Compatibility of the ForenSeq™ DNA Signature Prep Kit with laser microdissected cells: An exploration of issues that arise with samples containing low cell numbers.

Massively parallel sequencing is rapidly emerging as a valuable tool in forensic DNA analyses. As part of our validation of this technology, we established its compatibility with a laser microdissection cell collection method including a one-tube DNA extraction process. We also used the laser microdissector to explore the number of cells required to generate informative DNA sequence profiles and establish the limitations of the technology. Using the ForenSeq™ DNA Signature Prep Kit (Primer Mix B) and a MiSeq FGx™ sequencer, we successfully demonstrated the compatibility of MPS with a laser microdissection one-tube extraction method, with minor alterations made to the manufacturer's recommended library preparation protocol, including the addition of magnesium chloride to counteract the effect of dithiothreitol on amplification efficacy. This work highlighted several quality issues that may be encountered when preparing sequencing libraries from low quantity DNA samples, particularly that libraries prepared from low cell numbers showed high levels of adapter dimer compared to those prepared from more cells. To remediate this, we replaced the bead normalisation step with a qPCR normalisation method, whereby sequencing libraries are diluted based on their molarity as determined after library purification. The work presented here focuses on the results from the autosomal and Y STR markers as these could be directly compared to results obtained from traditional capillary electrophoresis techniques. Full autosomal STR DNA sequence profiles (27 loci) could be obtained from 50 epithelial cells and 100 spermatozoa (sperm cells). The limit of detection for the ForenSeq™ system was determined to be 25 epithelial and 25 sperm cells for both autosomal and Y STRs. Cells were dissected from both single source samples and mixtures of semen and saliva. There was no apparent difference in sensitivity, presence of contamination or PCR artefacts between libraries prepared from single source samples and libraries prepared from mixed source samples.

Forensic STR allele extraction using a machine learning paradigm.

We present a machine learning approach to short tandem repeat (STR) sequence detection and extraction from massively parallel sequencing data called Fragsifier. Using this approach, STRs are detected on each read by first locating the longest repeat stretches followed by locus prediction using k-mers in a machine learning sequence model. This is followed by reference flanking sequence alignment to determine precise STR boundaries. We show that Fragsifier produces genotypes that are concordant with profiles obtained using capillary electrophoresis (CE), and also compared the results with that of STRait Razor and the ForenSeq UAS. The data pre-processing and training of the sequence classifier is readily scripted, allowing the analyst to experiment with different thresholds, datasets and loci of interest, and different machine learning models.

The results of an experimental indoor hydroponic Cannabis growing study, using the 'Screen of Green' (ScrOG) method-Yield, tetrahydrocannabinol (THC) and DNA analysis.

The results of an indoor hydroponic Cannabis growth study are presented. It is intended that this work will be of assistance to those with an interest in determining an estimation of yield and value of Cannabis crops. Three cycles of six plants were grown over a period of 1 year in order to ascertain the potential yield of female flowering head material from such an operation. The cultivation methods used were selected to replicate typical indoor hydroponic Cannabis growing operations, such as are commonly encountered by the New Zealand Police. The plants were also tested to ascertain the percentage of the psychoactive chemical Δ-9 tetrahydrocannabinol (THC) present in the flowering head material, and were genetically profiled by STR analysis. Phenotypic observations are related to the data collected. The inexperience of the growers was evidenced by different problems encountered in each of the three cycles, each of which would be expected to negatively impact the yield and THC data obtained. These data are therefore considered to be conservative. The most successful cycle yielded an average of 881g (31.1oz) of dry, groomed female flowering head per plant, and over the whole study the 18 plants yielded a total of 12,360g (436.0oz), or an average of 687g (24.2oz) of dry head per plant. THC data shows significant intra-plant variation and also demonstrates inter-varietal variation. THC values for individual plants ranged from 4.3 to 25.2%. The findings of this study and a separate ESR research project illustrate that the potency of Cannabis grown in New Zealand has dramatically increased in recent years. DNA analysis distinguished distinct groups in general agreement with the phenotypic variation observed. One plant however, exhibiting a unique triallelic pattern at two of the five loci tested, while remaining phenotypically indistinguishable from three other plants within the same grow.