Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations
Abstract Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel...
Guardado en:
| Autores principales: | , , , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Nature Portfolio
2017
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/aca9a23e34c041d3a63565c43d988b71 |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| Sumario: | Abstract Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq). This method combines an ultra-sensitive single-stranded library construction with barcoding error correction, termed DEEPER-Library; and a DNA capture approach using RNA probes targeting both DNA strands, termed DEEPER-Capture. DEEPER-Seq can create NGS libraries from as little as 20 pg DNA with PCR error correcting capabilities, and capture target sequences at an average ratio of 29.2% by targeting both DNA strands simultaneously with an over 98.6% coverage. Our method tags and sequences each of the two strands of a DNA duplex independently and only scores mutations that are found at the same position in both strands, which allows us to identify mutations with allelic fractions down to 0.03% in a whole exome sequencing (WES) study with a background error rate of one artificial error per 4.8 × 109 nucleotides. |
|---|