A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis.

Brian Young,Luigi Armogida,Jonathan L King,Bruce Budowle,Ruslan Kalendar

doi:10.1371/journal.pone.0178005

Brian Young, Luigi Armogida + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0178005

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Abstract

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described.

Highlights

Since its inception, massively parallel sequencing (MPS) technology has had enormous impact on genomic characterization
The sub-sequence types exhibit a frequency spectrum that includes a few relatively abundant types corresponding to authentic alleles, along with many low-abundance types corresponding to background noise
The method provides an unambiguous definition for the DNA sub-sequence under analysis; and when common flanking sequence landmarks” (FSL) are applied consistently to all read sub-sequences, the sequence type count intensities for authentic alleles and background noise can be objectively linked for a given locus

Summary

Introduction

Massively parallel sequencing (MPS) technology has had enormous impact on genomic characterization. MPS technology is beginning to be applied to forensic DNA analyses. Sequencer instrumentation and forensic kit manufacturers are developing hardware, software, PCR primers and reagents necessary to support implementation of PCR-MPS systems as routine methods in forensic laboratories [1,2,3,4,5,6,7,8,9,10,11]. While currently considered as an adjunct or complementary system, PCR-MPS could supplement or possibly replace existing forensic analysis methods such as capillary electrophoresis (CE)-based DNA fragment analysis of short tandem repeat (STR) loci and Sanger sequencing of mitochondrial DNA (mtDNA) hypervariable regions.

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