Abstract
Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76–139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.
Highlights
Short tandem repeat (STR) profiling is a powerful, multiplex PCR-based assay that uses up to 16 tetranucleotide loci repeats [1,2]
Ethics Statement formalin-fixed paraffin embedded (FFPE) samples were taken as part of a larger next-generation sequencing (NGS) study of genomic prognostic factors within pre-treatment biopsies derived from prostate cancer patients with Institutional Review Board (IRB) approval and patient written consent (Canadian Prostate Cancer Genome Network (CPC-GENE) project; University Health Network-Research Ethics Board UHN06-0822-CE and UHN11-0024CE [16]
At least 80% or more of alleles are required to match in order to properly authenticate DNA samples using STR profiling [20]
Summary
STR profiling is a powerful, multiplex PCR-based assay that uses up to 16 tetranucleotide loci repeats [1,2]. Improvements to STR assays have been required for forensic analysis on highly-degraded DNA samples, namely reducing amplicon size to increase amplification efficiency [4,5]. Previous studies on degraded DNA have demonstrated that STR analysis using amplicons of 280 nt or less generates 48% more genotype profiles than would be possible with longer STRs [5]. STR analysis has been advocated by the American Type Culture Collection Standards Development Organization (ATCC SDO) Workgroup for human cell line authentication in general [6,7]. Monitoring sample quality at the beginning of a process is especially essential to ensure that cell lines and tissues are authenticated for correct input DNA in order to ensure validity of data output [8].
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