Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma
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 37x106-86x106 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.
|ISBN||1471-2164 (Electronic) 1471-2164 (Linking)|
|Authors||Warton, K. ; Lin, V. ; Navin, T. ; Armstrong, N. J. ; Kaplan, W. ; Ying, K. ; Gloss, B. ; Mangs, H. ; Nair, S. S. ; Hacker, N. F. ; Sutherland, R. L. ; Clark, S. J. ; Samimi, G.;|
|Responsible Garvan Author|
|Publisher Name||BMC Genomics|
|URL link to publisher's version||http://www.ncbi.nlm.nih.gov/pubmed/24929644|
|OpenAccess link to author's accepted manuscript version||https://publications.gimr.garvan.org.au/open-access/12482|