Introducing the PowerSeq® Whole Mito System–Coming Soon!

Ann MacPhetridge, Promega

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Mitochondrial DNA (mtDNA) sequencing has long been used as a tool for 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. Historically, using Sanger sequencing for mtDNA analysis has been labor intensive and time consuming, limiting its overall usefulness by many laboratories.

Massively parallel sequencing (MPS) addresses these challenges in mtDNA analysis by providing decreased hands-on time per sample, the ability to sequence multiple samples simultaneously, and increased resolution of mixtures and heteroplasmy¹. MPS can also provide increased haplotype discriminatory power by allowing analysis of the whole mitochondrial genome in a single reaction, rather than the control region alone.

The PowerSeq® Whole Mito System is designed to enable MPS analysis of the entire 16,569 bp human mitochondrial genome. The kit design includes a streamlined format that generates 161 amplicons in a single multiplex reaction and an integrated library prep workflow compatible with sequencing on the Illumina MiSeq® or Verogen MiSeq FGx® instruments. The small amplicon format with a size range of 92-254 base pairs (167 bp average) enables more efficient amplification from challenging, degraded samples where nuclear DNA analysis may be difficult. Promega has generated complete and accurate mitochondrial haplotypes with as little as 7.8pg of genomic DNA input.

Generated data can be analyzed using GeneMarker® HTS software (SoftGenetics) for the identification of variants².

Overview

The PowerSeq® Whole Mito System contains reagents for analysis of 100 samples, designed to amplify the entire 16,569 bp mitochondrial genome in a single PCR. It generates 161 small, overlapping amplicons with an average size of 167 bp. The use of small amplicons allows for robust analysis of degraded samples³,⁴. The recommended DNA template amount for amplification is 100pg, but lower input amounts can be used when 100pg is not available.

To make things easier for laboratories doing this type of work, we have included the reagents for sequencing library preparation and bead-based cleanups. The simplified library prep workflow consists of a single, 30- minute enzymatic step at room temperature followed by cleanup using size-selective bead chemistry.

Download the "Whole Mitochondrial DNA Sequencing for Human Identification with the PowerSeqTM Whole Mito System" scientific poster shared at the 32nd International Symposium on Human Identification earlier this year.

Workflow

The streamlined workflow enables laboratories to go from their sample to the sequencer in a single day (Figure 1).

Performance

The PowerSeq® Whole Mito System produces robust library prep yields from as little as 7.8pg of DNA template. As seen in Figure 2, a target library concentration of 4nM was achieved even with that low input amount. Additionally, no loss of coverage or accuracy was seen with as little as 8pg of input sample (Table 1).

Figure 2

Figure 2. Replicates of varying amounts of 2800M DNA were amplified and sequencing libraries were prepared using the PowerSeq® Whole Mito System and IDT for Illumina UD Index Adapters. Library prep yield was measured using the PowerSeq® Quant MS System on a 7500 real-time PCR instrument (Thermo Fisher Scientific). A target library concentration of 4nM is recommended for library pooling and sequencing on MiSeq® instruments.

Table 1. Varying input amounts of 2800M control DNA were amplified in replicates, with sequencing libraries prepared from each sample. Samples were sequenced on an Illumina MiSeq® instrument (v3 reagent kit) in a pool of 22 samples. Data were analyzed using GeneMarker HTS v2.5. Variant calls and read depth were consistent at every concentration tested.

For those laboratories looking to adopt MPS as a tool for their mitochondrial sequencing efforts, the PowerSeq® Whole Mito System is an ideal kit to incorporate into a forensic DNA workflow.

References

  1. Canale, L.C. et al. (2021) The time is now for ubiquitous forensic mtMPS analysis. WIREs. Forensic Sci. wfs2.1431
  2. Holland, M. and McElhoe, J. (2015) A custom software solution for forensic mtDNA analysis of MiSeq data. Forensic Sci. Int. Gene Suppl. 5, 614–616.
  3. Chaitanya, L. et al. (2015) Simultaneous whole mitochondrial genome sequencing with short overlapping amplicons suitable for degraded DNA using the Ion Torrent Personal Genome Machine. Hum. Mutat. 36, 1236–1247.
  4. Eichmann, C. and Parson, W. (2008) “Mitominis” multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples. Intl. J. Legal Med. 122, 385–388.