Investigation of DNA Methylation from Cell-Free DNA samples

Epigenetic-based research investigates the changes in gene expression which do not involve modifications to underlying DNA sequences. The origins of such modifications are established to be induced by environmental factors, age, as well as disease status1. DNA methylation is one of the most broadly studied and well-characterized modifications, and in 1983 methylation of DNA was first confirmed to occur in human cancer; many additional illnesses and disorders have since been linked to DNA methylation2. While the examination of methylome patterns can be carried out from many biospecimens, such as tissues, cells, and biofluids, the evaluation of circulating samples hold tremendous value for biomarker discovery and development because these samples can be collected using minimally– or non-invasive modalities.

Circulating cell-free DNA (cfDNA) is comprised of small DNA fragments (~170-500bp) found in biofluids and is thought to be derived from leakage of apoptotic and necrotic tumor cells and by active release by tumor cells3,4. Methylated DNA has been shown to have a slower degradation rate and to persist longer in blood than unmethylated DNA5, and in contrast to healthy human levels, diseased states are associated with higher levels of cfDNA6-8. The evaluation of DNA methylation using whole genome bisulfite sequencing has revealed that the circulating DNA in specific types of cancer is globally hypomethylated while showing hot-spots of hypermethylation associated with CpG islands or gene promoter regions9-10. The utility of methylome characterization of cfDNA extends beyond oncology; cfDNA methylation profiling can be employed to segregate lung cancer patients from interrelated pulmonary diseases; for example fibrotic interstitial lung disease (ILD) versus chronic obstructive pulmonary disease (COPD)11. Furthermore, the examination of cfDNA methylation may be a viable tool for non-invasive prenatal testing due to differences in DNA methylation between maternal peripheral blood and fetal or placental DNA12.

Advances in the preparation of bisulfite-converted DNA libraries and next-generation sequencing have enabled measurement of methylation levels from the low amounts of input DNA present in plasma or serum samples. Specific genomic regions of interest, such as CpG islands, can be monitored using bisulfite sequencing through the selection of specific DNA sequences in the prepared libraries using high density capture reagents from Roche, Illumina, and Agilent. Alternatively, it is possible to monitor global changes in methylation with affordable lower-depth sequencing through bioinformatic approaches that divide the genome into tiles of one million or more bases each. ORB has developed and validated protocols for both targeted and global methylation analysis by sequencing, and has optimized the isolation of cfDNA from liquid biopsy samples in order to minimize sample input requirements for this service. Please contact us about your specific application for low-input methylation sequencing; we look forward to the challenge of new projects and the application of this important method to biomarker discovery.

Sample Types Appropriate for ORB cfDNA Bisulfite Sequencing and Shipping Instructions

  • Whole Blood
  • Serum
  • Plasma
Sample Type Minimum Input Requirement Sample Submission Checklist
Cell-free DNA 10 ng cfDNA
Biofluid 4 ml Biofluid
ORB provides cfDNA isolation services from blood-based biofluid samples. ORB is a Biosafety Level 2 (BSL-2) laboratory, and can accept samples which contain agents associated with human disease.

When shipping, enclose a completed sample submission checklist in a separate dry compartment with the sample shipment. Also send a digital file version of the sample submission checklist to and provide notification of the sample shipment by calling 754-600-5128 on the day samples are shipped.

ORB’s cfDNA Bisulfite Sequencing Bioinformatic Deliverables

gDNA Read Summary

  • Detected sequence reads with genomic coordinates, counts per sample, and annotations.

Standard cfDNA Bisulfite Sequencing Report

  • Identification of methylated sites showing the methylation call and percentage methylation at each site.
  • Statistical analysis result showing methylated loci with significant differences in methylation levels between groups, annotation of nearest gene, and the relative position of the methylated site to the gene.
  • Maps of CpG-rich genomic regions indicating the distribution of differentially methylated regions across the chromosomes.
  • Distribution histograms of CpG coverage and CpG methylation levels.

Additional Raw Data

  • Compressed FASTQ files
  • Raw unique sequences mapped to genome (FASTA format)
  • Raw and normalized read counts, as well as reads mapping to each gDNA for each database queried

Please contact us by phone at 754-600-5128 or through the contact form to discuss your methylation analysis project or follow the links below to view information about related services.
Biofluid Profiling Illumina Sequencing Biomarker Discovery Companion Diagnostics Predictive Modeling


  1. Portela, A., & Esteller, M. (2010). Epigenetic modifications and human disease. Nature biotechnology28(10), 1057-1068.
  2. Karampini, E., & McCaughan, F. (2016). Circulating DNA in solid organ cancers—analysis and clinical application. QJM109(4), 223-227.
  3. Jung, K., Fleischhacker, M., & Rabien, A. (2010). Cell-free DNA in the blood as a solid tumor biomarker—a critical appraisal of the literature. Clinica Chimica Acta411(21), 1611-1624.
  4. Ivanov, M., Baranova, A., Butler, T., Spellman, P., & Mileyko, V. (2015). Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation. BMC genomics16(13), S1.
  5. Skvortsova, T. E., Bryzgunova, O. E., Lebedeva, A. O., Mak, V. V., Vlassov, V. V., & Laktionov, P. P. (2010). Methylated cell-free DNA in vitro and in vivo. In Circulating nucleic acids in plasma and serum (pp. 185-194). Springer Netherlands.
  6. Swarup, V., & Rajeswari, M. R. (2007). Circulating (cell-free) nucleic acids–a promising, non-invasive tool for early detection of several human diseases. FEBS letters581(5), 795-799.
  7. Schwarzenbach, H., Alix-Panabières, C., Müller, I., Letang, N., Vendrell, J. P., Rebillard, X., & Pantel, K. (2009). Cell-free tumor DNA in blood plasma as a marker for circulating tumor cells in prostate cancer. Clinical Cancer Research15(3), 1032-1038.
  8. Spindler, K. L. G., Pallisgaard, N., Vogelius, I., & Jakobsen, A. (2012). Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan. Clinical Cancer Research18(4), 1177-1185.
  9. Charlton, J., Williams, R. D., Weeks, M., Sebire, N. J., Popov, S., Vujanic, G., & Pritchard-Jones, K. (2014). Methylome analysis identifies a Wilms tumor epigenetic biomarker detectable in blood. Genome biology15(8), 434.
  10. Legendre, C., Gooden, G. C., Johnson, K., Martinez, R. A., Liang, W. S., & Salhia, B. (2015). Whole-genome bisulfite sequencing of cell-free DNA identifies signature associated with metastatic breast cancer. Clinical epigenetics7(1), 100.
  11. Wielscher, M., Vierlinger, K., Kegler, U., Ziesche, R., Gsur, A., & Weinhäusel, A. (2015). Diagnostic performance of plasma DNA methylation profiles in lung cancer, pulmonary fibrosis and COPD. EBioMedicine2(8), 929-936.
  12. Xiang, Y., Zhang, J., Li, Q., Zhou, X., Wang, T., Xu, M., & Zhao, X. (2014). DNA methylome profiling of maternal peripheral blood and placentas reveal potential fetal DNA markers for non-invasive prenatal testing. Molecular human reproduction20(9), 875-884.