Abstract
BackgroundFree 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.ResultsUse 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-476) contains supplementary material, which is available to authorized users.
Highlights
Free circulating DNA has many potential clinical applications, due to the non-invasive way in which it is collected
To eliminate the possibility that high molecular weight DNA released during blood processing was not detectable on a gel because it was degraded by active nucleases, serum and plasma blood collection tubes were pre-loaded with purified leukocyte genomic DNA prior to sample collection
The genomic DNA that we had pre-loaded in the plasma tube was recovered and visible as a high-molecular weight band co-migrating with purified genomic DNA (Figure 1B, Lane 5), while no corresponding band was visible in the serum isolated from the pre-loaded tube
Summary
Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. The size suggests that it originates from apoptotic cells, as it corresponds to the length of DNA wrapped around apparent. These data support the apoptotic rather than necrotic origin of fcDNA. Measurement of Y-chromosome DNA in the plasma of female patients receiving bone marrow from male donors showed that the DNA is predominantly of donor origin, derived from hematopoietic cells [3]. Another group excluded T-cells as a source of fcDNA based on lack of T-cell specific sequence rearrangement [1]. All fcDNA samples contained the germline sequence of T-cell receptor β-chain genes, while 18 of the 20 cancer fcDNA samples tested showed no amplification with the T-cell specific primers, indicating that T-cells are not a major source of fcDNA in cancer patients
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