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Open Access Highly Accessed Methodology article

Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma

Kristina Warton1, Vita Lin1, Tina Navin1, Nicola J Armstrong12, Warren Kaplan3, Kevin Ying3, Brian Gloss1, Helena Mangs4, Shalima S Nair1, Neville F Hacker5, Robert L Sutherland1, Susan J Clark16 and Goli Samimi16*

Author Affiliations

1 Garvan Institute and The Kinghorn Cancer Centre, 370 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia

2 School of Mathematics and Statistics, University of Sydney, Sydney, NSW 2006, Australia

3 Centre for Clinical Genomics, Garvan Institute and The Kinghorn Cancer Centre, Sydney, NSW 2010, Australia

4 The Ramaciotti Centre for Gene Function Analysis, University of New South Wales, Sydney, NSW 2052, Australia

5 School of Women’s and Children’s Health, University of New South Wales, and Gynaecological Cancer Centre, Royal Hospital for Women, Sydney, NSW 2052, Australia

6 St. Vincent’s Clinical School, Sydney, University of New South Wales, Sydney, NSW 2052, Australia

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BMC Genomics 2014, 15:476  doi:10.1186/1471-2164-15-476

Published: 15 June 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.

Keywords:
Free circulating DNA; Next-Generation Sequencing; Methylation; Biobanking; Blood