Selection of markers and primer design
The new multiplex panel includes the eleven markers DYS448, DYS456, DYS458, DYS635, Y-GATA H4, DYS576, DYS481, DYS549, DYS533, DYS570 and DYS643, which are contained in the PPY23 but absent in the PPY. Additionally, the loci DYS385a/b was included in the multiplex reaction as a traceability marker, as it is present in the aforementioned commercial kits.
Since the 12 Y-STR multiplex panel is an extension of the earlier one developed by Nuñez et al. (2017), for the eight markers that were already contained in that panel (DYS456, DYS576, DYS481, DYS549, DYS533, DYS570, DYS643 and DYS385a/b) the same primers described were used 15. For the remaining four Y-STR loci analyzed (DYS448, DYS458, DYS635 and Y-GATA H4), the primer design was performed using PerlPrimer v1.1.21 24. Potential primer cross-reactions were examined via this software, and the Y-chromosome specifity was evaluated using BLASTN aligment tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Forward primers were labeled with a fluorescent dye at the 5’ end (Supplementary Table S1).
Singleplex PCR reaction
Each primer pair was initially tested in a singleplex PCR reaction using the 2800M control DNA (Promega Corporation, Madison, WI). The singleplex reaction consisted of 12.5 µl of QIAGEN Multiplex PCR kit (Qiagen, Valencia, CA), 1 µl of primer (forward and reverse with an initial concentration of 10 µM), 1 ng of genomic DNA and 10.5 µl of Milli-Q water for a final reaction volume of 25 µl. PCR was performed in a GeneAmp 9800 (AB/LT/TFS) under the following cycle conditions: initial denaturation at 95ºC for 15 min was followed by 30 cycles of 94ºC for 30 s, 65ºC for 90 s, and 72ºC for 90 s, and a final extension at 72ºC for 10 min. Evaluation of amplification performance was conducted by electrophoresis on 1.50% agarose gels and visualized with GelRed (3.00% µL/ml) (Biotium Inc.,Hayward, USA) and UV light (UVItec Cambridge, USA or UK). PCR products were purified using 2 µl EXOSAP (0.5 µl EXO (Exonuclease I) and 2.5 µl SAP (Shrimp Alkaline Phosphatase) (Takara Bio Inc., Japan) to 5 µl of PCR product and sequencing was performed to confirm the specific amplification of each Y-STR. Sequencing was performed using the BigDye® TerminatorTM v1.1 Cycle Sequencing Kit (AB/LT/TFS: Applied Biosystems™, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA). Sequencing products were purified with BigDye® XTerminatorTM Purification Kit (AB/LT/TFS) and capillary electrophoresis was done on a 3130 Genetic Analyzer (AB/LT/TFS). Sequencing results were analyzed with the Sequencing Analysis software v5.2 (AB/LT/TFS).
Multiplex PCR reaction and capillary electrophoresis
The 12 Y-STR loci under study were typed using multiplex reactions containing 5 µl of QIAGEN Multiplex PCR kit (Qiagen, Valencia, CA), 0.8 µl of primermix (primer concentrations in the mix are described in Supplementary Table S1),1 ng of genomic DNA and Milli-Q water for a final reaction volumen of 10µl. PCR was performed in a GeneAmp 9800 (AB/LT/TFS) using the same foregoing cycle conditions. PCR products separation and detection were performed by capillary electrophoresis with an ABI3130 Genetic Analyzer (AB/LT/TFS) using the GeneScan 500 LIZ (AB/LT/TFS) as an internal size standard and fragment lengths were assessed with GeneMapper v4.0 (AB/LT/TFS). The analytical threshold was set at 50 RFUs for peak height minimum. Allelic nomenclature follows the recommendations of the International Society for Forensic Genetics (ISFG) (www.isfg.org).
To compare allele designation concordance of the new assay, a set of 50 male DNA samples from the resident population living in the Basque Country previously analyzed with PPY23 in our laboratory, was examined with the novel multiplex panel. These two assays have in common the 12 Y-STR markers included in the new multiplex.
Sensivity and stability studies
In order to evaluate the minimum amount of DNA required to obtain a complete Y-STR profile, 2800M DNA control (Promega, Madison, WI) was used for amplification in triplicates in ascending quantities: 25 pg/µl, 50 pg/µl, 100 pg/µl, 200 pg/µl, 400 pg/µl, 1 ng/µl, 1.6 ng/µl, and 10 ng/µl.
Stability studies were conducted by including different concentrations of two common inhibitors, haematin (Sigma-Aldrich Corporation, St. Louis, MO, USA) and humic acid (Sigma-Aldrich Corporation, St. Louis, MO, USA) to the amplification reaction mix containing 1 ng of 2800M DNA. This study was performed in duplicate using ascending concentrations of the PCR inhibitors: 100 µM, 150 µM, 300 µM, 500 µM, 750 µM, 1000 µM, 1500 µM, 3000 µM, and 5000 µM of haematin and 25 ng/µl, 50 ng/µl, 100 ng/µl, 200 ng/µl, 250 ng/µl, 300 ng/µl, 500 ng/µl, 1000 ng/µl, 2000 ng/µl, and 3000 ng/µl of humic acid.
Artificially degraded DNA samples were prepared to evaluate stability of the Y-STR panel. 1 µg of a DNA in-house control sample was digested with 1 µL of DNAse Reaction Buffer, 0.5 µL of DNAse I and Milli-Q water for a final reaction volumen of 10µl (DNase I, RNase-free, ThermoFisher Scientific, Waltham, MA, USA) for 15 min, 30 min, 1h, 2h, 4h and 16h at 37ºC. To inactivate DNAse I, 1 µL of 50 mM EDTA was added and then incubated at 65ºC for 10 min. These samples were amplified in duplicate.
To analyze the species specificity, DNA samples from bull, goat, sheep, pig, cock, rabbit, dog, cat and mouse were tested. All samples were provided by the Bank of DNA of the BIOMICs Group at the University of the Basque Country (UPV/EHU). Of each animal species, 1 ng of DNA was used for each amplification reaction.
Repeatability and reproducibility
The parameter of repeatability was evaluated by analysing whether the peaks of different replicas were always located in the position where the allele of the corresponding STRs should appear.
Reproducibility was checked by performing the entire analysis by three different operators and by running amplification PCRs on three different thermal cyclers: GeneAmp PCR System 9700 Gold (AB/LT/TFS), GeneAmp 9800 PCR System (AB/LT/TFS) and C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). Afterwards, the electropherograms resulting from these diverse analyses were compared to test the reproducibility of the results.
The capacity of our panel to detect an admixed contribution was tested with a male:male and male:female mixtures. Male:male DNA mixtures were prepared using 2800M DNA control and DNA in-house control simple with the following ratios of 19:1, 9:1, 3:1, 1:1, 1:3, 1:9 and 1:19. To perform the multiplex amplification reaction the total amount of mixed DNA input was mantained at 1 ng. Male:female DNA mixtures were established using a constant amount of 30 ng K562 DNA control (Promega) with a decreasing template of 2800M DNA control (1 ng, 400 pg, 200 pg, 100 pg, 50 pg and 25 pg). Each mixture analysis was repeated for two times.
Analysis of casework-type samples
To test the effectiveness of this panel with forensic-type samples, two samples from skeletal remains (bones) with degraded DNA and three casework samples from intercomparison exercises organized by the Spanish and Portuguese–Speaking Group (GHEP) of the International Society for Forensic Genetics (ISFG), were analyzed. Samples from skeletal remains (coded in this study as BADN1304 and BADN2049) belong to Spanish Civil War (1936–1939). These samples were selected as examples of forensic samples routinely analysed in our laboratory. The samples from GHEP were items M4 and M8 from exercise 2018 (coded in this study as M4-GHEP18 and M8-GHEP18) and item M4 from exercise 2020 (coded in this study as M4-GHEP20).
Population study and forensic parameters
A sample of 84 unrelated males from Ibiza (Balearic Islands, Spain), previously genotyped with the 12 Y-STR PPY 25 was combined with the new Y-STRs in order to assess the forensic parameters of the resulting 23 Y-STR haplotypes.
All donors gave their informed consent prior to inclusion in the study, following the ethical principles and guidelines of the Declaration of Helsinki for the protection of human subjects. Blood samples were collected under the approval of the the Danish local ethical committee (KF-01-037/03) and the Direcció General de R + D + I (Government of Balearic Islands, Spain) (procedure AAEE 12099/2003). DNA was extracted by using QIAamp spin columns (Qiagen, Hilden, Germany) following the manufacturer’s recommendations.
Allele frequencies, single-marker genetic diversity (GD), haplotype diversity (HD), as well as different, unique and population specific haplotypes, were assessed using the software Arlequin v18.104.22.168 26. For these calculations, the DYS385a/b alleles were treated as haplotypes. The global discrimination capacity (DC) was calculated by dividing the number of different haplotypes by the total number of individuals in the population sample.