Developmental Validation of a novel 5 dye Y-STR System comprising the 27 YfilerPlus loci

In this study, a new STRtyper-27 system, including the same Yfiler Plus loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA H4, DYS449, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627 and DYF387S1a/b), was established using a set of 5 fluorescent dye labels. Primers, internal size standard, allelic ladders and matrix standard set were designed and created in-house for this multiplex system. This paper describes the validation studies conducted with the STRtyper-27Y system using a 3130XL genetic analyzer for fragment length detection that included the analysis of the following parameters and aspects: sensitivity, species specificity, inhibition, haplotype concordance, precision, stutter, DNA mixtures, and stability studies with crime scene samples. The studies demonstrated, that the STRtyper-27Y system provided equivalent overall performance comparable to the latest Yfiler Plus kit, but with enhanced compatibility in terms of instrument platforms and software allowing forensic laboratories to conduct its forensic application and evaluate its performance, all in their own 5 dye Y-STR chemistry system /environment without software or instrument upgrades.

Eight among the chosen markers (DYS456, DYS576, DYS458, DYS460, DYS393, DYS391, DYS438, and DYS389I) were designed specifically for amplicons smaller than 220 bp for the detection of degraded DNA samples. All the primers were designed for this study using the Primer3 program (http:// bioinfo.ut.ee/primer3-0.4.0/primer3/input.htm). Each primer was checked for potential structures of the self dimmer using the AutoDimer v1.1 software and non-specific hybridizations in other genome regions using NCBI Basic Local Alignment Search Tool (BLAST). The four dyes used in the STRtyper-27 system to label primers are 6 FAM(blue), HEX(green), TAMRA(yellow), and ROX(red) dyes(Life Technologies). The fifth dye, SIZE-500 (orange)(Health GeneTech, China), was used to label the internal size standard. 27 loci were organized by expected amplicon size and assigned to the first four different fluorescent dyes in order to achieve an evenly balanced genotyping assay for a single PCR and electrophoretic separation. For details on the marker's repeat structure, genomic location, final concentration, observed allelic ranges, haplotypes of control DNA 9948, 007 and dye labeling refer to the Table 1.
The validation was performed with both 10 μ L and 25 μ L reaction systems, respectively. The 10 μ L reaction volume contained: 5.0 μ L PCR Master Mix, 2.5 μ L Primer Mix and 0.2~2 ng of template DNA. The 25 μ L reaction volume contained: 12.5 μ L PCR Master Mix, 6.25 μ L Primer Mix and 0.2~2 ng of template DNA. The PCR Master Mix included DMSO 10 mM, 125 mM Tris buffer, 125 mM KCl, and 65 mM (NH4) 2 SO 4 . The Primer Mix included appropriate concentration of primers, 7.5 mM MgCl 2 , 7.5 mM dNTPs, 2.5 mg/mL bovine serum albumin (BSA), and 5 U/μ L High Specificity Taq DNA polymerase (Dongsheng BioTech, China). The optimized formulation of the Master Mix and Primer Mix improve performance and support the direct amplification of a larger number of Y-STR markers for both casework and single-source samples. Amplification was performed in GeneAmp 9700 thermal cyclers (Life Technologies) and required 5 min of initial denaturation at 95 °C. Subsequent cycles of denaturation at 94 °C for 10 sec, annealing at 61 °C for 1 min and elongation at 70 °C for 30 sec were repeated 28~30 times according to DNA sample types, followed by 20 min of the final elongation at 60 °C to avoid splitting peaks.
Detection and genotyping. The Applied Biosystems 3130XL Genetic Analyzer (Life Technologies) was set with Dye set G5 to process the data from the five dyes 6 FAM (blue), HEX (green), TAMRA (yellow), ROX (red) and SIZE-500 (orange) after an appropriate matrix. Samples for capillary electrophoresis were prepared by mixing 1 μ L of PCR product, with 9 μ L of a 17:1 Hi-Di Formamide (Life Technologies) and SIZE-500 internal size standard (Health GeneTech). All prepared samples were separated on the ABI 3130XL Genetic Analyzer using 36 cm capillaries (Life Technologies). Standard run parameters involved: sample injection for 10 s at 3 kV and electrophoresis at 15 kV at 60 °C in POP-4 polymer (Life Technologies) as indicated in the HIDFragmentAnalysis36_ POP4_1 module. All genotyping was performed with GeneMapper ID v.3.2.1 software (Life Technologies) with in-house allelic ladder, programmed Panel and Bin sets for each marker. All alleles present in the allelic ladder were sequenced to confirm length and repeat unit structure, using Big Dye Terminator v. Assessing species specificity encompassed testing performance of the assay in amplifying 10 ng of non-human DNA from common male animal species (horse, dog, pig, cow, sheep, chick, duck, cat, fish, rabbit and mouse) and microorganisms pool (Escherichia coli, Micrococcus luteus, and Streptococcus salivarius). Three replicates for each species and microorganisms listed were tested.

Concordance, precision and stutter calculations. A total of 1225 sample already typed for Yfiler
Plus kit in a previous haplotype report [YHRD accession number: Henan, China[Han], YA004057] 11 were processed on the STRtyper-27 system. The results referring to the same 27 Y-STRs were compared for haplotype concordance.
A subset sample profiles from the concordance study were also used to measure the deviation of each sample allele size from the corresponding allele size in the allelic ladder and to calculate stutter. Peaks one repeat smaller or larger than the true allele (± 0.5 bp) were determined to be stutter peaks. The proportion of stutter product relative to the main allele (percent stutter) was measured by dividing the height of the stutter peak by the height of the associated allele peak 12 . In this study, the analytical threshold was lowered to 10 RFU and the stutter filters were set to 1% to detect the stutter peak heights.

Results and Discussion
Optimization of the STRtyper-27 typing system. A new STRtyper-27 multiplex system that allows co-amplification of the same 27 Y-STR loci included in the Yfiler Plus kit was developed and detected by existing instrument platforms and software so that forensic laboratories can conduct experiments, evaluate its performance, and compare results to the latest commercial available Y-STR kit, all in their own 5-dye Y-STR chemical environment. During the optimization process of PCR conditions, annealing temperatures between 57 °C and 69 °C were applied to the amplification of 1 ng positive control DNA 9948. The nonspecific peaks were relatively obvious at the annealing temperature of 57 °C. Full and clean profiles were obtained when PCR was performed with annealing temperatures between 59 °C and 63 °C, and fragment dropouts were observed at the annealing temperature of 65 °C (Supplementary Fig. S1). The best amplification profile was obtained at the annealing temperature of 61 °C (Fig. 1). Both 10 μ L reaction system and 25 μ L reaction system showed the same results.
Altering the number of PCR cycles can be used to adapt the reaction conditions to varying DNA template concentrations. First, we tested the low-copy-number DNA, 125 pg, 62.5 pg, and 31.25 pg positive control DNA 9948, with cycle numbers of 28, 29, and 30. As expected, in both 10 μ L and 25 μ L reaction system, the signal intensities of called alleles increase with higher cycle numbers. Afterwards, we tested the high-copy-number DNA, 10 ng and 20 ng positive control DNA 9948, using both 10 μ L and 25 μ L reaction systems. Robust amplifications and full profiles were obtained with cycle numbers of 28, 29, and 30 using a threshold of 50 RFU for allele calling. However, 30 cycle amplifications of 20 ng DNA gave rise to pull-up peaks when applying PCR products to analysis without prior dilution (data not shown).
After the optimization of the multiplex, 27 Y-STRs were successfully amplified in a single PCR reaction. The system produced peak height balanced haplotype results with amplicon sizes of 80-478 bp. Average peak heights ranged from 700 to 6000 RFUs, and balance between peaks amplified by the same pair of primers such as DYS385 or DYF387S1 was > 70%. The profile of allelic ladder and control DNA 9948 were presented in Fig. 2 and Supplementary Fig. S2, respectively. Sensitivity, species specificity, and inhibition studies. A novel system developed for forensic application needs to be able to produce a profile with sub-nanogram quantities of template DNA. To test the sensitivity of the STRtyper-27 system, positive control DNA 9948 was serially diluted from 4 ng to 62.5 pg per reaction. In the 10 μ L reaction system, occasional allele dropouts were found when ≤ 62.5 pg DNA was used as the template (Fig. 3), whereas in the 25 μ L reaction system, occasional allele dropouts were found when ≤ 125 pg DNA was used as the template (Fig. 4). As expected, the number of dropouts increases with decreasing DNA concentrations. Therefore, for severely degraded DNA samples, DNA template amount higher than 1 ng is recommended for reliable results.
No reproducible signal or characteristic human profiles could be obtained above 50 RFUs threshold for all of the domestic species and microbial pool genomic DNA samples tested demonstrating the low cross-reactivity with non-human species of the STRtyper-27 system (Supplementary Fig. S3) .
An inhibition study was also performed with hematin, humic acid, tannic acid, and calcium, respectively, added directly to the STR reaction to test the robustness of the STRtyper-27 system. For hematin the range of examined concentrations was 5-100 μ M with 50 μ M being the upper resistance value allowing successful amplification across all 27 loci (Supplementary Fig. S4). This result is much better than the one reported for Yfiler, which   Concordance, precision, and stutter calculations. All profiles from 1225 samples using the STRtyper-27 system were concordant with the Yfiler Plus kit reference profiles run on the required 3500 instrument and software. By comparison, the genotyping results of the same 27 Y-STRs were identical (data not shown). We compared the haplotype resolution of combined Y-STRs. 961 haplotypes were observed unique and the discrimination capacity of the 17 Y-STR loci was 86.94% with 1065 different haplotypes. By the addition of 10 Y-STRs (DYS449, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627 and DYSF387S1a/b), an improved discrimination capacity was obtained as 98.94% from 1212 observed haplotypes in 1225 Henan Han samples, indicating that the discrimination power of 27 Y-STR haplotypes in Henan Han is high for forensic and kinship casework.
Determining the sizing precision includes evaluation of measurement error and assessing the performance for accurate and reliable genotyping 12 . A subset of 200 extracted DNA and directed samples from the concordance study were used to measure the deviation of each sample allele from the corresponding allele size in the allelic ladder. All sample alleles tested were within ± 0.5 bp of the corresponding alleles in the allelic ladder demonstrating appropriate precision for sizing microvariants that differ by a single base ( Supplementary Fig. S5).
Stutter is characterized by the presence of an allelic-like signal that is typically one repeat shorter than the parent peak and is significantly less intense than the parent peak (< 20%) 16 . It is important to distinguish between stutter and a true allele in order to resolve mixtures of DNA. In this study, the analytical threshold of minimum stutter peak height was set to 10 RFUs for the accuracy of stutter file for GeneMapper ID v3.2.1. In Supplementary Table S1 the relative intensities of stutter signals are shown for each of the 27 Y-STR locus. The average stutter intensity of all loci tested in this study was 9.04 ± 2.8%. According to previous developmental validation study 17 , the percent stutter is usually more pronounced for shorter repeat motifs and generally increases with allele length. In this study the tri-nucleotide repeat locus DYS481 displayed the highest relative stutter average peaks at 18.19% of the main allele. The locus DYS449 and DYS518, which both contain longer allele ranges, displayed the higher relative stutter peaks at 15.70% and 14.42% of the main allele, respectively. The stutter range observed is similar to that seen with autosomal STRs and consequently stutter signals should normally be distinguishable from allele signals.
Mixture study. To Table S2). When the mixture ratio was increased to 18:2 and 2:18, an average of 88%, 77%, respectively, of the minor alleles was detected. And when the mixture ratio was increased to 19:1 and 1:19, an average of 51%, 37% respectively, of the minor alleles was called. The profile of 9948/007 DNA with ratio of 19:1 was showed in Fig. 5. The results showed that as the mixture ratio increased, the percentage of minor alleles detected decreased.
For the detection of female/male mixture, with known ratios (9947 and 9948 mixed at 1000:1, 100:1, 20:1 and 1:1), a series of samples were prepared in which 125 pg of 9948 DNA was admixed with varying quantities of 9947 DNA and the total admixture amplified in a single reaction. Full profiles were obtained when the male DNA component The sensitivity of the 25 μL system. Green represents peak height ratio > 60% within the same color. Yellow represents peak height ratio < 60% within the same color. Red represents allele drop-out. comprised 1/1 th, 1/10 th, 1/100 th, and 1/1000 th of the total. The results demonstrate that even in the presence of an excess of female DNA, it is possible to obtain a full profile of the male contributor with the STRtyper-27 system. Stability study. The STRtyper-27 system was also tested with DNA extracts obtained from crime scene samples such as blood, saliva, semen stains, and from different tissues (including hairs, skin, muscle, old bone and formalin fixed and paraffin embedded biopsy) to evaluate the effect of biological sample types and quantity, PCR inhibitors from background contaminants, and DNA degradation and the performance to obtain full or partial profiles compared with Yfiler Plus kit.
The STRtyper-27 typing results obtained from 24 biological stains (blood, saliva, and semen) showed full allelic concordance with the reference profiles previously obtained using Yfiler Plus kit. No allelic or locus drop out was observed when 500 pg of DNA was amplified, and several samples with less than 200 pg yielded full profiles. Degraded DNA obtained from 10 old bone and muscle samples (postmortem interval about 3-60 months), 6 hair roots and 4 FFPEB tissues were used to challenge the STRtyper-27 system against DNA degradation, in parallel with the Yfiler Plus kit. Seven degraded DNA samples rendered both full concordant Y-STR profiles or significant partial Y-STR profiles with both STRtyper-27 and Yfiler Plus kit. Supplementary Fig. S6 shown the STRtyper-27 electropherograms obtained from one degraded DNA bone sample.

Conclusion
This article outlines the development of a STRtyper-27 multiplex system that incorporates all loci of the latest available Yfiler Plus kit. Eight Y-STR loci (DYS456, DYS576, DYS458, DYS460, DYS393, DYS391, DYS438 and DYS389I) are less than 220 bp in length in this multiplex, helping to ensure that full profiles can be obtained with degraded DNA samples. The developmental validation studies demonstrated that the STRtyper-27 system generates minimal cross-reactivity, high quality, precise, accurate, and low level sensitive profiling STR data, even from a wide variety of forensic biological samples with sub-nanogram amounts of genomic DNA. The STRtyper-27 system provides an equivalent overall performance to the latest forensic 6-dye Yfiler Plus kit, but with significant compatibility in terms of existing instrument platforms and software that would be cost efficient especially for the local, provincial, and developing national ones.