Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions

Aim

To apply massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples.

Methods

A duplex polymerase chain reaction (PCR) assay targeting the mitochondrial DNA (mtDNA) hypervariable regions I/II (HVI/HVII) was developed for NGS analysis on the Roche 454 GS Junior instrument. Eight sets of multiplex identifier-tagged 454 fusion primers were used in a combinatorial approach for amplification and deep sequencing of up to 64 samples in parallel.

Results

This assay was shown to be highly sensitive for sequencing limited DNA amounts ( ~ 100 mtDNA copies) and analyzing contrived and biological mixtures with low level variants ( ~ 1%) as well as “complex” mixtures (≥3 contributors). PCR artifact “hybrid” sequences generated by jumping PCR or template switching were observed at a low level (<2%) in the analysis of mixed samples but could be eliminated by reducing the PCR cycle number.

Conclusion

This study demonstrates the power of NGS technologies targeting the mtDNA HVI/HVII regions for analysis of challenging forensic samples, such as mixtures and specimens with limited DNA.Limited and mixed DNA samples are often encountered in forensic cases and pose both technical and interpretation challenges. The highly polymorphic hypervariable regions I/II (HVI/II) of the mitochondrial genome are often successfully used to analyze limited and/or degraded DNA samples (1). However, there are some limitations to the current standard approaches used for mitochondrial DNA (mtDNA) sequence analysis when mixtures are encountered. In a five year retrospective study of mtDNA analysis of 691 casework hair samples, a mixture of mtDNA sequences attributed to a secondary source was observed in 8.7% of the hairs and sequence heteroplasmy was observed in 11.7% of the cases (2). While approaches that use capillary electrophoresis technologies for Sanger sequencing of mtDNA polymorphic regions allow for detection of mixtures, they do not allow for resolving individual sequences in a mixture (3-10). Mitochondrial DNA markers are ideal targets for detecting mixtures since, with few exceptions, a single sequence per contributor is the expected result due to its haploid nature. However, unlike short tandem repeats (STRs), peak areas or heights in sequence electropherograms are not necessarily indicative of the amount of DNA contributed to a mixture (9,11,12). As a result, peak height ratios for two bases cannot be used to determine the relative proportions of components of a mixture for mtDNA Sanger sequencing analysis. For this reason, Sanger sequencing does not allow for determining the individual mtDNA sequence haplotypes of mixed samples. Therefore, when mixed base calls are encountered during mtDNA Sanger sequence analysis of forensic specimens, most forensic laboratories choose not to interpret the result and categorize mtDNA mixture results as inconclusive for reporting purposes (13). Furthermore, Sanger sequencing cannot detect minor components present at less than 10% in a DNA mixture (9,12).The 454 genome sequencing technology is a scalable, clonal, and highly parallel pyrosequencing system that can be used for de novo sequencing of small whole genomes or direct sequencing of DNA products generated by polymerase chain reaction (PCR). The technology uses emulsion PCR (emPCR) to amplify a single DNA sequence to 10 million identical copies. The “clonal sequencing” aspect of the technology enables separation of individual components of a mixture as well as analysis of highly degraded DNA. The clonal sequencing approach used with the 454 GS technology and other next-generation sequencing (NGS) technologies provides a digital readout of the number of reads or individual sequences allowing for a quantitative determination of the components in a mixture (14). Recently, the potential value of using NGS technologies for forensic applications has been demonstrated (15-18). This article aims to describe a highly sensitive NGS method that uses PCR for targeted enrichment of the HVI/HVII regions of mtDNA for resolving simple and complex mixtures as well as detecting low levels of heteroplasmy.