Highlights
- •A conceptual analysis of DNA methylation (DNAm) profiling and its dependence on the amount of DNA was performed.
- •DNAm is a binary event at a given CpG, the DNAm level represents the methylation of all DNA molecules in the sample.
- •Expected variance is calculated using a binomial distribution providing important information for forensic applications.
- •The confidence interval of the DNAm measurement depends on the DNA amount in the sample.
- •The impact of the variance of the level of DNAm on the diagnostic accuracy depends on the application.
Abstract
Analysis of human DNA methylation (DNAm) can provide additional investigative leads
in crime cases, e.g. the type of tissue or body fluid, the chronological age of an
individual, and differentiation between identical twins. In contrast to the genetic
profile, the DNAm level is not the same in every cell. At the single cell level, DNAm
represents a binary event at a defined CpG site (methylated versus non-methylated).
The DNAm level from a DNA extract however represents the average level of methylation
of the CpG of interest of all molecules in the forensic sample. The variance of DNAm
levels between replicates is often attributed to technological issues, i.e. degradation
of DNA due to bisulfite treatment, preferential amplification of DNA, and amplification
failure. On the other hand, we show that stochastic variations can lead to gross fluctuation
in the analysis of methylation levels in samples with low DNA levels. This stochasticity
in DNAm results is relevant since low DNA amounts (1 pg – 1 ng) is rather the norm than the exception when analyzing forensic DNA samples.
This study describes a conceptual analysis of DNAm profiling and its dependence on
the amount of input DNA. We took a close look at the variation of DNAm analysis due
to DNA input and its consequences for different DNAm-based forensic applications.
As can be expected, the 95%-confidence interval of measured DNAm becomes narrower
with increasing amounts of DNA. We compared this aspect for two different DNAm-based
forensic applications: body fluid identification and chronological age determination.
Our study shows that DNA amount should be well considered when using DNAm for forensic
applications.
Keywords
To read this article in full you will need to make a payment
Purchase one-time access:
Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online accessOne-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:
Subscribe to Forensic Science International: GeneticsAlready a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
References
- Body fluid identification in forensics.BMB Rep. 2012; 45: 545-553
- DNA methylation-based forensic tissue identification.Forensic Sci. Int. Genet. 2011; 5: 517-524https://doi.org/10.1016/j.fsigen.2010.12.001
- Demonstration of DSI-semen—A novel DNA methylation-based forensic semen identification assay.Forensic Sci. Int. Genet. 2013; 7: 136-142https://doi.org/10.1016/j.fsigen.2012.08.009
- Development of a forensically useful age prediction method based on DNA methylation analysis.Forensic Sci. Int. Genet. 2015; 17: 173-179https://doi.org/10.1016/j.fsigen.2015.05.001
- Identification of spermatozoa by tissue-specific differential DNA methylation using bisulfite modification and pyrosequencing.Electrophoresis. 2014; 35: 3079-3086https://doi.org/10.1002/elps.201400175
- Assessing cell-to-cell DNA methylation variability on individual long reads.Sci. Rep. 2016; 6: 21317https://doi.org/10.1038/srep21317
- Single-cell genome-wide bisulfite sequencing uncovers extensive heterogeneity in the mouse liver methylome.Genome Biol. 2016; 17https://doi.org/10.1186/s13059-016-1011-3
- DNA methylation age of human tissues and cell types.Genome Biol. 2013; 14https://doi.org/10.1186/gb-2013-14-10-r115
- DNA methylation-based forensic age prediction using artificial neural networks and next generation sequencing.Forensic Sci. Int. Genet. 2017; 28: 225-236https://doi.org/10.1016/j.fsigen.2017.02.009
- Improved age determination of blood and teeth samples using a selected set of DNA methylation markers.Epigenetics. 2015; 10: 922-930https://doi.org/10.1080/15592294.2015.1080413
- The epigenetic tracks of aging.Biol. Chem. 2014; 395: 1307-1314https://doi.org/10.1515/hsz-2014-0180
- Potential forensic application of DNA methylation profiling to body fluid identification.Int. J. Legal Med. 2011; 126: 55-62https://doi.org/10.1007/s00414-011-0569-2
- Independent validation of DNA-based approaches for age prediction in blood.Forensic Sci. Int. Genet. 2017; 29: 250-256https://doi.org/10.1016/j.fsigen.2017.04.020
- Identification and evaluation of age-correlated DNA methylation markers for forensic use.Forensic Sci. Int. Genet. 2016; 23: 64-70https://doi.org/10.1016/j.fsigen.2016.03.005
- Epigenetic predictor of age.PLoS One. 2011; 6: e14821https://doi.org/10.1371/journal.pone.0014821
- Development of a methylation marker set for forensic age estimation using analysis of public methylation data and the Agena Bioscience EpiTYPER system.Forensic Sci. Int. Genet. 2016; 24: 65-74https://doi.org/10.1016/j.fsigen.2016.06.005
- Genome-wide methylation profiles reveal quantitative views of human aging rates.Mol. Cell. 2013; 49: 359-367https://doi.org/10.1016/j.molcel.2012.10.016
- Aging of blood can be tracked by DNA methylation changes at just three CpG sites.Genome Biol. 2014; 15https://doi.org/10.1186/gb-2014-15-2-r24
- Chronological age prediction based on DNA methylation: massive parallel sequencing and random forest regression.Int. Genet. 2017; 31: 19-28https://doi.org/10.1016/j.fsigen.2017.07.015
- The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing.Electrophoresis. 2012; 33: 1736-1745https://doi.org/10.1002/elps.201100711
- Examination of DNA methylation status of the ELOVL2 marker may be useful for human age prediction in forensic science.Forensic Sci. Int. Genet. 2015; 14: 161-167https://doi.org/10.1016/j.fsigen.2014.10.002
- A cautionary note on using binary calls for analysis of DNA methylation.Bioinformatics. 2015; 31: 1519-1520https://doi.org/10.1093/bioinformatics/btv090
- Allele-specific methylation in the human genome.Epigenetics. 2010; 5: 578-582https://doi.org/10.4161/epi.5.7.12960
- Epigenetic control of the genome—Lessons from genomic imprinting.Genes. 2014; 5: 635-655https://doi.org/10.3390/genes5030635
- Epigenome-wide association studies for common human diseases.Nat. Rev. Genet. 2011; 12: 529-541https://doi.org/10.1038/nrg3000
- Epigenetics and social behavior.2015https://doi.org/10.1093/oxfordhb/9780199357376.013.21
- Binomial confidence intervals and contingency tests: mathematical fundamentals and the evaluation of alternative methods.J. Quant. Linguist. 2013; 20: 178-208https://doi.org/10.1080/09296174.2013.799918
- Distribution of lung cell numbers and volumes between alveolar and nonalveolar tissue.Am. Rev. Respir. Dis. 1992; 146: 454-456https://doi.org/10.1164/ajrccm/146.2.454
- A rapid in situ deoxyribonucleic acid assay for determining cell number in culture and tissue.Anal. Biochem. 1982; 122: 338-344https://doi.org/10.1016/0003-2697(82)90292-5
- Quantitative analysis of human tissue-specific differences in methylation.Biochem. Biophys. Res. Commun. 2008; 376: 658-664https://doi.org/10.1016/j.bbrc.2008.09.044
- Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples.Forensic Sci. Int. Genet. 2016; 23: 55-63https://doi.org/10.1016/j.fsigen.2016.01.017
- Genome-wide methylation profiles reveal quantitative views of human aging rates.Mol. Cell. 2013; 49: 359-367https://doi.org/10.1016/j.molcel.2012.10.016
- The DNA methylome of human peripheral blood mononuclear cells.PLoS Biol. 2010; 8: e1000533https://doi.org/10.1371/journal.pbio.1000533
- Cyclical DNA methylation of a transcriptionally active promoter.Nature. 2008; 452: 45-50https://doi.org/10.1038/nature06544
- Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules.Proc. Natl. Acad. Sci. 2004; 101: 204-209https://doi.org/10.1073/pnas.2536758100
- Methylation and epigenetic fidelity.Proc. Natl. Acad. Sci. 2004; 101: 4-5https://doi.org/10.1073/pnas.0307781100
- A new method for accurate assessment of DNA quality after bisulfite treatment.Nucleic Acids Res. 2007; 35: e29https://doi.org/10.1093/nar/gkl1134
- A new approach to primer design for the control of PCR bias in methylation studies.BMC Res. Notes. 2008; 1: 54https://doi.org/10.1186/1756-0500-1-54
- Disclosing bias in bisulfite assay: methPrimers underestimate high DNA mMethylation.PLoS One. 2015; 10: e0118318https://doi.org/10.1371/journal.pone.0118318
- Quantitative sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at single-base resolution.Science. 2012; 336: 934-937https://doi.org/10.1126/science.1220671
- Single-molecule polymerase chain reaction reduces bias: application to DNA methylation analysis by bisulfite sequencing.Anal. Biochem. 2008; 377: 46-54https://doi.org/10.1016/j.ab.2008.02.026
- Single-cell, locus-specific bisulfite sequencing (SLBS) for direct detection of epimutations in DNA methylation patterns.Nucleic Acids Res. 2015; https://doi.org/10.1093/nar/gkv366
- A high-throughput DNA methylation analysis of a single cell.Nucleic Acids Res. 2011; 39: e44https://doi.org/10.1093/nar/gkq1357
- Methylation markers for the identification of body fluids and tissues from forensic trace evidence.PLoS One. 2016; 11: e0147973https://doi.org/10.1371/journal.pone.0147973
- Human age estimation from blood using mRNA, DNA, methylation, DNA rearrangement, and telomere length.Forensic Sci. Int. Genet. 2016; 24: 33-43https://doi.org/10.1016/j.fsigen.2016.05.014
Article info
Publication history
Published online: November 13, 2017
Accepted:
November 10,
2017
Received in revised form:
November 2,
2017
Received:
September 30,
2017
Identification
Copyright
© 2017 Elsevier B.V. All rights reserved.
