Screening of highly discriminative microhaplotype markers for individual identification and mixture deconvolution in East Asian populations

Microhaplotypes are forensic genetic markers that combine single nucleotide polymorphisms in close proximity to one another. Highly discriminative microhaplotype markers could be superior to short tandem repeats (STRs) in DNA mixture deconvolution investigations because they are not interfered by stutters. In this study, the effective number of alleles (A_e) and discrimination power values of microhaplotypes and STRs were compared. It was found that current microhaplotypes are not as discriminative as commonly used forensic STRs. Effective screening of highly discriminative microhaplotype markers were consequently conducted for East Asian populations. To satisfy different forensic application needs, four sets of microhaplotypes with A_e values ≥ 4 were screened for under different conditions that included marker length and physical distances between markers. While the four sets contained 703, 301, 337, and 190 microhaplotypes, their average A_e values reached 5.38, 6.30, 7.39, and 5.61, respectively. The microhaplotype group containing 301 markers (maximum length of 200 bp and separated by ≥ 5 million bases) was further investigated. The results showed that none of the 301 loci were exactly the same as those previously reported, while seven loci partially overlapped with known markers. While A_e values of 45 loci were ≥ 8, the A_e value of the mh17WL-008 locus reached a maximum of 93.57. Further analysis showed that the newly identified microhaplotype markers were also highly polymorphic in African, American, European, and South Asian populations.     

Evaluating the effects of whole genome amplification strategies for amplifying trace DNA using capillary electrophoresis and massive parallel sequencing

To draw robust conclusions when trace DNA samples are detected in complex cases, it is essential to successfully recover and genotype short tandem repeats (STRs) from trace DNA. However, obtaining complete STR profiles by the conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) method is generally difficult as trace DNA is often less than 100 pg. Previous studies have proven that through whole-genome amplification (WGA), the yield of DNA from trace DNA samples could be improved. In this study, we used two WGA kits, namely, REPLI-g® Single Cell kit and MALBAC® Single Cell DNA Quick-Amp Kit (hereafter referred to as REPLI and MALBAC), to amplify DNA samples with a series of dilutions (from 5.00 ng/μL to 0.391 pg/μL). Typing of STR markers in samples with and without WGA were then performed on a CE platform by the application of Goldeneye® DNA ID System 20 A kit, as well as directly calling sequences from massive parallel sequencing (MPS) for WGA samples with 1.00 ng, 125 pg and 25.0 pg as DNA inputs. Quantification results demonstrated that the yield of samples with WGA could reach the microgram level. The amplification fold was at least > 2000 and > 200 for REPLI and MALBAC, respectively. CE results showed that the number of correctly called loci was improved for trace DNA after WGA when the DNA inputs were lower than 25.0 pg for REPLI and 6.25 pg for MALBAC, respectively. WGA remarkably improved the percentage of called loci with DNA inputs lower than 50.0 pg, although poor performance in repeatability was observed. MPS results suggested that the correctly called loci calculated by MPS reads were mostly more than those calculated by CE, particularly for those of short length, implying MPS of samples after WGA is worth testing in the future. In conclusion, WGA has the potential usability for forensic trace DNA analysis at the single-cell level with good fidelity, although its repeatability requires further improvement.     

Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™

Next generation sequencing (NGS) offers the opportunity to analyse forensic DNA samples and obtain massively parallel coverage of targeted short sequences with the variants they carry. We evaluated the levels of sequence coverage, genotyping precision, sensitivity and mixed DNA patterns of a prototype version of the first commercial forensic NGS kit: the HID-Ion AmpliSeq™ Identity Panel with 169-markers designed for the Ion PGM™ system. Evaluations were made between three laboratories following closely matched Ion PGM™ protocols and a simple validation framework of shared DNA controls. The sequence coverage obtained was extensive for the bulk of SNPs targeted by the HID-Ion AmpliSeq™ Identity Panel. Sensitivity studies showed 90–95% of SNP genotypes could be obtained from 25 to 100 pg of input DNA. Genotyping concordance tests included Coriell cell-line control DNA analyses checked against whole-genome sequencing data from 1000 Genomes and Complete Genomics, indicating a very high concordance rate of 99.8%. Discordant genotypes detected in rs1979255, rs1004357, rs938283, rs2032597 and rs2399332 indicate these loci should be excluded from the panel. Therefore, the HID-Ion AmpliSeq™ Identity Panel and Ion PGM™ system provide a sensitive and accurate forensic SNP genotyping assay. However, low-level DNA produced much more varied sequence coverage and in forensic use the Ion PGM™ system will require careful calibration of the total samples loaded per chip to preserve the genotyping reliability seen in routine forensic DNA. Furthermore, assessments of mixed DNA indicate the user’s control of sequence analysis parameter settings is necessary to ensure mixtures are detected robustly. Given the sensitivity of Ion PGM™, this aspect of forensic genotyping requires further optimisation before massively parallel sequencing is applied to routine casework.     

Forensic nanopore sequencing of microhaplotype markers using QitanTech’s QNome

In recent years, extraordinary progress has been made in genome sequencing technologies, which has led to a decrease in cost and an increase in the diversity of sequenced genomes. Nanopore sequencing is one of the latest genome sequencing technologies. It aims to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions, and provides a new approach for forensic genetics to detect longer markers in real time. To date, multiple studies have been conducted to sequence forensic markers using MinION from Oxford Nanopore Technologies (ONT), and the results indicate that nanopore sequencing holds promise for forensic applications. Qitan Technology (QitanTech) recently launched its first commercial nanopore genome sequencer, QNome. It could achieve a read length of more than 150 kbp, and could generate approximately 500 Mb of data in 8 h. In this pilot study, we explored and validated this alternative nanopore sequencing device for microhaplotype (MH) profiling using a custom set of 15 MH loci. Seventy single-contributor samples were divided into 7 batches, each of which included 10 samples and control DNA 9947A and was sequenced by QNome. MH genotypes generated from QNome were compared to those from Ion Torrent sequencing (Ion S5XL system) to evaluate the accuracy and stability. Twelve samples randomly selected from the last three batches and Control DNA 9947A were also subjected to ONT MinION sequencing (with R9.4 flow cell) for parallel comparison. Based on MHtyper, a bioinformatics workflow developed for automated MH designation, all MH loci can be genotyped and reliably phased using the QNome data, with an overall accuracy of 99.83% (4 errors among 2310 genotypes). Three occurred near or in the region of homopolymer sequences, and one existed within 50 bp of the start of the sequencing reaction. In the last 15 samples (12 individual samples and 3 replicates of control DNA 9947A), two SNPs located at 4-mer homopolymers failed to obtain reliable genotypes on the MinION data. This study shows the potential of state-of-the-art nanopore sequencing methods to analyze forensic MH markers. Given the rapid pace of change, sporadic and nonrepetitive errors presented in this study are expected to be resolved by further developments of nanopore technologies and analysis tools.     

Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics

SNPs that are molecularly very close (<10 kb) will generally have extremely low recombination rates, much less than 10⁻⁴. Multiple haplotypes will often exist because of the history of the origins of the variants at the different sites, rare recombinants, and the vagaries of random genetic drift and/or selection. Such multiallelic haplotype loci are potentially important in forensic work for individual identification, for defining ancestry, and for identifying familial relationships. The new DNA sequencing capabilities currently available make possible continuous runs of a few hundred base pairs so that we can now determine the allelic combination of multiple SNPs on each chromosome of an individual, i.e., the phase, for multiple SNPs within a small segment of DNA. Therefore, we have begun to identify regions, encompassing two to four SNPs with an extent of <200 bp that define multiallelic haplotype loci. We have identified candidate regions and have collected pilot data on many candidate microhaplotype loci. Here we present 31 microhaplotype loci that have at least three alleles, have high heterozygosity, are globally informative, and are statistically independent at the population level. This study of microhaplotype loci (microhaps) provides proof of principle that such markers exist and validates their usefulness for ancestry inference, lineage–clan–family inference, and individual identification. The true value of microhaplotypes will come with sequencing methods that can establish alleles unambiguously, including disentangling of mixtures, because a single sequencing run on a single strand of DNA will encompass all of the SNPs.     

A forensic DNA profiling system for Northern European brown bears (Ursus arctos)

A set of 13 dinucleotide STR loci (G1A, G10B, G1D, G10L, MU05, MU09, MU10, MU15, MU23, MU26, MU50, MU51, MU59) were selected as candidate markers for a DNA forensic profiling system for Northern European brown bear (Ursus arctos). We present results from validation of the markers with respect to their sensitivity, species specificity and performance (precision, heterozygote balance and stutter ratios). All STRs were amplified with 0.6 ng template input, and there were no false bear genotypes in the cross-species amplification tests. The validation experiments showed that stutter ratios and heterozygote balance was more pronounced than in the tetranucleotide loci used in human forensics. The elevated ratios of stutter and heterozygote balance at the loci validated indicate that these dinucleotide STRs are not well suited for interpretation of individual genotypes in mixtures. Based on the results from the experimental validations we discuss the challenges related to genotyping dinucleotide STRs in single source samples. Sequence studies of common alleles showed that, in general, the size variation of alleles corresponded with the variation in number of repeats. The samples characterized by sequence analysis may serve as standard DNA samples for inter laboratory calibration. A total of 479 individuals from eight Northern European brown bear populations were analyzed in the 13 candidate STRs. Locus MU26 was excluded as a putative forensic marker after revealing large deviations from expected heterozygosity likely to be caused by null-alleles at this locus. The remaining STRs did not reveal significant deviations from Hardy–Weinberg equilibrium expectations except for loci G10B and MU10 that showed significant deviations in one population each, respectively. There were 9 pairwise locus comparisons that showed significant deviation from linkage equilibrium in one or two out of the eight populations. Substantial genetic differentiation was detected in some of the pairwise population comparisons and the average estimate of population substructure (F_ST) was 0.09. The average estimate of inbreeding (F_IS) was 0.005. Accounting for population substructure and inbreeding the total average probability of identity in each of the eight populations was lower than 1.1 × 10^?9 and the total average probability of sibling identity was lower than 1.3 × 10^?4. The magnitude of these measurements indicates that if applying these twelve STRs in a DNA profiling system this would provide individual specific evidence.     

A mixture detection method based on separate amplification using primer specific alleles of INDELs-a study based on two person's DNA mixture

Samples containing unbalanced DNA mixtures from individuals often occur in forensic DNA examination and clinical detection. Because of the PCR amplification bias, the minor contributor DNA is often masked by the major contributor DNA when using traditional STR or SNP typing techniques. Here we propose a method based in allele-specific Insertion/Deletion (INDEL) genotyping to detect DNA mixtures in forensic samples. Fourteen INDELs were surveyed in the Chinese Han population of Shanxi Province. The INDELs were amplified using two separate primer-specific reactions by real-time PCR. The difference Ct value of the 2 reactions (D-value) were used for determination of the single source DNA. INDELs types and further confirmed by electrophoresis separation. The minor allele frequency (MAF) was above 0.2 in 10 INDELs. The detection limit was 0.3125 ng–1.25 ng template DNA for real-time PCR in all 14 INDEL markers. For single source 10 ng DNA, the average D-value was 0.31 ± 0.14 for LS type, 6.96 ± 1.05 for LL type and 7.20 ± 1.09 for SS type. For the series of simulated DNA mixture, the Ct value varied between the ranges of single source DNA, depending on their INDEL typing and mixture ratios. This method can detect the specific allele of the minor DNA contributor as little as 1:50 in rs397782455 and rs397696936; 1:100 in rs397832665, rs397822382 and rs397897230; the detection limit of the minor DNA contributor was as little as 1:500–1:1000 in the rest INDEL markers, a much higher sensitivity compared with traditional STR typing. The D-value variation depended on the alternation of dilution ratio and INDEL types. When the dilution was 1:1000, the maximum and minimum D-values were 8.84 ± 0.11 in rs397897230 and 4.27 ± 0.19 in rs397897239 for LL and SS type mixture, the maximum and minimum D-values were 9.32 ± 0.54 in rs397897230 and 4.38 ± 0.26 in rs 397897239 for LL(SS) and LS type mixture, separately. Any D-value between 0.86 and 5.11 in the 14 INDELs indicated the presence of mixture. The separate amplification strategy based on real-time PCR provides a promising and convenient method for detection of unbalanced DNA mixture for Chinese Han population.     

The Qiagen Investigator® Quantiplex HYres as an alternative kit for DNA quantification

The Investigator^® Quantiplex HYres kit was evaluated as a potential replacement for dual DNA quantification of casework samples. This kit was determined to be highly sensitive with a limit of quantification and limit of detection of 0.0049 ng/μL and 0.0003 ng/μL, respectively, for both human and male DNA, using full or half reaction volumes. It was also accurate in assessing the amount of male DNA present in 96 mock and actual casework male:female mixtures (various ratios) processed in this exercise. The close correlation between the male/human DNA ratios expressed in percentages derived from the Investigator^® Quantiplex HYres quantification results and the male DNA proportion calculated in mixed AmpFlSTR^® Profiler^® Plus or AmpFlSTR^® Identifiler^® Plus profiles, using the Amelogenin Y peak and STR loci, allowed guidelines to be developed to facilitate decisions regarding when to submit samples to Y-STR rather than autosomal STR profiling. The internal control (IC) target was shown to be more sensitive to inhibitors compared to the human and male DNA targets included in the Investigator^® Quantiplex HYres kit serving as a good quality assessor of DNA extracts. The new kit met our criteria of enhanced sensitivity, accuracy, consistency, reliability and robustness for casework DNA quantification.     

An integrated system of ABO typing and multiplex STR testing for forensic DNA analysis

A new amplification system for ABO and STR genotyping in a single reaction has been successfully developed. Two types of information can be obtained from a biological sample at one time. One is the classical information of ABO blood group typing for screening suspects and the other is STR information for individual identification. The system allows for the simultaneous detection of 15 autosomal STR loci (containing all CODIS STR loci as well as Penta D and Penta E), six ABO genotypes (O/O, B/B, A/A, A/O, A/B, and B/O) and the gender-determining locus Amelogenin. Primers are designed so that the amplicons are distributed ranging from 75 bp to 500 bp within a four-dye fluorescent design, leaving a fourth dye for the internal size standard. With 30 cycles, the results showed that the optimal amount of DNA template for this multiplex ranges from 250 pg to 2 ng and the lowest detection threshold is 125 pg (as low as 63pg for ABO loci). For the DNA template outside the optimal detection range, we could adjust the number of cycles to obtain the robust profiles. Mixture studies showed that over 83% of minor alleles were detected at 1:9 ratios. The full profiles were still observed when 4 ng of degraded DNA was digested by DNase I and 1 ng undegraded DNA was added to 40 μM haematin. Polymerase chain reaction (PCR)-based conditions including the concentrations of primers, magnesium and the Taq polymerase as well as volume, cycle numbers and annealing temperature were examined and optimised. In addition, the system was validated by 364 bloodstain samples and 32 common casework samples. According to the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, our system demonstrates good detection performance and is an ideal tool for forensic DNA typing with potential application.     

Forensic molecular biomarkers for mixture analysis

The deconvolution of DNA mixtures has gathered the attention of forensic DNA scientists for over two decades. To enhance mixture deconvolution capabilities, a new generation of sensitive DNA-typing approaches has been recently proposed. In this review, we describe novel, forensically relevant multi-SNP loci (i.e., microhaplotypes or microhaps), compound markers (i.e., DIP-STRs, SNP-STRs and DIP-SNPs) and lineage markers (i.e., rapidly mutating Y chromosome STRs) that improve the deconvolution of two and more than two-person mixtures typed using conventional STR, binary and non-binary loci. We explore the features and applications of these emerging molecular biomarkers with respect to their ability to forensically detect same-or-opposite sex donors. Finally, we discuss the impact of initial massively parallel sequencing (MPS) investigations of STR, microhaplotype and SNP/indel assays for DNA mixture profiling.     

Genotyping polymorphic microhaplotype markers through the Illumina® MiSeq platform for forensics

Microhaplotype markers are emerging forensic genetic markers that have received broad attention in forensics and may supplement existing genetic marker panels. Short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs) are the general genetic markers at present. Stutter and the high mutation rate of STR markers and the low polymorphism of SNP markers obstruct the solving of certain cases. Kidd proposed microhaplotype markers that encompass 2–4 SNPs. In this study, we screened microhaplotype loci through three criteria, and chose the Illumina^® MiSeq platform to sequence the new markers. A new nomenclature was proposed and Perl-based tool FLfinder was designed to genotype the microhaplotype marker. After counting the number of haplotypes in samples that were sequenced and calculating common forensic parameters, 13 loci with high polymorphism were reported. Twelve of the 13 loci had an average allele coverage ratio (ACR) of 0.72 to 0.92. Structure analysis showed that 2504 samples (1000 genome project) could be divided into 5 groupings of populations, and each one representing a continental origin. The finding indicates that microhaplotype markers could be used for individual identification and ancestry inference, and a new choice is provided for forensic practice in the future.     

Typing of semen-containing mixtures using ARMS-based semen-specific CpG-InDel/STR markers

Mixed traces are common biological materials found at crime scenes, and their identification remains a significant challenge in the field of forensic genetics. In recent years, DNA methylation has been considered as a promising approach for body fluid identification, and length polymorphic loci are still the preferred markers for personal identification. In this study, we used tissue-specific CpG sites with linked insertion or deletion (InDel) or short tandem repeat (STR) markers (CpG-InDel/STR) for both body fluid and individual identification. The tissue-specific CpG loci, which were all selected from the previous reports, were analyzed using a combination of bisulfite conversion and amplification refractory mutation system-multiprimer-PCR technology. InDels or STRs, which were selected within 400 bp upstream or downstream of the semen-specific CpG loci, were analyzed using a capillary electrophoresis platform. Eventually, we successfully constructed a panel containing 17 semen-specific CpG-InDel/STR compound markers compassing 21 InDels/STRs, 3 body-fluid positive controls (vaginal secretion-, saliva-, and blood-specific CpG), and 1 gender identification locus. Using this panel, full genotyping of individuals could be obtained successfully with 50 ng DNA input. Semen stains stored at room temperature for 7 months and degraded samples that were heat treated for up to 6 h were still identified efficiently. For semen containing mixed stains, it is also useful when the semen content is as low as 3.03%. Moreover, the cumulative discrimination power of this panel is 0.9999998. In conclusion, it is a robust panel enabling the validation of both the tissue source and individual identification of semen containing mixed stains and can be employed as an alternative solution for forensic case investigation.

     

Efficiency analysis of VersaPlex™ 27PY system in Central Indian Population: First report from Indian population

In the current scenario, DNA typing is the need of forensic science field due to its ability to provide results in much shorter time. In view of advancement of forensic DNA typing and incensement in the number of STRs markers, Promega offered a new VersaPlex™ 27PY system with 27 loci (23 autosomal STR loci, Amelogenin, DYS391 and two rapidly mutating Y-STR loci (DYS570 and DYS576)). In this study, the efficacy of “23 autosomal STR loci” for paternity testing and personal identification was demonstrated in Indian population. For this, 217 central Indians were tested and all the statistical parameters of forensic and population genetic interest were calculated. In addition, sensitivity of the kit was also tested for forensic casework. During investigation with VersaPlex™ 27PY system, allele 11 at locus TPOX was observed to be most frequent with the highest allelic frequency 0.432. Studied 23 loci showed valuable together with highest value of combined power of discrimination (CPD = 1), combined power of exclusion (CPI = 0.9999999989) and lowest value of combined matching probability (CPM = 7.92x10^-28).     

Developmental validation of a forensic rapid DNA-STR kit: Expressmarker 16

DNA-STR analysis is widely used in the forensic science field and has important requirements on the analysis time to obtain faster inspections. The developed forensic STR kit, referred to as Expressmarker 16 (EX16), could shorten the amplification time to a minimum of 35 min. It enables 16 STR loci to be co-detected, including 13 CODIS loci, D2S1338, D6S1043 and Amelogenin loci. The kit is validated by a series of tests formed by DNA mixtures, stutter ratios, optimized PCR protocols based on annealing temperature research, species specificities, inhibitors, sensitivity, and parallel tests according to FBI QAS (2009/2011) (QAS, 2009; SWGDAM, 2010). The results demonstrated that EX16 was a useful tool for rapid criminal investigation.     

Development of specific and rapid detection of human DNA by recombinase polymerase amplification assay for forensic analysis

The determination of human-derived samples is very important in forensic investigations and case investigation in order to determine vital information on the suspect and the case. In this study, we established a recombinase polymerase amplification (RPA) assay for rapid identification of human-derived components. The sensitivity of the assay was 0.003125 ng, with excellent species specificity, and human-derived DNA could be detected in the presence of non-human-derived components at a ratio of 1:1000. Moreover, the RPA assay had a strong tolerance to inhibitors, in the presence of 800 ng/μL humic acid, 400 ng/μL tannic acid, and 8000 ng/μL collagen. In forensic investigation, common body fluids (blood, saliva, semen, vaginal secretions) are all applicable, and the presence of DNA can be detected from samples after simple alkaline lysis, which greatly shortens the detection time. Four simulation and case samples (aged bones, aged bloodstains, hair, touch DNA) were also successfully applied. The above research results show that the RPA assay constructed in this study can be fully applied to forensic medicine to provide high sensitivity and applicability detection methods.     

Development and validation of a new STR 25-plex typing system

In this study, a new STR 25-plex typing system, including 23 autosomal STRs (D1S1656, D2S1338, D2S441, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, vWA) and a Y-STR locus of DYS391 and amelogenin, was developed. The included 24 STRs belonged to the main international DNA databases (CODIS, ISSL, ESS-extended, UCL, GCL and NCIDD) except D6S1043 (specially chosen for Chinese population). Developmental validation indicated that the STR 25-plex typing system was reproducible, accurate, sensitive and robust. The sensitivity testing of the system was such that a full profile was obtainable even with 125 pg of human DNA. Specificity testing was demonstrated by the lack of cross-reactivity with a variety of commonly encountered animal species and microbial pool. For the stability testing, full profiles can been obtained with humic acid concentration ≤ 60 ng/μL and hematin < 500 μM. Also, this multiplex system is suitable for mixture study. All of the minor alleles were called for ratios of 1:1, 1:3 and 3:1 of the mixture with the system. In addition, the whole PCR amplification can finish within 1 h, making the system suitable for fast-detection. For the forensic evaluation of the multiplex system, 23 autosomal STRs included followed the Hardy–Weinberg equilibrium. A total of 268 alleles were detected for the 23 autosomal STR loci among 200 individuals. Since 23 autosomal STRs were independent from each other, CMEC_duo was 0.99999916563607 and CMEC_tri was 0.99999999986525. All the forensic efficiency parameters demonstrated that this multiplex system is highly polymorphic and informative in the Han population of China.     

Exploring of new Y-chromosome SNP loci using Pyrosequencing and the SNaPshot methods

The single nucleotide polymorphisms on the Y chromosome (Y-SNP) have been considered to be important in forensic casework. However, Y-SNP loci were mostly population specific and lacked biallelic polymorphisms in the Asian population. In this study, we developed a strategy for seeking and genotyping new Y-SNP markers based on both Pyrosequencing and the SNaPshot methods. As results, 34 new biallelic markers were observed to be polymorphic in the Chinese Han population by estimation of allele frequencies of 103 candidate’s Y-SNP loci in DNA pools using Pyrosequencing technology. Then, a multiplex system with 20 Y-SNP loci was genotyped using the SNaPshot? multiplex kit. Twenty Y-SNP loci defined 56 different haplotypes, and the haplotype diversity was estimated to be 0.9539. Our result demonstrated that the strategy could be used as an efficient tool to search and genotype biallelic markers from a large amount of candidate loci. In addition, 20 Y-SNP loci constructed a multiplex system, which could provide supplementary information for forensic identification.     

Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens

Though the utility of next-generation sequencing (NGS) technologies for forensic short tandem repeat (STR) typing has been evident for several years, commercially available assays and software solutions developed specifically to meet forensic needs have only recently become available. One of these, the ForenSeq™ DNA Signature Prep Kit (Illumina, Inc.) sequences 27 autosomal STR (aSTR) and 24 Y chromosome STR (Y-STR) loci (concurrent with additional nuclear markers) per multiplexed sample, with automated secondary and tertiary analyses of the data accomplished via the associated ForenSeq™ Universal Analysis Software (UAS). In this study we investigated the performance of the ForenSeq system for aSTR and Y-STR typing by examination of 151 sample libraries developed from high quality DNAs amplified at the target 1 ng template. Utilizing PCR Primer Mix B, greater than 99.5% of aSTR loci and 97.0% of Y-STR loci were recovered when 42 or fewer sample libraries were pooled for sequencing. A direct comparison of UAS developed fragment length results to capillary electrophoresis (CE) based data identified only two allele call discrepancies when no UAS quality flag was triggered. Review of the ForenSeq data indicated that most samples with total sequence read counts exceeding 40,000 could be interpreted to develop nearly complete aSTR genotypes or Y-STR haplotypes. However, markers D22S1045 and DYS392 produced poor or inconsistent results even when sample read counts were greater than 85,000. Excluding these two loci, analyst-interpreted aSTR and Y-STR ForenSeq profiles were 99.96% and 100% concordant, respectively, with CE data. In addition to demonstrating concordance on par with other CE kit to kit comparisons, the results from this study will assist laboratories seeking to develop workflows for high volume processing and analysis of aSTRs and Y-STRs from reference-type specimens using the ForenSeq system.     

Assessment of application value of 19 autosomal short tandem repeat loci of GoldenEyeTM 20A kit in forensic paternity testing

This study was carried out to assess the application value of 19 autosomal short tandem repeat (STR) loci of GoldenEye^TM 20A kit, in which 13 combined DNA index system core STR loci and PentaE, PentaD, D2S1338, D19S433, D12S391, and D6S1043 of six STR loci could be used in forensic paternity testing in Chinese population. We amplified the genomic DNA from blood samples on FTA paper of 289 paternity testing cases by using the GoldenEye^TM 20A kit. The amplified products were detected by capillary electrophoresis, and then the genotypes of 20 genetic markers including 19 STR loci as well as Amelogenin for sex determination were analyzed by GeneMapper v3.2 and GeneMarker HID Software. The results of genotypes were compared to the three commonly used commercial kits including AmpF?STR Identifiler^TM, PowerPlex^TM16, and AmpF?STR Sinofiler^TM kits. Compared to the three other common commercial kits, the GoldenEye^TM 20A kit had higher value of combined paternity index in certainty of paternity or non-exclusion paternity cases, and more numbers of STR loci were excluded in exclusionary paternity cases. Our data in this study showed that the GoldenEye^TM 20A kit has a higher application value in forensic paternity testing and will be of help for kinship analysis.     

Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids

Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5–1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5–9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10⁻²², and their combined use can provide robust information for Japanese forensics.