A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA.

To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can be important for determining when and how best to employ bacterial DNA analysis, especially when there is insufficient human DNA for successful short tandem repeat (STR) typing. The goal of this work was to develop a quantitative PCR (qPCR) assay that simultaneously quantifies human and bacterial DNA that would be simple and cost-effective for laboratories to implement.

A New Tool for Probabilistic Assessment of MPS Data Associated with mtDNA Mixtures.

Mitochondrial (mt) DNA plays an important role in the fields of forensic and clinical genetics, molecular anthropology, and population genetics, with mixture interpretation being of particular interest in medical and forensic genetics. The high copy number, haploid state (only a single haplotype contributed per individual), high mutation rate, and well-known phylogeny of mtDNA, makes it an attractive marker for mixture deconvolution in damaged and low quantity samples of all types. Given the desire to deconvolute mtDNA mixtures, the goals of this study were to (1) create a new software, MixtureAceMT™, to deconvolute mtDNA mixtures by assessing and combining two existing software tools, MixtureAce™ and Mixemt, (2) create a dataset of in-silico MPS mixtures from whole mitogenome haplotypes representing a diverse set of population groups, and consisting of two and three contributors at different dilution ratios, and (3) since amplicon targeted sequencing is desirable, and is a commonly used approach in forensic laboratories, create biological mixture data associated with two amplification kits: PowerSeq™ Whole Genome Mito (Promega™, Madison, WI, USA) and Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific by AB™, Waltham, MA, USA) to further validate the software for use in forensic laboratories.

Routine Mitogenome MPS Analysis from 1 and 5 mm of Rootless Human Hair.

While hair shafts are a common evidence type in forensic cases, they are often excluded from DNA analysis due to their limited DNA quantity and quality. Mitochondrial (mt) DNA sequencing is the method of choice when working with rootless hair shaft fragments due to the elevated copy number of mtDNA and the highly degraded nature of nuclear (n) DNA. Using massively parallel sequencing (MPS) of the mitochondrial (mito) genome, we studied the impact of hair age (time since collection) and physical characteristics (hair diameter, medullary structure, and length of hair tested) on mtDNA recovery and MPS data quality.

Exploring statistical weight estimates for mitochondrial DNA matches involving heteroplasmy.

Massively parallel sequencing (MPS) of mitochondrial (mt) DNA allows forensic laboratories to report heteroplasmy on a routine basis. Statistical approaches will be needed to determine the relative frequency of observing an mtDNA haplotype when including the presence of a heteroplasmic site. Here, we examined 1301 control region (CR) sequences, collected from individuals in four major population groups (European, African, Asian, and Latino), and covering 24 geographically distributed haplogroups, to assess the rates of point heteroplasmy (PHP) on an individual and nucleotide position (np) basis.

Damage patterns observed in mtDNA control region MPS data for a range of template concentrations and when using different amplification approaches.

Massively parallel sequencing (MPS) of mitochondrial (mt) DNA allows practitioners the ability to fully resolve heteroplasmic sites. In forensic DNA analysis, identifying heteroplasmy (a naturally occurring mixture of two mtDNA profiles) can provide additional mtDNA profile information which can lead to an increase in the discrimination potential of an mtDNA match between an evidentiary sample and reference source. Forensic samples such as hair and skeletal remains, especially older, more compromised samples, can often exhibit DNA damage.

Characterization of background noise in MiSeq MPS data when sequencing human mitochondrial DNA from various sample sources and library preparation methods.

Improved resolution of massively parallel sequencing (MPS) allows for the characterization of mitochondrial (mt) DNA heteroplasmy to levels previously unattainable with traditional sequencing approaches. An essential criterion for the reporting of heteroplasmy is the ability of the MPS method to distinguish minor sequence variants (MSVs) from system noise, or error. Therefore, an assessment of the background noise in the MPS method is desirable to identify the point at which reliable data can be reported.

Recovery of mtDNA from unfired metallic ammunition components with an assessment of sequence profile quality and DNA damage through MPS analysis.

Recovery of suitable amounts of quality DNA from copper and brass surfaces, like those encountered in ammunition, has been a challenge for the forensic community. The ability of copper ions to rapidly facilitate oxidative damage leading to fragmentation of DNA significantly reduces the pool of templates for PCR amplification. We compared two methods for recovering mitochondrial (mt) DNA from the surface of unfired copper projectiles, brass casings, and aluminum casings, and found that using a cotton swab moistened with 0.

Deep-Coverage MPS Analysis of Heteroplasmic Variants within the mtGenome Allows for Frequent Differentiation of Maternal Relatives.

: Distinguishing between maternal relatives through mitochondrial (mt) DNA sequence analysis has been a longstanding desire of the forensic community. Using a deep-coverage, massively parallel sequencing (DCMPS) approach, we studied the pattern of mtDNA heteroplasmy across the mtgenomes of 39 mother-child pairs of European decent; haplogroups H, J, K, R, T, U, and X. Both shared and differentiating heteroplasmy were observed on a frequent basis in these closely related maternal relatives, with the minor variant often presented as 2-10% of the sequencing reads.

Assessing heteroplasmic variant drift in the mtDNA control region of human hairs using an MPS approach.

Resolution of mitochondrial (mt) DNA heteroplasmy is possible when applying a massively parallel sequencing (MPS) approach. However, interpretation criteria for matching heteroplasmic sequences will need to be established that address a number of important topics, including the drift of variants in sample types such as human hair shafts. Prior to MPS analysis, we compared three different DNA extraction methods for hair using a custom mtDNA quantitative PCR (mtqPCR) assay, and found that a method involving bead capture significantly outperformed methods currently in place in forensic laboratories.

Evaluation of GeneMarker HTS for improved alignment of mtDNA MPS data, haplotype determination, and heteroplasmy assessment.

Existing software has not allowed for effective alignment of mitochondrial (mt) DNA sequence data generated using a massively parallel sequencing (MPS) approach, combined with the ability to perform a detailed assessment of the data. The regions of sequence that are typically difficult to align are homopolymeric stretches, isolated patterns of SNPs (single nucleotide polymorphisms), and INDELs (insertions/deletions). A custom software solution, GeneMarker HTS, was developed and evaluated to address these limitations, and to provide a user-friendly interface for forensic practitioners and others interested in mtDNA analysis of MPS data.

MPS analysis of the mtDNA hypervariable regions on the MiSeq with improved enrichment.

The non-coding displacement (D) loop of the human mitochondrial (mt) genome contains two hypervariable regions known as HVR1 and HVR2 that are most often analyzed by forensic DNA laboratories. The massively parallel sequencing (MPS) protocol from Illumina (Human mtDNA D-Loop Hypervariable Region protocol) utilizes four sets of established PCR primer pairs for the initial amplification (enrichment) step that span the hypervariable regions. Transposase adapted (TA) sequences are attached to the 5'-end of each primer, allowing for effective library preparation prior to analysis on the MiSeq, and AmpliTaq Gold DNA polymerase is the enzyme recommended for amplification.

Considering DNA damage when interpreting mtDNA heteroplasmy in deep sequencing data.

Resolution of mitochondrial (mt) DNA heteroplasmy is now possible when applying a massively parallel sequencing (MPS) approach, including minor components down to 1%. However, reporting thresholds and interpretation criteria will need to be established for calling heteroplasmic variants that address a number of important topics, one of which is DNA damage. We assessed the impact of increasing amounts of DNA damage on the interpretation of minor component sequence variants in the mtDNA control region, including low-level mixed sites.

Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA.

The manifestation of mitochondrial DNA (mtDNA) diseases depends on the frequency of heteroplasmy (the presence of several alleles in an individual), yet its transmission across generations cannot be readily predicted owing to a lack of data on the size of the mtDNA bottleneck during oogenesis. For deleterious heteroplasmies, a severe bottleneck may abruptly transform a benign (low) frequency in a mother into a disease-causing (high) frequency in her child. Here we present a high-resolution study of heteroplasmy transmission conducted on blood and buccal mtDNA of 39 healthy mother-child pairs of European ancestry (a total of 156 samples, each sequenced at ∼20,000× per site).

Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq.

The development of molecular tools to detect and report mitochondrial DNA (mtDNA) heteroplasmy will increase the discrimination potential of the testing method when applied to forensic cases. The inherent limitations of the current state-of-the-art, Sanger-based sequencing, including constrictions in speed, throughput, and resolution, have hindered progress in this area. With the advent of next-generation sequencing (NGS) approaches, it is now possible to clearly identify heteroplasmic variants, and at a much lower level than previously possible.