Massively Parallel Sequencing of Whole Mitochondrial Genome
Anupama Gopalakrishnan, Promega Corporation
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In situations where highly degraded nuclear DNA or skeletal remains are the only samples available for identification, mitochondrial DNA (mtDNA) sequencing provides an alternate route. In contrast to the current Sanger sequencing method that is manual, laborious and low-throughput, the PowerSeq® Whole Mito System yields higher quality sequencing data with a much higher throughput and greater sensitivity for heteroplasmy detection in degraded samples recovered in missing persons cases and ancient DNA, enabling maternal inheritance-based kinship testing.
Pros and Cons of Mitochondrial Sequencing for Forensics
Autosomal STRs, present in two copies per nuclear genome, provide a significantly higher power of discrimination and continues to be the preferred method for human identification. Human mitochondrial DNA consisting of ~16,569 base pairs is present in 3–10 copies per mitochondrion and 100s to 1000s of copies per cell. Therefore, in cases where evidence was exposed to harsh environmental elements that can degrade nuclear DNA, the high copy number of mtDNA improves the chances of obtaining a DNA result. Examples include skeletal remains, fingernails, and naturally shed hair that lack root tissue from cold cases or anthropological studies. Also, since mitochondrial DNA is inherited maternally, barring mutation, an individual’s mitochondrial DNA type is not unique to that individual as it is shared with all maternal relatives. However, in cases involving missing persons or mass disasters, this expands the pool of potential reference samples in which maternal family members provide reference specimens for comparison to unidentified remains.
Therefore, while not ideal for unequivocal human identification, the higher copy number of mtDNA, along with a relatively high mutation rate, makes it ideal for typing applications where samples are degraded or of low quality and not suitable for STR analysis.
Sanger vs Massively Parallel Sequencing
While traditional Sanger method is a simpler workflow and sequences a single DNA fragment at a time generating only limited amounts of data, it is much more labor intensive and time-consuming as compared to massively parallel sequencing (MPS). MPS, while complex and requiring a multi-step workflow, sequences millions of fragments of DNA simultaneously per run generating a significantly greater amount of data for the same DNA input. For example, to analyze the whole mitochondrial genome, hundreds of Sanger sequencing reactions are needed to generate the amount of data generated in a single MPS workflow. Sequencing data generated from Sanger and MPS workflows can be thought of as analog and digital respectively. For example, while both workflows enable identification of mtDNA heteroplasmies, quantification of heteroplasmy levels is possible with MPS sequence data.
Resolving individual sequences in a mixture sample is not possible with Sanger sequencing of mtDNA polymorphic regions as peak heights from sequencing electropherograms are not quantitative and cannot be used to determine relative proportions of components of a mixture. MPS provides a digital readout of the number of individual sequences which allows for a quantitation of the component sequences and therefore, enable haplotype determination.
PowerSeq® Whole Mito System
The PowerSeq® Whole Mito System contains amplification and library preparation reagents to use in a workflow as described below to enable sequencing of the whole mitochondrial genome on the MiSeq® instrument. 161 amplicons are generated in the short amplicon format allowing analysis of highly degraded forensic samples.
Figure 1: PowerSeq® Whole Mito System Workflow.
PowerSeq® Whole Mito System Performance Data
The following figures demonstrate that the PowerSeq® Whole Mito System provides superior sensitivity and inhibitor tolerance.
Figure 2: Sensitivity. Varying input amounts of positive control 2800M DNA were amplified and sequenced using PowerSeq® Whole Mito System and MiSeq® sequencing reagents and analyzed using GeneMarker® HTS software, version 2.5. Each concentration was sequenced in duplicate and 22 libraries were pooled in a single run. Table shows % major variant, minimum and average coverage across whole mitochondrial genome.
Figure 3: Inhibitor Tolerance. 100pg of 2800M control DNA was amplified in the presence of varying concentrations of CaCl2 and Humic Acid. These amplicons were used for library preparation, sequenced on a MiSeq® instrument and data generated was analyzed using GeneMarker® HTS v2.5. Percentage of reads less than 100 are shown in the left Y-axis and minimum coverage reads are shown on the right Y-axis.
Use with Real-World Samples
Use of a prototype version of PowerSeq® Whole Mito System in forensically relevant samples has been shown in the generation of mitogenome sequences from rootless hair shaft stored over varying time periods (Canale et al. 2022).
In an in-house experiment, two teeth samples from a sibling pair were processed using Bone DNA Extraction Kit and quantified using PowerQuant® System. While the quantification results showed extensive DNA degradation (high [Auto]/[Deg] ratios), MPS using Prototype PowerSeq® Whole Mito System yielded good coverage enabling haplogroup determination.
Figure 4: Bone DNA Extraction Kit. Mitochondrial DNA from 25-year old teeth samples were prepared for massively parallel sequencing using the Prototype PowerSeq® Whole Mito System and sequenced on the MiSeq® instrument. Data generated was analyzed using GeneMarker® HTS and coverage data (%>100 reads, min and average reads) are shown in the table. The resultant variant calls were queried using Mitomaster for haplogroup determination.
In contrast to the current Sanger sequencing method that is manual, laborious and low-throughput, the PowerSeq® Whole Mito System yields higher quality sequencing data with a much higher throughput and greater sensitivity for heteroplasmy detection in degraded samples recovered in missing persons cases and ancient DNA, enabling maternal inheritance-based kinship testing.
- Canale LC, McElhoe JA, Dimick G, DeHeer KM, Beckert J, Holland MM. Routine Mitogenome MPS Analysis from 1 and 5 mm of Rootless Human Hair. Genes (Basel). 2022 Nov 18;13(11):2144.