5-Methyl C and 5-Hydroxymethyl C Reduce Contamination in mtDNA NGS
June 2014
Next-Generation Sequencing (NGS) has transformed the landscape of genome sequencing making it quicker, cheaper and more accurate. Several NGS platforms exist and they often use universal primers (primers that are complementary to common sequences in a specific set of DNA molecules or plasmids). While universal primers offer a clear advantage, they can lead to contamination and amplification of unwanted products. In a recent paper, Expanded Genetic Codes in Next-Generation Sequencing Enable Decontamination and Mitochondrial Enrichment, McKernan and colleagues present a method that ensures serial amplification steps can be performed without contamination, even without physical isolation of lab equipment. Simultaneously, this method allows for enrichment of mitochondrial DNA. The team smartly utilizes the unique properties of two restriction enzymes (one of which was just recently introduced by NEB) and their ability to preferentially cleave the methylated and hydroxymethylated nucleic acids. They refer to the PCR amplification scheme as Déjà vu PCR.
Current decontamination methods utilize the properties of dUTP and uracil DNA glycosylase. However, uracilated DNA amplifies poorly. Here, the authors utilize the 5th and 6th bases, 5-methylcytosine (5-me-dC) and 5-hydroxymethylcytosine (5-hme-dC) (both available from TriLink) and the unique restriction enzymes, MspJI and AbaSI. MspJI preferentially digests heavily methylated double stranded DNA, while AbaSI digests 5-hme-C. They selectively use 5-me-dC and 5-hme-dC in each amplification step, thus creating unique PCR products that can be differentially digested. Additionally, they maintain robust amplification.
To test the proposed method, the authors chose 16 kb long range PCR (LR-PCR) to amplify the whole mitochondrial genome. They then constructed a transposon-mediated library suitable for a secondary 12-cycle amplification step (Nexteraâ„¢ PCR reaction). Importantly, LR-PCR is sensitive to heteroplasmy and detecting large deletions. However this can be compromised by nuclear mitochondrial sequences (NUMT). To eliminate contamination by NUMT, the authors first digest the target DNA with MspJI under the premise that NUMT is methylated, while mtDNA is not. (It should be disclosed that there is still debate on this topic.) As an example of other potential contaminants in this system, the authors note that the Nexteraâ„¢ libraries contain mitochondrial DNA inserts. A small portion of these are complementary to the LR-PCR primers, thus providing a source of possible contamination. With traditional methods, this produces unwanted background and impairs the ability of the assay to detect heteroplasmy.
The overall scheme proposed involves an initial amplification with 5-me-dCTP. The secondary amplification utilizes 5-hme-dCTP. Decontamination with AbaSI between the first and second amplification digests any potential contaminating hydroxymethylated secondary amplification products from previous samples. Using this method, they observed a 100 fold reduction in background hydroxymethylated DNA. Because the incorporation of 5-me-dCTP significantly increases the melting temperature (Tm), the authors had to test various DMSO concentrations to lower the Tm and prevent hydrolytic damage. They showed that 4% DMSO was optimal in that it lowered the Tm and still provided robust sequencing coverage as gauged by SNP detection. Then, they demonstrated that a target allele can be efficiently amplified when spiked with a contaminant from a different mitochondrial haplogroup at a ratio of 1:1. This effect was shown across 8 different haplogroups. Finally, the authors tested for mitochondrial enrichment by sequencing the entire mitochondrial genome and several nuclear genes after treatment with MspJI. Upon amplification, they determined that the ratio of mitochondrial:genomic reads was ~25:1. Taken together, the authors demonstrate that the proposed method can efficiently decontaminate and reduce background while providing robust mitochondrial enrichment.
While 5-me-dCTP and 5-hme-dCTP are typically associated with epigenetic studies, in this study the nucleotides were utilized to develop a method to improve mitochondrial DNA sequencing through the removal of common contaminants. Additionally, this method can be utilized in the clinic setting to safeguard genetic privacy. The sequences of many universal primers are public knowledge and widely available. Theoretically, this allows for the ability to independently sequence DNA and gain access to personal medical information. This method improves the ability to specifically digest patient sensitive material without destruction of relevant clinical samples. While many factors need to be considered for each independent experiment (i.e. those that are targeting CpG islands), this method is suited to a wide variety of samples. TriLink currently offers both 5-me-dCTP and 5-hme-dCTP as well as many other modified nucleic acids for your sequencing needs.
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