ForenSeq MainstAY offers an easy transition point to include next-generation sequencing (NGS) into your operational workflow. NGS supports the simultaneous typing of various categories of STRs in a single reaction, enabling the recovery of the maximum information from a forensic sample. NGS also combines the discriminatory power of STRs with additional sequence-level variation, which can resolve isoalleles within an STR. The ForenSeq MainstAY Kit contains reagents to amplify 27 autosomal and 25 Y-STR loci as small amplicons and generate sequencing data on the MiSeq FGx Sequencing System using the MiSeq FGx Reagent Micro Kit. With this workflow, both sequence and allele length polymorphism in the Au-STRs and Y- STRs can be identified in a single amplification. This eliminates the need to run multiple workflows and maximizes the informative value of a forensic sample, thereby increasing the statistical power of inclusion. NGS also eliminates the problem of overlapping alleles, allowing users to interrogate more alleles in the multiplex.
- The combination of STR loci and Amelogenin is an effective tool for human identification
- Maintains compatibility with existing databases worldwide
- Low input recommendations of 1 ng gDNA deliver a reproducibly high call rate
- Analyze sequence data in a familiar and easy-to-use interface with the MainstAY Analysis Module
- Generate reports in less than 1.5 days
Including 27 Au-STRs, 25 Y-STRs and Amelogenin in a single reaction provides a higher expected likelihood ratio than most commercially available autosomal STR kits and enables direct comparisons of single-source profiles (e.g., in database searches and international data sharing) regardless of the assay used to type the comparative sample. The genetic profile generated by ForenSeq MainstAY enables casework searches with Au-STRs as well as haplotype searches with a Y-STR database. The Y-STRs produce a haplotype profile from male DNA which makes them extremely useful when working with male/female mixtures. In the presence of only one male contributor, it can be used to exclude potential male contributors.
Markers in the ForenSeq MainstAY Kit
|D1S1656 vWA CSF1PO D20S482
||DYF387S1 DYS43 DYS393 DYS570
|TPOX D12S391 D6S1043 D21S11
||DYS19 DYS448 DYS437 DYS576
|D2S441 D13S317 D7S820 PentaD
||DYS385a-b DYS460 DYS438 DYS612
|D2S1338 PentaE D8S1179 D22S1045
||DYS389I DYS481 DYS635 Y-GATA-H4
|D3S1358 D16S539 D9S1122 TH01
||DYS389II DYS505 DYS643
|D5S818 D19S433 D10S1248
The maximum number of libraries that can be simultaneously sequenced depends upon the number of reads or the depth of coverage (DoC) desired per locus. Balanced read coverage across amplicons is also critical to locus and allele recovery. The ForenSeq MainstAY Kit has a small number of markers multiplexed in a single sequencing reaction resulting in high overall coverage. Interlocus balance was determined using MainstAY libraries from 1 ng of input DNA from 46 male Coriell samples, including a positive amplification control, 49 female Coriell samples, and an NTC. Average coverage across the Au-STRs and Y-STRs for male samples was 669 and 469 reads, respectively. Average coverage for the Au-STRs for female samples was 1065 reads. The fold difference between the Au-STRs with highest and lowest depth of coverage was 15x, while the fold difference between the Y-STRs with highest and lowest reads was 4.6x (see figure “Average DoC for markers included in the ForenSeq MainstAY Kit”).The median interlocus balance for male samples, expressed as a percentage of total reads, was 1.9% (Au-STRs: 2.2% and Y-STRs: 1.6%). For female samples, the interlocus balance for the Au-STRs was 3.7% (see figure “Interlocus balance for genetic markers included in the ForenSeq MainstAY Kit”).
The intralocus balance (ILB) or heterozygous balance (HB) was calculated as a ratio of the lower number of allele reads to the higher number of allele reads for a heterozygote pair. Compared to CE-based STRs, NGS shows a larger range for the ILB due to amplification biases for smaller amplicons during library preparation and sequencing. ILB for heterozygotes and isometric genotype pairs was evaluated using the 1-ng DNA samples in this study for each sample and locus. All markers in the panel exhibited ILB values above 60% with a median ILB of 86%. The coverage and low variation of the ForenSeq MainstAY Kit indicates that amplicons are well balanced, which reduces the likelihood of locus or allelic dropout and improves the identification of minor contributors in a mixture (see figure ”Intralocus balance for genetic markers included in the ForenSeq MainstAY Kit with 1-ng DNA input”).
The ability to generate genotypes and haplotypes across a range of inputs was evaluated using serially diluted gDNA in template amounts of 1 ng, 500 pg, 250 pg, 125 pg, 62.5 pg, 31.25 pg, 15.625 pg, and 7.82 pg, in triplicate. 96 libraries were simultaneously sequenced using the MiSeq FGx Reagent Micro Kit. The ForenSeq MainstAY Kit offers a high level of sensitivity, generating full profiles (no allele loss) and 100% call rate with as little as 62.5 pg of input DNA when 96 libraries were simultaneously sequenced. To determine allele call rate and comparative accuracy of the sequencing-based genotype data, the genotypes and haplotypes were generated with the MainstAY Analysis Module in the Universal Analysis Software using the default settings and compared to orthogonal genotyping data from conventional genotyping methods (CE fragment length detection for STRs). Au-STRs demonstrated 100% concordance down to 62.5 pg, with a concordance of 95% even at 31 pg. Y-STRs demonstrated 100% concordance down to 125 pg, with 91% concordance down to 31 pg. All discordant calls were the result of stutter that exceeded the default stutter filter set for 1 ng samples. (see figure “Sensitivity study with serially diluted gDNA samples amplified in triplicate”).
To evaluate the concordance of the genotypes and haplotypes generated by the ForenSeq MainstAY Kit, 31 libraries including the NIST SRM 2391d standards, Coriell DNAs, a positive amplification control, and three NTCs were prepared and sequenced in triplicate. Data was analyzed using the MainstAY analysis module in the Universal Analysis Software with default analysis thresholds and stutter filters. Accuracy of the genotypes and haplotype alleles was determined by comparing it to orthogonal CE data. Precision and call rates were determined using repeatability and reproducibility experiments where libraries were prepared and sequenced from 15 control DNA samples in quadruplicate with one positive amplification control and three NTCs by three operators on three different MiSeq FGx instruments. High accuracy was observed in Au-STRs (99.82%) and Y-STRs (100%). Six discordant alleles were detected out of 3261 Au-STR alleles. No discordant alleles were detected for the 774 Y-STRs alleles. Similarly, high precision rates of 99.87% and 99.42% were observed for Au-STRs and Y-STRs, respectively. All discrepancies were attributed to stutter alleles exceeding the default stutter filters.
To evaluate the ability of the ForenSeq MainstAY Kit to detect minor alleles in low level contributors, mixtures of control female and male samples were generated at 1 ng, with the female sample as the major contributor. The percentage of the minor contributor in the mixture ranged from 50% to 0.2%. The number of unique, unshared minor allele contributor alleles in the mixture was assessed using the default analytical and interpretation thresholds, as well as stutter filters in the MainstAY analysis module in the Universal Analysis Software (see figure “Mixtures of female:male gDNA control samples at serially diluted levels of the minor contributor”). At 50% minor contributor, 31 unique minor autosomal alleles and 27 Y alleles were detected. Approximately 15 unique minor autosomal alleles (50% unique Au-STR alleles) and 26 Y alleles were detectable at 5% minor contribution. At 1% minor contribution, over 10 Y-STR loci were detectable. ForenSeq MainstAY enables efficient identification of minor contributors in a mixture even at low level contributions.
The ability to type genotypes and haplotypes of degraded DNA samples with the ForenSeq MainstAY workflow was assessed using partially degraded gDNA that mimics forensic samples exposed to environmental and chemical stresses. Libraries from 1 ng of degraded blood from 30 samples, 1 positive amplification control and NTC, in triplicate were prepared and sequenced using the Miseq FGx Reagent Micro Kit. Degradation index (DI) was used to assess the sample quality of the libraries. Samples with a DI of 1–4 were considered not-degraded, samples with a DI of 4–10 were considered moderately degraded, and those with a DI >10 were considered severely degraded. The samples tested with MainstAY had a DI that ranged from 1 to 56, spanning low, moderate, and highly degraded samples. All STRs were accurately typed in samples with no degradation. Samples with moderate degradation showed >85% call rate of accurately typed STRs, while samples that were severely degraded had a call rate >71%, showing minimal loss of amplifiable STRs. The sample with the highest DI of 56 showed 60% recovery of alleles (40 of 74). ForenSeq MainstAY enables the detection of a high number of alleles even from severely degraded samples (see figure “Genomic DNA extracted from degraded blood samples”).
|DNA input recommendation
||1 ng per sample from gDNA, buccal swabs, FTA card; 1.2 mm per sample from FTA card punch
||96 reaction kit and 384 reaction kit
|Recommended multiplexing capacity
||8–96 samples per run
|Locus multiplexing capacity
||Simultaneous analysis of 52 genetic markers
|Primer mix content
||27 global autosomal STRs including CODIS and European Standard set; 25 Y-STRs including minimum set for the Y Haplotype Reference Database (YHRD)
||Mean: 235 bp; Maximum: 481 bp
|Low level mixture detection
||Detects minor contributors at 5%
|Total prep time
||Library: 7 hours and 15 minutes; Hands-on: 1 hour and 30 minutes
|Total sequencing time
||About 28 hours with the MiSeq FGx Reagent Kit; about 22 hours with the MiSeq FGx Reagent Micro Kit