This article describes an approach for establishing objective analytical thresholds suitable for multiplexed amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) methods, and a definition is proposed for PCR-MPS method background noise, along with a description of an analytical threshold based on background noise.
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. (publisher abstract modified)
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