Journal article
Methylation-capture and Next-Generation sequencing of free circulating DNA from human plasma
BMC Genomics, Vol.15(1), pp.476-489
2014
Abstract
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 37×106-86×106 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.
Details
- Title
- Methylation-capture and Next-Generation sequencing of free circulating DNA from human plasma
- Authors/Creators
- K. Warton (Author/Creator) - The Kinghorn Cancer CentreV. Lin (Author/Creator)T. Navin (Author/Creator) - The Kinghorn Cancer CentreN.J. Armstrong (Author/Creator) - The Kinghorn Cancer CentreW. Kaplan (Author/Creator) - The Kinghorn Cancer CentreK. Ying (Author/Creator) - The Kinghorn Cancer CentreB. Gloss (Author/Creator) - The Kinghorn Cancer CentreH. Mangs (Author/Creator) - UNSW SydneyS.S. Nair (Author/Creator) - The Kinghorn Cancer CentreN.F. Hacker (Author/Creator) - Royal Hospital for WomenR.L. Sutherland (Author/Creator) - The Kinghorn Cancer CentreS.J. Clark (Author/Creator) - The Kinghorn Cancer CentreG. Samimi (Author/Creator) - The Kinghorn Cancer Centre
- Publication Details
- BMC Genomics, Vol.15(1), pp.476-489
- Publisher
- BioMed Central
- Identifiers
- 991005541250107891
- Copyright
- © 2014 Warton et al
- Murdoch Affiliation
- Murdoch University
- Language
- English
- Resource Type
- Journal article
UN Sustainable Development Goals (SDGs)
This output has contributed to the advancement of the following goals:
Source: InCites
Metrics
140 File views/ downloads
37 Record Views
InCites Highlights
These are selected metrics from InCites Benchmarking & Analytics tool, related to this output
- Collaboration types
- Domestic collaboration
- Citation topics
- 1 Clinical & Life Sciences
- 1.199 Lung Cancer
- 1.199.1633 Circulating Tumor Biomarkers
- Web Of Science research areas
- Biotechnology & Applied Microbiology
- Genetics & Heredity
- ESI research areas
- Molecular Biology & Genetics