Figure 1.
DREAM PCR and Déjà vu PCR makes use of what we have termed a “DNA diode” where enzymes that specifically digest 5th and 6th bases respectively are leveraged to ensure complex serial amplification steps can be performed contamination free without physical isolation of lab equipment.
Both enzymes are heat inactivated and do not show activity post PCR. Any hmeC products cannot contaminate the Nextera reaction setup as AbaSI is present to selectively digest hmeC-DNA while leaving the target meC DNA intact. Likewise, any Nextera DNA contaminating the LR-PCR setup will be digested by MspJI since it that targets both forms of methylation.
Figure 2.
Deleted Mitochondrial DNA hyper-amplifies with LR-PCR.
Observed vs Expected coverage of two unique haplogroup mtDNA samples pooled prior to LR-PCR amplification. One 4.5 kb Kearns-Sayre homozygous deleted mtDNA (12.1 kb, KSS mtDNA) sample is mixed with a known wild type mtDNA (16.6 kb, NA12878 mtDNA) sample with a different haplogroup. The KSS mtDNA sample has a unique haplogroup that creates heteroplasmies at expected loci when mixed with a full length mtDNA control. After sequencing the mixtures to 10,000× mean coverage on an Illumina MiSeq V2 system, allele frequencies are measured across a barcoded dilution series where the deleted sample alleles are expected to be seen at 5%,10%,15%,25%,50%,75% of the reads. Plotted is the expected coverage of the KSS mtDNA alleles versus the observed ratio (Y-Axis) of the control mtDNA alleles. This is measured by mapping reads with Bowtie and counting allele frequencies at the haplogroup specific loci. This result is expected in that a multiplexed PCR containing 12.1 kb and16.6 kb molecules will selectively amplify the smaller template. The selective amplification was still observed despite 15 minute extension times applied in PCR. This also highlights the pronounced sensitivity for detecting large deletions in mtDNA samples using LR-PCR.
Figure 3.
Quantitative PCR of digested and undigested Déjà vu libraries.
120 minute digestion of AbaSI at 25°C on methylated DNA and hydroxymethylated DNA. A 100 fold reduction in background hydroxymethylated DNA is obtained with a 2 hr 25°C digestion with 0.3Units of Enzyme.
Figure 4.
DMSO impact on sequencing methylated libraries.
Use of DMSO is estimated to lower the Tm 0.6°C per % according to Von Ashen et al. The use of 4%DMSO improves the C1, C10, C20 and C100 sequencing metrics. All samples were deprecated to 400× coverage to normalize read depth. BEDtools was utilized to calculate C1-C100s coverage statistics. The use of 4% DMSO in PCR with 5mCTP improves the C20 coverage of targets in sequencing panels.
Figure 5.
Decontamination effectiveness.
To measure decontamination potential we mixed equimolar 5me-dCTP amplified mtDNA into non-methylated Target mtDNA. Methylated and non-methylated DNA were from mtDNA haplogroups differing in 8 loci. Each haplogroup mtDNA sample was barcoded with unique DNA barcodes prior to pooling, decontamination and amplification. Complete decontamination was measured via sequencing the mixed libraries to 10,000× coverage and measuring heteroplasmy levels with and without MspJI decontamination. MspJI digestion removed 100% of expected heteroplasmy contaminants(red) suggesting it can decontaminate up to equimolar contamination events. Undigested pooled libraries were sequenced as a control (blue) and exhibited 35–65% heteroplasmy levels. These artificial heteroplasmies were produced by pooling a methylated mitochondrial Long Range PCR product from a different haplogroup into a non methylated product. This haplogroup is completely removed by the decontamination methods described.
Figure 6.
Approximately 2.9 million 250 bp reads were sequenced for each condition. The ratio of Mitochondrial reads to Nuclear reads (M:N ratio) is displayed using Methyl digestion prior to Haloplex capture of targets. X axis displays increasing units of MspJI producing increasing M:N ratios. To confirm the lack of methylation with mtDNA we also performed haloplex capture on a Methyl enrichment fraction derived from Methyl Binding Domain conjugated magnetic particles. (EpiMark, New England Biolabs). Methyl enriched DNA shows a near equimolar 1:1 read ratio despite Control samples showing a 12.3 M:N and MspJI treatment delivering a 25:1 M:N ratio.
Figure 7.
Confirmation of mtDNA copy number with qPCR.
SYBR Green Real Time PCR of mtDNA genes ND1 and ND6 estimates mitochondrial copy number at 428 copies next to diploid genes BECN1 and NEB.