Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories.
Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines. A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or "barcodes" are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR inhibitors. Calculations from ForenSeq™ STR and SNP repeatability and reproducibility studies (1ng template) indicate 100.0% accuracy of the MiSeq FGx™ System in allele calling relative to CE for STRs (1260 samples), and >99.1% accuracy relative to bead array typing for SNPs (1260 samples for iiSNPs, 310 samples for aiSNPs and piSNPs), with >99.0% and >97.8% precision, respectively. Call rates of >99.0% were observed for all STRs and SNPs amplified with both ForenSeq™ primer mixes. Limitations of the MiSeq FGx™ System are discussed. Results described here demonstrate that the MiSeq FGx™ System meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities.
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: ajager@illumina.com.
Anne C
AC
Jäger
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: malvarez1@illumina.com.
Michelle L
ML
Alvarez
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: cdavis1@illumina.com.
Carey P
CP
Davis
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: eguzman@illumina.com.
Ernesto
E
Guzmán
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: yhan@illumina.com.
Yonmee
Y
Han
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: lway@illumina.com.
Lisa
L
Way
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: pwalichiewicz@illumina.com.
Paulina
P
Walichiewicz
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: dsilva@illumina.com.
David
D
Silva
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: mpham@illumina.com.
Nguyen
N
Pham
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: gcaves@illumina.com.
Glorianna
G
Caves
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: jbruand@illumina.com.
Jocelyne
J
Bruand
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: fschlesinger@illumina.com.
Felix
F
Schlesinger
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: spond@illumina.com.
Stephanie J K
SJK
Pond
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: jvarlaro@illumina.com.
Joe
J
Varlaro
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: kmstephens@illumina.com.
Kathryn M
KM
Stephens
Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address: cholt@illumina.com.
Cydne L
CL
Holt
D053523:Amelogenin / Q000235:genetics; D000818:Animals; D016172:DNA Fingerprinting; D005260:Female; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation*; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D013045:Species Specificity
D053523:Amelogenin
DNA typing; ForenSeq™ DNA Signature Prep Kit; ForenSeq™ Universal Analysis Software (UAS); Forensic DNA; Forensic genomics; Massively parallel sequencing (MPS); MiSeq FGx™ System; Next-Generation Sequencing (NGS); SWGDAM validation; Short tandem repeat (STR); Single nucleotide polymorphism (SNP)