Assessing the performance of the micro version of the ForenSeq™ protocol with the MiSeq FGx™ System

Abstract

Massively parallel sequencing (MPS) is an emerging technique for forensic DNA analysis that gives benefits in sequencing multiple genetic markers with high sequencing throughput, compared to the more commonly used standard DNA profiling method, PCR amplification of short tandem repeat (STR) sequences followed by capillary electrophoresis. The MiSeq FGx™ System accompanied with the ForenSeq™ DNA Signature prep kit allows for the generation of autosomal, X and Y chromosome STR sequences at the same time as sequencing markers informative for ancestry, eye colour and hair colour. This panel offers DNA identification and investigative lead information that can be used as intelligence for criminal investigations. DNA sequencing of small batches of crime scene samples is often required, rendering the ForenSeq™ standard protocol inefficient in the use of the technology for this purpose. This thesis evaluated the performance of the recently released ForenSeq™ micro flow cell that is optimised to sequence fewer samples with the MiSeq FGx™ System. A range of validation studies were applied to test the performance of the ForenSeq™ micro flow cell and included sensitivity, repeatability, reproducibility, mixtures, partial DNA degradation, PCR inhibition and casework-type samples. Bioinformatic analysis was completed with the associated ForenSeq™ Universal Analysis Software. Profiling analysis of the micro version of the ForenSeq™ protocol was carried out in parallel to the ForenSeq™ standard protocol for the purpose of comparison. Full profiles were obtained when sequencing samples containing as low as 50 pg of DNA. Reliable, reproducible, and consistent results were achieved in terms of allele detection across a range of DNA amounts sequenced. Both two-person mixtures at 1:49 and three-person mixtures at 1:1:10 ratios were resolved with sufficient alleles being detected specific to the minor contributor. However, limitations arose in detecting the minor contributor phenotype across all mixtures. Sequence recovery from partially degraded DNA was successful with STR markers being more prone to dropout relative to SNP markers. Investigating the impact of severely degraded DNA could provide benefits in determining the detection limits. Inhibitor concentrations of up to 50 ng/μL hematin and humic acid, and 25 ng/μL tannic acid was tolerated with partial profiles generated. Casework-type samples (bloodstains present on cotton, polyester, denim, wallpaper, and leaves) produced full DNA profiles. However, sequencing minimal amounts of touch DNA generated only a partial DNA profile. The research carried out demonstrated successful compatibility in sequencing fewer samples with the ForenSeq™ micro flow cell, ultimately making it sequencing efficient and cost-effective. Integrating the methodology of the ForenSeq™ standard protocol with the micro version of the ForenSeq™ protocol also provided insight into the ForenSeq™ Systems limitations and possible improvements that can be made for future advancements.