BENCHMARKS A B
for fragmentation in Tables 1 and 2 and Figure 2 (A-, B-, and C-). As expected, the samples that were kept at room temperature for 3 days appeared more fragmented than the freshly extracted ones (Figure 2). When the methylation levels of these
Figure 1. Assessment of DNA fragmentation in genomic DNA samples from 10 individuals. Genomic DNA was run on 1% agarose gels. Highly fragmented samples had higher HpaII/MspI ratios as determined by LUMA (see Supplementary Material for details), indicating lower methylation levels.
MspI peaks, and since the number of HpaII sites is always smaller than the number of MspI sites, it will tend to increase the HpaII/MspI ratio, resulting in a false decrease in global methylation levels (8) (Supplementary Material). In order to test this hypothesis exper-
imentally, we ran several genomic DNA samples on agarose gels to assess DNA integrity and also measured their global methylation levels by LUMA. As predicted, the fragmented samples had much higher HpaII/MspI ratios than the intact ones (Figure 1; samples from 10 individuals numbered from 1 to 10). In order to prove that the altered ratios
resulted solely from fragmentation, it was necessary to demonstrate (i) that the ratios are altered in fragmented DNA when compared with non-fragmented DNA from the same individual and (ii) that there is no true change in methylation level either as a result of fragmentation
A- A+
or as a result of the processes that lead to fragmentation. We collected two sets of blood
samples each from three healthy individuals denoted as A, B, and C. Samples were processed in the Genomics Core Facility at Stony Brook University Medical Center under an institutional review board (IRB) approved protocol. Each blood sample was dispensed into two identical EDTA-containing tubes. One tube from each of these individuals was kept at room temperature for 3 days prior to DNA extraction in order to allow time for fragmentation (resulting from endogenous nuclease activity). These samples are referred to as old and (+) for fragmentation in Tables 1 and 2 and Figure 2 (A+, B+, and C+). Another tube of blood from each of these individuals was extracted immediately. These samples are referred to as fresh and (-)
Table 1. HPLC derived values for non-methylated and methylated deoxycytosines. HPLC values dC
B- dmC dC/(dC+dmC)
18.92 0.92 0.95
18.83 0.94 0.95
18.67 0.95 0.95
B+
18.77 0.90 0.95
C-
19.03 0.89 0.96
C+
18.53 0.95 0.95
HPLC derived values for non-methylated (dC) and methylated (dmC) deoxycytosines in genomic DNA extracted from the blood of three individuals: A, B, and C. The DNA samples were extracted immediately from blood (fresh, (-) for fragmentation) or after being kept at room temperature for 3 days (old, (+) for fragmentation). dC and dmC values are the percentages of total nucleotides normalized against an internal control.
Table 2. Corrected and uncorrected LUMA HpaII/MspI ratios. HpaII/MspI
samples were calculated using standard LUMA, it was found that the HpaII/ MspI ratios were higher in the more fragmented sample—this is similar to findings reported previously (9,10). In addition, the methylcytosine levels of these samples were measured using HPLC in order to control for methylation changes. The results demonstrated that the methylcytosine levels were similar in both fragmented and non-fragmented samples, eliminating the possibility that the methylation levels of the samples had changed as a result of being kept at room temperature for 3 days (Figure 2 and Table 1). These results demonstrate that the changes in the ratios are likely to be due solely to fragmentation. (For further discussion on the comparison of HPLC and LUMA results, please see the Supplementary Material). We considered different methods to
A- A+ B- B+ C- C+ 3 7 8 9 10
Uncorrected 0.37 0.62 0.29 0.39 0.22 0.34 0.65 0.21 0.25 0.23 0.77 Corrected
0.35 0.46 0.27 0.34 0.21 0.28 -0.04 0.19 0.20 0.21 0.55
Uncorrected and corrected (with undigested gDNA) HpaII/MspI ratios for the samples from eight individuals: A, B, C, 3, 7, 8, 9, 10. Blood samples from A, B, and C were DNA extracted either im- mediately (-) or after being kept at room temperature for 3 days (+). Uncorrected and corrected ratios remained similar for less fragmented samples but decreased dramatically for the fragmented samples.
Vol. 58 | No. 5 | 2015 263
overcome the effects of DNA fragmen- tation on LUMA measurements and chose a modification that simply involves adding a third reaction to the usual two (EcoRI+HpaII and EcoRI+MspI), namely direct pyrosequencing of undigested genomic DNA. Through this addition, it is possible to quantify and take into account the signal obtained from the undigested DNA sample in the HpaII/ MspI ratio calculation. Comparison of the pyrograms of undigested sheared and unsheared DNA samples clearly shows the incorporation of nucleo- tides in the sheared sample but not in the unsheared one (Supplementary Figure S1). In order to control for the amount of DNA in the undigested reaction, LUMA was performed with three sets of undigested DNAs for each sample, and their average was taken. The average value for the undigested samples was subtracted from those in the digested samples on a nucleotide- by-nucleotide basis, and the HpaII/ MspI ratio were subsequently calcu- lated. While the ratios of intact DNA samples remained the same after this correction, the ratios of the fragmented samples decreased dramatically. (See
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