Abstract:
Nanopore sequencing for forensic analyses provides several advantages including portability,
real-time analysis, and reduced costs, but this has yet to be optimised for low-input samples
commonly encountered in forensic casework. Part one of this study aimed to develop a forensic
STR analysis workflow able to provide correct genotyping results using nanopore sequencing.
Optimisations to the workflow included enrichment using whole genome amplification (WGA) to
improve DNA yield of low-input samples. Different library preparation methods, flow cells, and
bioinformatic tools were also investigated. Once a STR analysis workflow was developed, part two
of this study evaluated the workflows further using more challenging samples, including low-input,
forensic-like, and mixture samples.
Part one of this study demonstrated correct genotyping of 12 out of 14 STR loci using no
enrichment techniques, with FGA and CD4 being identified as loci prone to incorrect genotyping
due to homopolymer sequences in the repeat structures. All three whole genome amplification
methods evaluated provided sufficient increases in DNA yield, with the REPLI-g® Single Cell Kit
generating accurate profiles comparable to no enrichment samples. R10.3 flow cells could not be
fully assessed for their improvement in sequencing homopolymers potentially due to
incompatibility of the sequencing chemistry with the library preparation method chosen. Two MPS
tools, Fragsifier and STRait Razor, were evaluated for STR sequence extraction using nanopore
data, with Fragsifier generating profiles with improved read counts with higher accuracy.
Part two of this study showed that the no enrichment workflow can generate full STR profiles from
as low as 100 pg of starting material, with partial profiles using 50 pg. The use of WGA on lowinput
samples introduced additional challenges due to the higher levels of stochastic effects
observed such as amplification bias and heterozygote imbalance. These effects were exacerbated
in mixture samples using either the no enrichment or enrichment with WGA workflow, and the
observed mixture ratio could not be used to accurately predict the expected mixture ratio in any
sample analysed. This study highlights the limitations of this sequencing platform and further
improvement is required before nanopore sequencing can be considered for a forensic purpose.