title: Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma.
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 × 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.
authors.forename: Kristina
authors.initials: K
authors.lastname: Warton
authors.forename: Vita
authors.initials: V
authors.lastname: Lin
authors.forename: Tina
authors.initials: T
authors.lastname: Navin
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Armstrong
authors.forename: Warren
authors.initials: W
authors.lastname: Kaplan
authors.forename: Kevin
authors.initials: K
authors.lastname: Ying
authors.forename: Brian
authors.initials: B
authors.lastname: Gloss
authors.forename: Helena
authors.initials: H
authors.lastname: Mangs
authors.forename: Shalima S
authors.initials: SS
authors.lastname: Nair
authors.forename: Neville F
authors.initials: NF
authors.lastname: Hacker
authors.forename: Robert L
authors.initials: RL
authors.lastname: Sutherland
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Clark
authors.affiliation: Garvan Institute and The Kinghorn Cancer Centre, 370 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia. g.samimi@garvan.org.au.
authors.forename: Goli
authors.initials: G
authors.lastname: Samimi
mesh_terms: D000368:Aged; D001482:Base Composition; D004247:DNA / Q000097:blood* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D059018:DNA Contamination; D019175:DNA Methylation; D005260:Female; D015723:Gene Library; D064368:Healthy Volunteers; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008875:Middle Aged; D015203:Reproducibility of Results; D012307:Risk Factors; D017422:Sequence Analysis, DNA
chemical_list: D004247:DNA