A new implementation of a semi-continuous method for DNA mixture interpretation.

A new calculation module within the PopStats module of the CODIS software package, based on the underlying mathematics presented in the MixKin software package, has been developed for assigning the Likelihood Ratio (LR) of DNA mixture profiles. This module uses a semi-continuous model that allows for population structure and allelic drop-out and drop-in but does not require allelic peak heights or other laboratory-specific parameters. This new implementation (named SC Mixture), like MixKin, does not specify or estimate a probability of drop-out. Instead, each contributor to a mixture has an independent drop-out rate, and the probability of the mixture profile for a specified proposition concerning the contributors is integrated over the range of possible drop-out rates. The allelic drop-in rate and the population structure parameter, theta, used by the software are specified by the user. The user can examine up to five contributors to a mixture, however, conditioning on assumed contributors and limiting the number of unknowns in both numerator and denominator hypotheses greatly improves performance. We report results from an extensive validation study performed for ten mixtures with each of one (single source), two, three, four, or five contributors, with four combinations of drop-in rate and a population structure parameter. Each mixture was run as a complete profile or with the random removal of alleles to simulate drop-out. All 1620 combinations were evaluated with PopStats, MixKin, and LRmix and considerable consistency was found among the results with all three packages.

Massively parallel sequencing of forensic STRs: Considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements.

The DNA Commission of the International Society for Forensic Genetics (ISFG) is reviewing factors that need to be considered ahead of the adoption by the forensic community of short tandem repeat (STR) genotyping by massively parallel sequencing (MPS) technologies. MPS produces sequence data that provide a precise description of the repeat allele structure of a STR marker and variants that may reside in the flanking areas of the repeat region. When a STR contains a complex arrangement of repeat motifs, the level of genetic polymorphism revealed by the sequence data can increase substantially. As repeat structures can be complex and include substitutions, insertions, deletions, variable tandem repeat arrangements of multiple nucleotide motifs, and flanking region SNPs, established capillary electrophoresis (CE) allele descriptions must be supplemented by a new system of STR allele nomenclature, which retains backward compatibility with the CE data that currently populate national DNA databases and that will continue to be produced for the coming years. Thus, there is a pressing need to produce a standardized framework for describing complex sequences that enable comparison with currently used repeat allele nomenclature derived from conventional CE systems. It is important to discern three levels of information in hierarchical order (i) the sequence, (ii) the alignment, and (iii) the nomenclature of STR sequence data. We propose a sequence (text) string format the minimal requirement of data storage that laboratories should follow when adopting MPS of STRs. We further discuss the variant annotation and sequence comparison framework necessary to maintain compatibility among established and future data. This system must be easy to use and interpret by the DNA specialist, based on a universally accessible genome assembly, and in place before the uptake of MPS by the general forensic community starts to generate sequence data on a large scale. While the established nomenclature for CE-based STR analysis will remain unchanged in the future, the nomenclature of sequence-based STR genotypes will need to follow updated rules and be generated by expert systems that translate MPS sequences to match CE conventions in order to guarantee compatibility between the different generations of STR data.

An improved method for post-PCR purification for mtDNA sequence analysis.

Mitochondrial DNA (mtDNA) analysis of forensic samples typically is performed when the quantity and quality of DNA are insufficient for nuclear DNA analysis or when maternal relatives may be the only reference source. Many of the steps required in the analytical process are both lengthy and labor intensive. Therefore, improvements in the process that reduce labor without compromising the quality of the data are desirable. The current procedure requires purification of the amplicons by centrifugal filtration after PCR and prior to cycle sequencing. Because this method requires several manipulations to perform, alternate cleanup procedures were investigated. These include the use of 1) Qiagen QlAquick PCR Purification columns, 2) Concert Rapid PCR Purification columns, and 3) ExoSAP-IT reagent. When the yield of purified amplicons, quality of the sequence profile, and ease of assay were evaluated, the use of ExoSAP-IT reagent for post-amplification purification was chosen to replace the filtration method.