Characterisation of identity-informative genetic markers in the Australian population with European ancestry.

Identity-informative single nucleotide polymorphisms (iiSNPs) are valuable genetic markers for human identification and kinship testing in forensic casework, especially when the quality and quantity of DNA evidence is not suitable for routine short tandem repeat (STR) profiling. This study analysed 105 buccal samples representing the Australian population with European ancestry in order to assign allele frequencies and conduct population genetic analyses for 94 iiSNPs and 20 STRs. The markers were assessed by calculating relevant forensic statistics and testing for deviations from Hardy-Weinberg and linkage equilibrium.

Comparison of commercial targeted amplicon sequencing assays for human remains identification casework.

Targeted amplicon sequencing (TAS) facilitates the genotyping of forensically informative single nucleotide polymorphisms (SNPs) using massively parallel sequencing (MPS). For human remains identification, where any extracted DNA is likely to be degraded, TAS may succeed when short tandem repeat (STR) profiling using capillary electrophoresis fails. Further, as well as yielding identity information, SNPs can provide information about ancestry, phenotype, kinship and paternal lineage (Y chromosome haplotypes).

Operationalisation of the ForenSeq® Kintelligence Kit for Australian unidentified and missing persons casework.

Single nucleotide polymorphism (SNP) genotyping technologies can generate investigative leads for human remains identification, including estimation of biological sex, biogeographical ancestry (BGA), externally visible characteristics (EVCs), identity, uniparental lineage and extended kinship. The ForenSeq® Kintelligence Kit provides forensic laboratories with the ability to apply this suite of genetic tools to forensic samples using one panel targeting 10,230 SNPs (including 56 ancestry-informative, 24 phenotype-informative, 94 identity-informative, 106 X chromosome, 85 Y chromosome and 9867 kinship-informative SNPs) sequenced on the MiSeq FGx® Sequencing System. The ForenSeq® Kintelligence Kit has been internally validated, optimised and operationalised by the Australian Federal Police National DNA Program for Unidentified and Missing Persons (AFP Program) for coronial casework.

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes.

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information.

Recovery of whole mitochondrial genome from compromised samples via multiplex PCR and massively parallel sequencing.

In forensic casework, compromised samples often possess limited or degraded nuclear DNA, rendering mitochondrial DNA a more feasible option for forensic DNA analyses. The emergence of massively parallel sequencing (MPS) has enabled the recovery of extensive sequence information from very low quantities of DNA. We have developed a multiplex PCR method that amplifies the complete mitochondrial genome in a range of forensically relevant samples including single cells, cremated remains, bone, maggot and hairs isolated from dust bunnies.

Successful nuclear DNA profiling of rootless hair shafts: a novel approach.

Historically, rootless hair shaft samples submitted to a forensic laboratory for DNA analysis are reserved for mitochondrial DNA (mtDNA) analysis due to the presence of highly degraded as well as insufficient amounts of nuclear DNA. Although mtDNA has been very successful in obtaining results from rootless hair, this system has its limitations, namely, it is a lineage marker that cannot differentiate between maternally related genotypes. Given the high incidence of hairs as forensic evidence, there is a need for the use of a nuclear DNA test system capable of producing reliable results for hair shaft forensic evidence.

Linear amplification of target prior to PCR for improved low template DNA results.

Forensic analysis of genetic material is often limited by the quantity and quality of DNA available for examination. Stochastic effects associated with low amounts of starting template can lead to a reduction in the quality of the result, making interpretation difficult. This paper presents an amplification method to copy target DNA in a linear fashion prior to short tandem repeat (STR) analysis to increase the available starting template without introducing the amplification bias seen in other methods used to increase the sensitivity of PCR.

Comment on Kokshoorn, B, and Blankers, BJ 'Response to Grisedale, KS and van Daal, A: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method'.

Kokshoorn and Blankers responded to our recent article by saying that replicate analysis and consensus profiling of low template samples was best in terms of reliability and objectivity. We agree that the consensus approach has benefits, particularly in eliminating non-repeating spurious alleles from the final profile. However, with the development of statistical models that can accommodate stochastic effects and allele drop in, it may be beneficial to perform a single amplification with three times the amount of template, since much information is lost from the profile using the consensus approach.

Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method.

Background: The consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification.