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.

Sequencing the hypervariable regions of human mitochondrial DNA using massively parallel sequencing: Enhanced data acquisition for DNA samples encountered in forensic testing.

Mitochondrial DNA testing is a useful tool in the analysis of forensic biological evidence. In cases where nuclear DNA is damaged or limited in quantity, the higher copy number of mitochondrial genomes available in a sample can provide information about the source of a sample. Currently, Sanger-type sequencing (STS) is the primary method to develop mitochondrial DNA profiles. This method is laborious and time consuming. Massively parallel sequencing (MPS) can increase the amount of information obtained from mitochondrial DNA samples while improving turnaround time by decreasing the numbers of manipulations and more so by exploiting high throughput analyses to obtain interpretable results. In this study 18 buccal swabs, three different tissue samples from five individuals, and four bones samples from casework were sequenced at hypervariable regions I and II using STS and MPS. Sample enrichment for STS and MPS was PCR-based. Library preparation for MPS was performed using Nextera® XT DNA Sample Preparation Kit and sequencing was performed on the MiSeq™ (Illumina, Inc.). MPS yielded full concordance of base calls with STS results, and the newer methodology was able to resolve length heteroplasmy in homopolymeric regions. This study demonstrates short amplicon MPS of mitochondrial DNA is feasible, can provide information not possible with STS, and lays the groundwork for development of a whole genome sequencing strategy for degraded samples.

Massively parallel sequencing of forensically relevant single nucleotide polymorphisms using TruSeq™ forensic amplicon.

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).

High-quality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq.

Mitochondrial DNA typing in forensic genetics has been performed traditionally using Sanger-type sequencing. Consequently sequencing of a relatively-large target such as the mitochondrial genome (mtGenome) is laborious and time consuming. Thus, sequencing typically focuses on the control region due to its high concentration of variation. Massively parallel sequencing (MPS) has become more accessible in recent years allowing for high-throughput processing of large target areas. In this study, Nextera(®) XT DNA Sample Preparation Kit and the Illumina MiSeq™ were utilized to generate quality whole genome mitochondrial haplotypes from 283 individuals in a both cost-effective and rapid manner. Results showed that haplotypes can be generated at a high depth of coverage with limited strand bias. The distribution of variants across the mitochondrial genome was described and demonstrated greater variation within the coding region than the non-coding region. Haplotype and haplogroup diversity were described with respect to whole mtGenome and HVI/HVII. An overall increase in haplotype or genetic diversity and random match probability, as well as better haplogroup assignment demonstrates that MPS of the mtGenome using the Illumina MiSeq system is a viable and reliable methodology.

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.

Single nucleotide polymorphism typing with massively parallel sequencing for human identification.

The Ion AmpliSeq™ HID single nucleotide polymorphism (SNP) panel, a primer pool of 103 autosomal SNPs and 33 Y-SNPs, was evaluated using the Ion 314™ Chip on the Ion PGM™ Sequencer with four DNA samples. The study focused on the sequencing of DNA at three different initial target quantities, related interpretation issues, and concordance of results with another sequencing platform, i.e., Genome Analyzer IIx. With 10 ng of template DNA, all genotypes at the 136 SNPs were detected. With 1 ng of DNA, all SNPs were detected and one SNP locus in one sample showed extreme heterozygote imbalance on allele coverage. With 100 pg of DNA, an average of 1.6 SNP loci were not detected, and an average of 4.3 SNPs showed heterozygote imbalance. The average sequence coverage was 945-600× at autosomal SNPs and 465-209× at Y-SNPs for 10 ng-100 pg of DNA. The average heterozygote allele coverage ratio was 89.6-61.8 % for 10 ng-100 pg of DNA. At 10 ng of DNA, all genotypes of the 95 SNPs shared between the two different sequencing platforms were concordant except for one SNP, rs1029047. The error was due to the misalignment of a flanking homopolymer. Overall, the data support that genotyping a large battery of SNPs is feasible with massively parallel sequencing. With barcode systems, better allele balance, and specifically designed alignment software, a more comprehensive rapid genotyping and more cost-effective results may be obtained from multiple samples in one analysis than are possible with current typing and capillary electrophoresis systems.

STRait Razor: a length-based forensic STR allele-calling tool for use with second generation sequencing data.

Recent studies have demonstrated the capability of second generation sequencing (SGS) to provide coverage of short tandem repeats (STRs) found within the human genome. However, there are relatively few bioinformatic software packages capable of detecting these markers in the raw sequence data. The extant STR-calling tools are sophisticated, but are not always applicable to the analysis of the STR loci commonly used in forensic analyses. STRait Razor is a newly developed Perl-based software tool that runs on the Linux/Unix operating system and is designed to detect forensically-relevant STR alleles in FASTQ sequence data, based on allelic length. It is capable of analyzing STR loci with repeat motifs ranging from simple to complex without the need for extensive allelic sequence data. STRait Razor is designed to interpret both single-end and paired-end data and relies on intelligent parallel processing to reduce analysis time. Users are presented with a number of customization options, including variable mismatch detection parameters, as well as the ability to easily allow for the detection of alleles at new loci. In its current state, the software detects alleles for 44 autosomal and Y-chromosome STR loci. The study described herein demonstrates that STRait Razor is capable of detecting STR alleles in data generated by multiple library preparation methods and two Illumina(®) sequencing instruments, with 100% concordance. The data also reveal noteworthy concepts related to the effect of different preparation chemistries and sequencing parameters on the bioinformatic detection of STR alleles.

Prototype PowerPlex® Y23 System: A concordance study.

The Prototype PowerPlex(®) Y23 System (Promega Corporation, Madison, WI) is a polymerase chain reaction-based amplification kit that targets the 23 Y STR loci DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and Y-GATA-H4. A total of 951 samples from six populations were typed to evaluate the kit and examine concordance for 17 of the loci that are in common with those that can be typed using the AmpFlSTR(®) Yfiler™ kit (Life Technologies, Carlsbad, CA). A total of 16,167 loci were analyzed for each multiplex, and overall concordance was observed. Because of different kit designs, and although concordant for the genetic type, discordant calls can occur due to a deletion at the DYS448 locus. Users should take into consideration such nomenclature anomalies when comparing Y STR profiles. This new kit allows a large battery of Y STR loci to be analyzed using the same basic technologies already employed in forensic laboratories.

A case of Amelogenin Y-null: a simple primer binding site mutation or unusual genetic anomaly?

A thirteen year old boy was murdered by a gunshot wound to the head. In order to confirm identity of the boy, samples were sent to the Instituto de Ciencias Forenses de Puerto Rico (PR-ICF) DNA laboratory. Autosomal DNA results exhibited only an X at the Amelogenin locus, whereas the autopsy results reported the child to be anatomically male. The sample was amplified with four separate YSTR marker systems. While a full Y-STR profile for the father of the boy was obtained, the boy only amplified at STR markers on the p arm of the Y chromosome. Theories that could account for this large absence of Y-STR results include an X-Y translocation or Yp isochromosome.

Maternity exclusion with a very high autosomal STRs kinship index.

This paper reports a maternity testing case to assess the biological relationship between a woman and the boy she was adopting. For all 46 tested autosomal STR loci, the adopting woman and the boy shared at least one allele at each locus, which supported that the woman could be the biological mother of the boy. The pairwise kinship indices (KIs) were calculated for various identity-by-descent distributions. Motherson was the most likely relationship with a very high KI (i.e., 6.91E+08) based on 35 independent autosomal STR loci, but KIs of other pairwise relationships (e.g., aunt-nephew, full sib, etc.) were also high. Further testing of X-STRs and mtDNA excluded the maternity relationship between woman and boy, in which 13 out of 20 X-STR loci were inconsistent and 18 nucleotide mismatches were observed at hypervariable regions I and II of the mtDNA. However, a more distant relationship (e.g., aunt-nephew) cannot be excluded. This case reinforces that possible false identifications can occur in kinship analysis cases yielding very high KIs.

Variants observed for STR locus SE33: a concordance study.

Discordance of STR typing results can be expected between kits that employ different primers for amplification. The complex motif of the SE33 locus and its flanking regions can contribute to the degree of discordant results. Sequence-dependent conformational changes can manifest as length differences under certain electrophoretic conditions and/or use of different primers. The AmpFlSTR® NGM SElect™ PCR Amplification Kit (Life Technologies, Carlsbad, CA), PowerPlex® ESX 17 system (Promega Corporation, Madison, WI), and PowerPlex® ESI 17 system (Promega Corporation) were compared for concordance of allele calls for the SE33 marker in selected samples. A total of 16 samples were identified that were discordant at one of the SE33 alleles by an apparent one nucleotide in size. While the ESX 17 and NGM SElect™ kits yielded concordant results for these 16 samples, the ESI 17 kit generated alleles that differed. The discordant alleles were observed in individuals of African and European descent. Sequence analysis revealed that the one-base difference in size is not due to an indel but is instead the result of a single nucleotide polymorphism (SNP) in the flanking region of the SE33 repeat region. Three different SNPs were observed, one of which is novel. Although these migration anomalies were observed only with the ESI 17 kit, one cannot preclude that a similar phenomenon may occur with the other kits as data sets increase. The type and degree of discordance of STR allele calls among STR kits is an important issue when comparing STR profiles among laboratories and when determining search parameters for identifying candidate associations in national databases.

Extraction platform evaluations: a comparison of AutoMate Express™, EZ1® Advanced XL, and Maxwell® 16 Bench-top DNA extraction systems.

The DNA extraction performance of three low-throughput extraction systems was evaluated. The instruments and respective chemistries all use a similar extraction methodology that involves binding DNA to a coated magnetic resin in the presence of chaotropic salt, washing of the resin to remove undesirable compounds, and elution of DNA from the particles in a low-salt solution. The AutoMate Express™ (Life Technologies Corporation, Carlsbad, CA), EZ1® Advanced XL (Qiagen Inc., Valencia, CA), and Maxwell® 16 (Promega Corporation, Madison, WI) were compared using a variety of samples including: blood on swabs, blood on denim, blood on cotton, blood mixed with inhibitors (a mixture of indigo, hematin, humic acid, and urban dust) on cotton, blood on FTA® paper, saliva residue on cigarette butt paper, epithelial cells on cotton swabs, neat semen on cotton, hair roots, bones, and teeth. Each instrument had a recommended pre-processing protocol for each sample type, and these protocols were followed strictly to reduce user bias. All extractions were performed in triplicate for each sample type. The three instruments were compared on the basis of quantity of DNA recovered (as determined by real-time PCR), relative level of inhibitors present in the extract (shown as shifts in the C(T) value for the internal PCR control in the real-time PCR assay), STR peak heights, use of consumables not included in the extraction kits, ease of use, and application flexibility. All three systems performed well; however extraction efficiency varied by sample type and with the preprocessing protocol applied to the various samples.

Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes.

With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus™ STR reactions alone and in combination with AmpliTaq(®) Gold. All reactions included the additional step of a post-PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.

Y-STR loci diversity in native Alaskan populations.

Y chromosome short tandem repeat (Y-STR) loci are important genetic markers for forensic biological evidence analyses. However, paternal inheritance, reduced effective population size, and lack of independence between loci can reduce Y-STR diversity and may yield greater population substructure effects on a locus-by-locus basis compared with the autosomal STR loci. Population studies are necessary to assess the genetic variation of forensically relevant markers so that proper inferences can be made about the rarity of DNA profiles. This study examined 16 Y-STRs in three sampled populations of Native Americans from Alaska: Inupiat, Yupik, and Athabaskan. Population genetic and statistical issues addressed were: (1) the degree of diversity at locus and haplotype levels, (2) determination of the loci that contribute more so to haplotype diversity, and (3) the effects of population substructure on forensic statistical calculations of the rarity of a Y-STR profile. All three population samples were highly polymorphic at the haplotype level for the 16 Y-STR markers; however, the Native Americans demonstrated reduced genetic diversity compared with major US populations. The degree of substructure indicated that the three populations were related and admixed in terms of paternal lineage. The examination of more polymorphic loci may be needed to increase the power of discrimination of Y-STR systems in these populations.