Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma.
BMC Genomics
; 15: 476, 2014 Jun 15.
Article
en En
| MEDLINE
| ID: mdl-24929644
BACKGROUND: Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality. RESULTS: Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37 × 10(6)-86 × 10(6) unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing. CONCLUSIONS: Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
ADN
/
Metilación de ADN
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Secuenciación de Nucleótidos de Alto Rendimiento
Tipo de estudio:
Etiology_studies
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Guideline
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Risk_factors_studies
Límite:
Aged
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Female
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Humans
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Middle aged
Idioma:
En
Revista:
BMC Genomics
Asunto de la revista:
GENETICA
Año:
2014
Tipo del documento:
Article