerebelosa/genética , Femenino , Genoma Humano/genética , Genómica/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Humanos , Lactante , Masculino , Nanotecnología , Pancitopenia/diagnóstico , Pancitopenia/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Supresoras de Tumor/genética , Secuenciación Completa del Genoma/instrumentaciónRESUMEN
Identifying variants from RNA-seq (transcriptome sequencing) data is a cost-effective and versatile alternative to whole-genome sequencing. However, current variant callers do not generally behave well with RNA-seq data due to reads encompassing intronic regions. We have developed a software programme called Opossum to address this problem. Opossum pre-processes RNA-seq reads prior to variant calling, and although it has been designed to work specifically with Platypus, it can be used equally well with other variant callers such as GATK HaplotypeCaller. In this work, we show that using Opossum in conjunction with either Platypus or GATK HaplotypeCaller maintains precision and improves the sensitivity for SNP detection compared to the GATK Best Practices pipeline. In addition, using it in combination with Platypus offers a substantial reduction in run times compared to the GATK pipeline so it is ideal when there are only limited time or computational resources available.
