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

Abstract

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.

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