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.

Fragile X syndrome , which is caused by expanded CGG repeats of the FMR1 gene , is associated with a broad spectrum of clinical involvement and is the most common inherited form of intellectual disability. Early diagnosis and intervention are likely to lead to improved outcome for children with fragile X syndrome , but such strategies require better estimates of the frequencies of expanded alleles of the FMR1 gene. In this study , we report the results of a newborn screening study of 5267 male blood spots collected from the Northwest region of Spain as part of the national newborn screening program. The blood spots were screened using a rapid polymerase chain reaction-based method that is capable of identifying the presence of all expanded alleles for both males and females. The screened samples included 199 gray zone alleles , 21 premutation alleles , and two full mutation alleles (1 in 2633). The frequency of premutation alleles was three times higher (1 in 251) than the quoted value of 1 in 813 from a Canadian population and is fully consistent with the results of large-scale Israeli screening studies. Our results demonstrate that newborn screening for the presence of expanded FMR1 alleles is an effective means for defining the distribution of expanded FMR1 alleles in newborn populations; as such , this method is suitable for large-scale newborn screening.

Forensic mitochondrial DNA (mtDNA) analysis conducted using next-generation sequencing (NGS), also known as massively parallel sequencing (MPS), as compared to Sanger-type sequencing brings modern advantages, such as deep coverage per base (herein referred to as read depth per base pair (bp)), simultaneous sequencing of multiple samples (libraries) and increased operational efficiencies. This report describes the design and developmental validation, according to forensic quality assurance standards, of end-to-end workflows for two multiplexes, comprised of ForenSeq mtDNA control region and mtDNA whole-genome kits the MiSeq FGxTM instrument and ForenSeq universal analysis software (UAS) 2.0/2.1. Polymerase chain reaction (PCR) enrichment and a tiled amplicon approach target small, overlapping amplicons (60–150 bp and 60–209 bp for the control region and mtGenome, respectively). The system provides convenient access to data files that can be used outside of the UAS if desired. Studies assessed a range of environmental and situational variables, including but not limited to buccal samples, rootless hairs, dental and skeletal remains, concordance of control region typing between the two multiplexes and as compared to orthogonal data, assorted sensitivity studies, two-person DNA mixtures and PCR-based performance testing. Limitations of the system and implementation considerations are discussed. Data indicated that the two mtDNA multiplexes, MiSeq FGx and ForenSeq software, meet or exceed forensic DNA quality assurance (QA) guidelines with robust, reproducible performance on samples of various quantities and qualities.

The TruSeq™ Forensic Amplicon library preparation protocol, originally designed to attach sequencing adapters to chromatin-bound DNA for chromatin immunoprecipitation sequencing (TruSeq™ ChIP-Seq), was used here to attach adapters directly to amplicons containing markers of forensic interest. In this study, the TruSeq™ Forensic Amplicon library preparation protocol was used to detect 160 single nucleotide polymorphisms (SNPs), including human identification SNPs (iSNPs), ancestry, and phenotypic SNPs (apSNPs) in 12 reference samples. Results were compared with those generated by a second laboratory using the same technique, as well as to those generated by whole genome sequencing (WGS). The genotype calls made using the TruSeq™ Forensic Amplicon library preparation protocol were highly concordant. The protocol described herein represents an effective and relatively sensitive means of preparing amplified nuclear DNA for massively parallel sequencing (MPS).

Background: Because fragile X syndrome (FXS) is prevalent, it has become the subject of newborn and high-risk screening efforts. International screening, however, can be financially and logistically prohibitive, particularly in countries where resources may be scarce. Recently, we have developed a screening test on blood spot that can detect expanded alleles from the normal through the full mutation range in both males and females. It is accurate, rapid, inexpensive, and applicable on blood spots and therefore ideal for international screening. The use of this blood spot screening technique was piloted in ''a high-risk screening'' study of individuals in Guatemala. Methods: One hundred and five blood spots from subjects from Guatemala were screened for the Fragile X Mental Retardation 1 mutation. They were classified as ''high-risk'' through placement into one of the following five categories: (a) relatives of someone with a previous FXS diagnosis, (b) individuals with confirmed autism, (c) individuals with confirmed intellectual disability, (d) individuals with Parkinson's-like presentation, and (e) individuals with a family history of intellectual disability but no confirmed cases of FXS. Results: Fifteen of the individuals tested yielded an expanded allele, 10 premutations and 5 full mutations. All 15 expansions were found in individuals with a relative with a confirmed FXS diagnosis. No expansions were found in the other clinical groups. Conclusions: Blood spot polymerase chain reaction screening is an effective, cost-efficient method to conduct cascade testing in families with a known history of FXS, even in small screening cohorts.

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Background: As our knowledge of how DNA alterations can drive cancer progression increases, assays that can simultaneously detect multiple types of variants in a simple and cost-effective manner are becoming increasingly crucial. This holds true of copy number variations (CNVs), where evaluation of this type of variant is an important and necessary feature of any solid tumor profiling assay. Conventional methods for detecting CNVs such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and quantitative PCR (qPCR) are limited to detecting only one gene amplification at a time. This can be a significant drawback with FFPE samples, where the DNA is of low abundance and often heavily degraded, while the number of gene amplifications known to be important in cancer continues to grow. Additionally, different tumor types may express amplifications at different rates and expression may be heterogeneous within the tumor, potentially with irregular staining patterns - all of which illustrate the need for new approaches to CNV detection. Methods: Next-generation sequencing (NGS) offers the ability to assess variants in multiple genes using one sample. To that end, Illumina is developing a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes for sequencing on the NextSeq1 platform. The assay consists of a DNA workflow for the identification of single-nucleotide variants (SNVs), small insertions and deletions (indels), as well as an RNA workflow for the identification of splice variants and gene fusions. In addition, using the DNA workflow, a novel analysis pipeline, and CNV caller, CNVs from 57 different genes can be simultaneously assessed all by sequencing of a single sample. Results: Here we present data on both cell lines and FFPE samples of SNVs and indels down to 5% allele fraction and CNVs down to ∼2-fold amplification, all from 40 ng of DNA. To demonstrate the accuracy and precision of our CNV detection method, we tested 7 samples for CNVs using orthogonal CNV detection methods. The Illumina NGS assay detected ERBB2 amplifications in 4 out of 7 samples. Of the 4 Illumina NGS positive samples, 3 samples were positive by FISH and all 4 were positive by droplet digital PCR (ddPCR) and the Illumina TruSight Tumor 15 panel. The 3 samples that were negative for ERBB2 amplifications by the Illumina NGS assay were also negative by both FISH and ddPCR. Within these samples we also found a previously unknown FGFR1 amplification. Conclusions: The novel Illumina NGS library preparation method is an innovative and useful tool to find multiple CNVs, along with other variant types, within a single sample. The assay can detect multiple CNVs within a single FFPE sample and identify previously uncharacterized CNVs that could be important in finding the correct treatment for a cancer patient. Citation Format: Chia-Ling Hsieh, Clare Zlatkov, Byron Luo, Chen Zhao, Kathryn Stephens, Han-Yu Chuang, Lisa Kelly, Katherine Chang, Rachel Liang, Jianli Cao, Scott Lang, Ashley Adams, Naseem Ajili, Laurel Ball, Glorianna Caves, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Sarah Dumm, Ridwana Ekram, Yonmee Han, Anne Jager, Suzanne Johansen, Li Teng, Jenn Lococo, Jaime McLean, Juli Parks, Jason Rostron, Jennifer Sayne, Jennifer Silhavy, June Snedecor, Mckenzi Toh, Stephanie Tong, Elizabeth Upsall, Paulina Walichiewicz, Xiao Chen, Amanda Young, Ali Kuraishy, Karen Gutekunst, Matt Friedenberg, Charles Lin. Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3624.