TY - JOUR
T1 - Assessment of human nuclear and mitochondrial DNA qPCR assays for quantification accuracy utilizing NIST SRM 2372a
AU - Cropper, Emily
AU - Coble, Michael D.
AU - Kavlick, Mark F.
N1 - Funding Information:
This is publication number 21–27 of the Laboratory Division of the Federal Bureau of Investigation (FBI). The authors would like to acknowledge Quentin Gauthier, Lara Adams, Susannah Kehl, and Thomas Callaghan for their critical review of this manuscript and helpful comments. This research was supported in part by an appointment to the Federal Bureau of Investigation (FBI) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the Federal Bureau of Investigation.
Publisher Copyright:
© 2022
PY - 2022/7
Y1 - 2022/7
N2 - In forensic DNA casework, a highly accurate real-time quantitative polymerase chain reaction (qPCR) assay is recommended per the Scientific Working Group on DNA Analysis Methods (SWGDAM) (SWGDAM Validation Guidelines for DNA Analysis Methods [1]) to determine whether a DNA sample is of sufficient quantity and robust quality to move forward with downstream short tandem repeats (STR) or sequencing analyses. Most of these assays rely on a standard curve, referred to herein and traditionally as absolute qPCR, in which an unknown is compared, relative to that curve. However, one fundamental issue with absolute qPCR is the quantifiable concentration of commercial assay standards can vary depending on (1) origin, i.e., whether from a cell line or a human subject, (2) supplier, (3) lot number, (4) shipping method, etc. In 2018, the National Institute for Standards and Technology (NIST) released a human DNA standard reference material for evaluating qPCR quantification standards, Standard Reference Material (SRM) 2372a, Romsos et al. (2018) [2] which contains three well-characterized human genomic DNA samples: Component A) a single male1 donor, Component B) a single female1 donor, and Component C) a 1:3 male2:female2 donor, each with certification data for nDNA and informational mitochondrial DNA(mtDNA)/nuclear DNA (nDNA) ratio data. The SRM 2372a was used to assess four qPCR assays: (1) Quantifiler Trio (Thermo Fisher Scientific, Waltham, MA) for nDNA quantification, (2) NovaQUANT (EMD Millipore Corporation, San Diego, CA) for nDNA and mtDNA quantification, (3) a custom duplex mtDNA assay, and (4) a custom triplex mtDNA assay. Additionally, extracts from eighteen (18) skeletal remains were tested with the latter three assays for concordance of DNA concentration and with assays (2) and (3), for the degradation state. Our assessment revealed that an accurate, efficient, and reproducible qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the qPCR chemistry, and (3) the specific primers, and probes (if applicable), used in an assay. Our findings indicate qPCR assays may not always quantify as expected and that performance of each lot should be verified using a well-characterized DNA standard such as the NIST SRM 2372a and adjusted if warranted.
AB - In forensic DNA casework, a highly accurate real-time quantitative polymerase chain reaction (qPCR) assay is recommended per the Scientific Working Group on DNA Analysis Methods (SWGDAM) (SWGDAM Validation Guidelines for DNA Analysis Methods [1]) to determine whether a DNA sample is of sufficient quantity and robust quality to move forward with downstream short tandem repeats (STR) or sequencing analyses. Most of these assays rely on a standard curve, referred to herein and traditionally as absolute qPCR, in which an unknown is compared, relative to that curve. However, one fundamental issue with absolute qPCR is the quantifiable concentration of commercial assay standards can vary depending on (1) origin, i.e., whether from a cell line or a human subject, (2) supplier, (3) lot number, (4) shipping method, etc. In 2018, the National Institute for Standards and Technology (NIST) released a human DNA standard reference material for evaluating qPCR quantification standards, Standard Reference Material (SRM) 2372a, Romsos et al. (2018) [2] which contains three well-characterized human genomic DNA samples: Component A) a single male1 donor, Component B) a single female1 donor, and Component C) a 1:3 male2:female2 donor, each with certification data for nDNA and informational mitochondrial DNA(mtDNA)/nuclear DNA (nDNA) ratio data. The SRM 2372a was used to assess four qPCR assays: (1) Quantifiler Trio (Thermo Fisher Scientific, Waltham, MA) for nDNA quantification, (2) NovaQUANT (EMD Millipore Corporation, San Diego, CA) for nDNA and mtDNA quantification, (3) a custom duplex mtDNA assay, and (4) a custom triplex mtDNA assay. Additionally, extracts from eighteen (18) skeletal remains were tested with the latter three assays for concordance of DNA concentration and with assays (2) and (3), for the degradation state. Our assessment revealed that an accurate, efficient, and reproducible qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the qPCR chemistry, and (3) the specific primers, and probes (if applicable), used in an assay. Our findings indicate qPCR assays may not always quantify as expected and that performance of each lot should be verified using a well-characterized DNA standard such as the NIST SRM 2372a and adjusted if warranted.
KW - DNA degradation
KW - DNA quantification
KW - DNA standard
KW - MIQE guidelines
KW - NIST SRM 2372a
KW - mtDNA
KW - nDNA
UR - http://www.scopus.com/inward/record.url?scp=85130141665&partnerID=8YFLogxK
U2 - 10.1016/j.fsigen.2022.102711
DO - 10.1016/j.fsigen.2022.102711
M3 - Article
C2 - 35576790
AN - SCOPUS:85130141665
SN - 1872-4973
VL - 59
JO - Forensic Science International: Genetics
JF - Forensic Science International: Genetics
M1 - 102711
ER -