Reviewer's report
Title:Quantification of epigenetic biomarkers: an evaluation of established and
emerging methods for DNA methylation analysis
Version:1Date:28 October 2014
Reviewer number:2
Reviewer's report:
In the present manuscript, Redshaw and colaborators performed a
comprehensive analysis based on PCR amplification and DNA sequencing
techniques currently used to quantify DNA methylation levels. The authors used
commercially available methylated/unmethylated DNA samples mixed at
pre-determined ratios. DNA methylation changes have been consistently
associated to human diseases, specially cancer, neurological, and immunological
disorders. Further, there is no doubt that disease-specific methylation aberrations
can potentially be used as a target in clinical management of these conditions.
Validation of analytical procedures is one of the most important parts of assay
development for clinical use. The authors compared the results obtained after a
combination of protocols used for single-locus methylation analysis and provide
new insights into interpretation of DNA methylation in terms of linearity, accuracy
and precision. The study is relevant and should be useful to future investigators
making decisions about which method is most suitable for the purposes of
desired experiments.
Major Compulsory Revisions
None
Minor Essential Revisions
1) Abstract section, lines 30-31 – the authors should use an acronym for
bisulfite-treated amplicon sequencing by Next Generation Sequencing such as
BSAS (as previously reported by Masser et al. Focused, high accuracy
5-methylcytosine quantitation with base resolution by benchtop next-generation
sequencing. Epigenetics Chromatin. 2013 Oct 11;6(1):33).
As there are differences between the method we have used and that used by
Masser et al., we thought it might be misleading to use the same acronym.
However, we have modified the text to refer to ‘bisulfite amplicon NGS’
throughout to distinguish between this and other NGS methods.
2) Page 3, Background Section, line 86....by comparison of “expression” levels
with a mock digested template.... the word expression should be changed to
amplification levels.
This has been changed.
3) Page 4, line 94 – [32-26] or [32-36]?
This has been corrected.
4) Page 4, lines 100-101 – idem 1, the authors should suggest a acronym to
differentiate general Next Generation Sequencing (NGS) from Whole Genome or
Amplicon-based Next Generation Sequencing after bisulfite treatment.
As with comment 1), we now refer to ‘bisulfite amplicon NGS’ that we use in
this study to distinguish it from general NGS.
5) Page 5, Results section, lines 122-123, the phrase “To assess the accuracy
and precision of DNA methylation quantification technologies, five [commonly]
used methods” should be reviewed since dPCR and bisulfite amplicon
sequencing by NGS are emerging methods as suggested in the title of this
manuscript.
This has been modified.
6) Page 8, line 236, the abbreviation MID should be defined when first cited. The
same is valid to RE (page 3, line 88).
This has been corrected for MID. The abbreviation for RE is already defined in
lines 69-70.
7) Page 14, Materials and Methods section, line 446. The nucleotide position of
the target region, according to the UCSC genome browser, should be provided.
The genomic location of all assays has been provided in Additional File 6. The
methods section has been modified to clarify this.
Discretionary Revisions
None
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests: I declare that I have no competing interests.