TY - JOUR
T1 - Developmental validation of a MPS workflow with a PCR-based short amplicon whole mitochondrial genome panel
AU - Cihlar, Jennifer Churchill
AU - Amory, Christina
AU - Lagacé, Robert
AU - Roth, Chantal
AU - Parson, Walther
AU - Budowle, Bruce
N1 - Funding Information:
Funding: Thermo Fisher Scientific provided reagents necessary to perform this study. This work was supported in part by the European Union, grant agreement number 779485-STEFA-ISFP-2016-AG-IBA-ENFSI and the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice, grant number 2016-DN-BX-K001. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the U.S. Department of Justice.
Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2020/11
Y1 - 2020/11
N2 - For the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology—Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge—has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra-and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.
AB - For the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology—Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge—has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra-and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.
KW - Developmental validation
KW - Ion Torrent
KW - Massively parallel sequencing
KW - Mitochondrial DNA
KW - SWGDAM guidelines
UR - http://www.scopus.com/inward/record.url?scp=85096062747&partnerID=8YFLogxK
U2 - 10.3390/genes11111345
DO - 10.3390/genes11111345
M3 - Article
C2 - 33202822
AN - SCOPUS:85096062747
SN - 2073-4425
VL - 11
SP - 1
EP - 31
JO - Genes
JF - Genes
IS - 11
M1 - 1345
ER -