Developmental validation of the Microreader™ 20A ID system

The Microreader? 20A ID system is designed for forensic applications such as personal identification, parentage testing, and research. It includes 13 combined DNA index system (CODIS) short tandem repeat (STR) loci (CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11), three expanded CODIS STR loci (D12S391, D19S433, and D2S1338), three non‐CODIS STR loci (D6S1043, Penta D, and Penta E), and the amelogenin locus in one reaction with a six‐dye fluorescent (FAM, HEX, TAMAR, ROX, PUR, and QD550) analysis system. In this study, the Microreader? 20A ID system was validated according to the Scientific Working Group on DNA Analysis Methods validation guidelines for forensic DNA Analysis methods and Chinese national standard, including PCR‐based studies, sensitivity study, precision, and accuracy evaluation, stutter calculation, inhibitor tests, species specificity, and DNA mixture studies. Our results suggest that the Microreader? 20A ID system is a useful tool for personal identification and parentage testing.     

Allele frequency of 19 autosomal STR loci in the Bai population from the southwestern region of mainland China

The aim of this study was to investigate a 19 STR loci database using the Bai population from China. This multiplex amplification kit included 13 CODIS STR markers and six plus STR markers (D19S433, Penta E, D2S1338, Penta D, D6S1043, and D12S391) that were successfully analyzed by using 1158 DNA samples from the Bai population from the southwestern part of mainland China. These results indicate that this multiplex amplification kit may provide significant polymorphic information for kinship testing and relationship investigations.     

Development and validation of a forensic six-dye multiplex assay with 29 STR loci

This paper describes the development and validation of a novel 31-locus, six-dye STR multiplex system, which is designed to meet the needs of the rapidly growing Chinese forensic database. This new assay combines 20 extended-CODIS core loci (D3S1358, D5S818, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D8S1179, D18S51, D16S539, D13S317, FGA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045), nine highly polymorphic loci in Chinese Han population (D3S3045, D6S1043, D6S477, D8S1132, D10S1435, D15S659, D19S253, Penta D, and Penta E), and two gender determining markers, amelogenin and Y-Indel, which could amplify DNA from extracts, as well as direct amplification from substrates. To demonstrate the suitability for forensic applications, this system was validated by precision and accuracy evaluation, concordance tests, case sample tests, sensitivity, species specificity, stability, stutter calculation, and DNA mixtures, according to the guidelines described by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and regulations published by the China Ministry of Public Security. The validation results indicate the robustness and reliability of this new system, and it could be a potentially helpful tool for human identification and paternity testing in the Chinese population, as well as facilitating global forensic DNA data sharing.     

Development and validation of a novel SiFaSTRTM 23‐plex system

Human identification and paternity testing are usually based on the study of STRs depending on their particular characteristics in the forensic investigation. In this paper, we developed a sensitive genotyping system, SiFaSTR^? 23‐plex, which is able to characterize 18 expanded Combined DNA Index System STRs (D3S1358, D5S818, D2S1338, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D10S1248, D8S1179, D1S1656, D18S51, D12S391, D19S433, D16S539, D13S317, and FGA), three highly polymorphic STRs among Chinese people (Penta D, Penta E, and D6S1043), one Y‐chromosome Indel and amelogenin using a multiplex PCR; the PCR amplified products were analyzed using the Applied Biosystems 3500 Genetic Analyzer. Full genotyping profiles were obtained using only 31.25 pg of control DNA; various case‐type specimens, as well as ten‐year‐old samples were also successfully genotyped. Additionally, in the validation studies, this multiplex was demonstrated to be human‐specific and compatible with the interference of multiple PCR inhibitors. The system also enabled the detection of mixtures, and complete profiles could be obtained at the mixed ratios of 1:1, 1:3, and 3:1. The development and validation study here illustrated that the SiFaSTR^? 23‐plex system is accurate, powerful, and more sensitive than many other systems. What's more, the population data in our study not only illustrated that this 23‐plex typing system was straightforward and efficient but also expanded the Chinese STR database, which could facilitate the appropriate application of the 23 genetic markers in forensic genetics, especially in the Chinese population.     

Developmental validation of the Microreader Group Y Direct ID System: A novel six-dye typing system with 54 Y-chromosomal loci for forensic application

Y-chromosome, as a gender-determined biological marker, is inherited only between fathers and sons. The Y-chromosome short tandem repeats (Y-STRs) play an essential role in paternity lineage tracing as well as sexual assault cases. The Microreader Group Y Direct ID System as a six-dye multiplex amplification kit, including 53 Y-STR and one Y-Indel locus, would improve performance and aid in obtaining more information through a greater number of loci with high polymorphism. In the present study, to verify the accuracy and efficiency of the kit, developmental validation was conducted by investigating sensitivity, species specificity, PCR inhibition, male–male and male–female mixtures, and reproducibility. The kit was tested using 311 male samples from Han and Qiang populations in Sichuan Province. The results showed that this kit had fairly high power for forensic discrimination (Han: haplotype diversity [HD] = 1, Qiang: HD = 0.999944). Additionally, 44 confirmed father–son pairs were also genotyped, among which 69 distinct haplotypes could be obtained. These father–son pairs cannot be distinguished by commonly used Y-STR panels, indicating that adding these extra Y-STRs to a single panel can achieve better discrimination performance. Collectively, the Microreader Group Y Direct ID System is robust and informative for forensic applications.     

Genetic polymorphisms of 20 short tandem repeat loci from the Han population in Henan,China

The aim of this study was to investigate the genetic polymorphism of 20 short tandem repeat (STR) loci including D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA in Han population of Henan, China and to assess its value in forensic science. Genomic DNA was extracted from 274 blood samples of unrelated healthy individuals in the Henan Han population. Alleles were amplified with PowerPlex® 21 system kit and PCR products were detected with ABI3130 genetic analyzer (Applied Biosystems) and the data were analyzed with modified PowerStats v1.2. A total of 229 alleles were observed in this Han population and the allelic frequencies ranged from 0.0020 to 0.5090 in the present study. Observed genotype distributions for each locus do not show deviations from Hardy–Weinberg equilibrium expectations (p < 0.05). The combined power of discrimination, combined power of exclusion, and combined matching probability of this 20 STR loci were 0.999999999, 0.999999994603, and 4.0433 × 10^?24, respectively. The 20 STR loci are highly polymorphic in the Han population of Henan, China and they may be of great value in forensic science and human population genetics.     

Analysis of five rare alleles at the STR loci D1S1656, D12S391, D13S317, Penta D,and D2S441

Short tandem repeat (STR) automatic typing technology is extensively used in forensic laboratories with commercial kits, in rare cases genotyping misinterpretations or mislabeling may occur due to unexpected rare alleles. This study refers to the investigation of several rare alleles observed from routine cases. Besides cross-kit verification with Goldeneye 25A (Beijing PeopleSpot Inc, China) and Huaxia platinum (Thermo Fisher Scientific, USA) kits, the next-generation sequencing technology by MiSeq FGx System (Illumina, USA) was applied to further validation. To solve the inconsistent outcomes reached by the above mentioned approaches at D2S441 locus, single gene amplification, gene cloning, and genetic sequencing was also performed. As a result, five rare alleles were detected. Two novel alleles of allele 3 at the D13S317 locus and allele 5 at the D2S441 locus were found; three previously reported alleles of allele 9 at D1S1656 locus, allele 19 at Penta D locus, and allele 28 at D12S391 locus in STRBase were initially supplemented with sequence information. We, therefore, propose that such uncommon observations with rare events should be carefully investigated and interpreted.     

"Paterniplex", a highly discriminative decaplex STR multiplex tailored for investigating special problems in paternity testing

The goal of the study was to develop a STR multiplex ("Paterniplex") that is--as supplement to commercially available multiplex kits like the Identifiler kit (Applied Biosystems, Foster City, CA)--suitable for solving complex paternity cases such as deficiency cases or cases with mutations. The Paterniplex comprises the nine highly polymorphic STRs D8S1132, D7S1517, D10S2325, D12S391, Se33, D17S976, Penta E, Penta D and FGA in addition to Amelogenin as sex determination marker. The loci were selected because of their high degree of polymorphism (higher than that of the widely used TH01 marker). Only one locus, FGA, is shared with the Identifiler kit to avoid sample mix up. The study further gives details on the population genetics of the loci in a German Caucasian population (allelic distribution, Hardy-Weinberg Equilibrium and forensic efficiency markers such as the Discriminating Power) and three examples for cases that could not be solved using commercially available kits alone, but using the Paterniplex in addition to a commercial kit.     

Genetic polymorphism of 13 non‐CODIS STR loci in three national populations from China

The aim of this study was to investigate a 13 non‐CODIS STR loci database using three national populations from China. A new multiplex PCR system that simultaneously amplified 13 loci in the same PCR reaction was developed. This multiplex system included the 13 STR markers (D3S2402, D3S2452, D3S1766, D3S4554, D3S2388, D3S3051, D3S3053, D4S2364, D4S2404, AC001348A, AC001348B, D17S975, and D17S1294), which were successfully analyzed by using 441 DNA samples from three national populations in China (154 Mongol, 177 Kazakh, and 110 Uigur). Allele frequencies and mutation rates of the 13 non‐CODIS STR loci were investigated. A total of 4–10 alleles at each locus were observed and altogether 84, 88, and 87 alleles for the all selected loci were found in the Mongol, Kazakh, and Uigur, respectively. Eight mutations were detected from the 13 selected loci in 9880 meioses in kinship cases. These results indicate that this multiplex system may provide significant polymorphic information for kinship testing and relationship investigations.     

Independent development and validation of a novel six-color fluorescence multiplex panel including 61 diallelic DIPs and 2 miniSTRs for forensic degradation sample

Current forensic DNA profiles are obtained based on analyses of PCR product sizes or DNA sequence polymorphisms. Sometimes routine forensic analysis using short tandem repeat (STR) generates unsuccessful DNA testing result if the biological sample encountered is excessively degraded and low-template DNA. Herein, a new six-color fluorescence labeling system, including 59 autosomal diallelic deletion or insertion polymorphisms (DIPs), 2 miniSTRs, 2 Y-chromosome DIPs, and 1 Amelogenin gene with the amplicon sizes of less than 200 bp, was self-developed. According to the validation guidelines for DNA analysis methods formulated by the Scientific Working Group on DNA Analysis Methods, the validation studies have also been carried out for the multiplex system. This novel panel possessed the features of strong stability, high sensitivity, and good specificity, which was especially suitable for the forensic degraded and mixed sample detections. The cumulative power of exclusion and cumulative matching probability of the system were 0.9999978 and 9.833E-28, respectively, in Han Chinese in Hunan, China. Moreover, this system will be an effective new tool that can be independently applied to forensic personal identification and paternity testing in the populations from the East Asia region, even from the South Asia, America, and Europe regions. The system can also contribute to population phylogenetic affinity and genetic structure analyses among different populations.     

Developmental validation of the AGCU 21+1 STR kit: A novel multiplex assay for forensic application

In this study, we describe the developmental validation assay performed on a novel designed STR multiplex system, AGCU 21+1 STR kit. This kit contains a sex‐determining locus amelogenin and 21 noncombined DNA index system STR loci, that are, D6S474, D12ATA63, D22S1045, D10S1248, D1S1677, D11S4463, D1S1627, D3S4529, D2S441, D6S1017, D4S2408, D19S433, D17S1301, D1GATA113, D18S853, D20S482, D14S1434, D9S1122, D2S1776, D10S1435, and D5S2500. The 21+1 kit was validated by a series of tests including optimized PCR conditions, sensitivity, precision and accuracy, stutter ratio, DNA mixture, inhibitors, and species specificity according to the revised validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Our results in this study show that the kit is a useful tool for forensic application.     

Systematic assessment of the performance of Illumina's MiSeq FGx™ forensic genomics system

This study assesses the performance of Illumina's MiSeq FGx System for forensic genomics by systematically analyzing single source samples, evaluating concordance, sensitivity and repeatability, as well as describing the quality of the reported outcomes. DNA from 16 individuals (9 males/7 females) in nine separate runs showed consistent STR profiles at DNA input ≥400 pg, and two full profiles were obtained with 50 pg DNA input. However, this study revealed that the outcome of a single sample does not merely depend on its DNA input but is also influenced by the total amount of DNA loaded onto the flow cell from all samples. Stutter and sequence or amplification errors can make the identification of true alleles difficult, particularly for heterozygous loci that show allele imbalance. Sequencing of 16 individuals’ STRs revealed genetic variations at 14 loci at frequencies suggesting improvement of mixture deconvolution. The STR loci D1S1656 and DXS10103 were most susceptible to drop outs, and D22S1045 and DYS385a‐b showed heterozygote imbalance.  Most stutters were typed at TH01 and DYS385a‐b, while amplification or sequencing errors were observed mostly at D7S820 and D19S433. Overall, Illumina's MiSeq FGx System produced reliable and repeatable results.  aSTRs showed fewer drop outs than the Y‐ and X‐STRs.     

Short tandem repeat analysis in Japanese population

Short tandem repeats (STRs), known as microsatellites, are one of the most informative genetic markers for characterizing biological materials. Because of the relatively small size of STR alleles (generally 100-350 nucleotides), amplification by polymerase chain reaction (PCR) is relatively easy, affording a high sensitivity of detection. In addition, STR loci can be amplified simultaneously in a multiplex PCR. Thus, substantial information can be obtained in a single analysis with the benefits of using less template DNA, reducing labor, and reducing the contamination. We investigated 14 STR loci in a Japanese population living in Sendai by three multiplex PCR kits, GenePrint PowerPlex 1.1 and 2.2. Fluorescent STR System (Promega, Madison, WI, USA) and AmpF/STR Profiler (Perkin-Elmer, Norwalk, CT, USA). Genomic DNA was extracted using sodium dodecyl sulfate (SDS) proteinase K or Chelex 100 treatment followed by the phenol/chloroform extraction. PCR was performed according to the manufacturer's protocols. Electrophoresis was carried out on an ABI 377 sequencer and the alleles were determined by GeneScan 2.0.2 software (Perkin-Elmer). In 14 STRs loci, statistical parameters indicated a relatively high rate, and no significant deviation from Hardy-Weinberg equilibrium was detected. We apply this STR system to paternity testing and forensic casework, e.g., personal identification in rape cases. This system is an effective tool in the forensic sciences to obtain information on individual identification.     

Differential pre‐amplification of STR loci for fragmented forensic DNA profiling

DNA profiling of short tandem repeats (STR) has been successfully used for the identification of individuals in forensic samples, accidents and natural disasters. However, STR profiling of DNA isolated from old crime scenes and damaged biological samples is difficult due to DNA degradation and fragmentation. Here, we show that pre‐amplification of STR loci using biotinylated primers for the STR loci is an efficient strategy to obtain STR profiling results from fragmented forensic samples. Analysis of STR loci with longer amplicon sizes is generally hampered, since these relatively long loci are vulnerable to DNA fragmentation. This problem was overcome by using reduced or increased primer concentrations for loci with shorter or longer amplicon sizes, respectively, in our pre‐amplification strategy. In addition, pre‐amplification of STR loci into two groups of short or long amplicon size increases the efficiency of STR profiling from highly fragmented forensic DNA samples. Therefore, differential pre‐amplification of STR loci is an effective way to obtain DNA profiling results from fragmented forensic samples.     

Considering the flanking region variants of nonbinary SNP and phenotype-informative SNP to constitute 30 microhaplotype loci for increasing the discriminative ability of forensic applications

The flanking region variants of nonbinary SNPs and phenotype-informative SNPs (piSNPs) have been observed, which may greatly improve the discriminative ability after constituting microhaplotype. In this study, 30 microhaplotype loci based on the nonbinary SNPs and piSNPs (shown to be related to phenotypes such as hair and eye color) were selected. Genotyping were conducted on 100 unrelated northern Han Chinese, and the 26 populations from the 1000 Genome Project were also included for comparison of populations differentiation. The simulated study was conducted for evaluating the efficiency of kinship testing. These 30 microhaplotype loci we selected had good polymorphism, with a mean effective number of alleles (Ae) of 3.46. The average Ae increase was 1.27 compared with the target SNPs. The populations from the five regions worldwide could also be distinguished using these loci. The results of kinship testing showed that these microhaplotype loci had the similar ability as 15 STR loci of AmpFlSTR^R Identifiler^R PCR Amplification Kit to identify the biological parent and a stronger ability to exclude the nonbiological parents. So, these 30 microhaplotype loci may be multifunctional for forensic application, including the ability of personal identification and kinship testing equivalent to 15 STR loci, and the power of ancestry inference for distinguishing the main intercontinental population. Moreover, our selected phenotypic microhaplotype loci may theoretically have phenotype prediction capabilities. But the phenotype prediction efficiency of these phenotypic microhaplotype loci may be worse than that of piSNPs and the detailed prediction accuracy of different populations needs to be further studied.     

A powerful,novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations

A new multiplex system for six tetranucleotide short tandem repeat (STR) loci was devised based on multicolor dye technology. Six loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), each with high discriminating power (each unbiased estimates of expected heterozygosity, Exp. Hz, > 0.80 in a preliminary test), were selected from more than 100 tetranucleotide STRs included in a commercially available primer set. These loci were also selected so as not to link with general STRs in commercially released kits including the combined DNA index system (CODIS) 13 core STRs. The primers were newly designed in the present study, in which each amplicon size had a range of less than 200 base pairs (bp), in order to genotype from highly degraded template DNA. Using this system, we genotyped 270 Honshu (mainland)-Japanese and 187 Okinawa-Japanese. From these allele frequencies, we performed three tests, a homozygosity test, a likelihood ratio test and an exact test for Hardy-Weinberg equilibrium (HWE), and no significant deviations (p < 0.05) from HWE were observed. We also compared the allele distributions at six STRs between both populations, and they were significantly different (p < 0.05) at three loci (D6S2439, D9S1118 and D4S2639). Furthermore, the Exp. Hz and the power of discrimination (PD) at all loci in the Honshu-Japanese population were higher than 0.80 and 0.93, respectively. These statistical values for discriminating power in the Honshu-Japanese were almost the same as in the Okinawa-Japanese. This novel, multiplex polymerase chain reaction (PCR) amplification and typing system for six STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field.     

A modular microfluidic system for deoxyribonucleic acid identification by short tandem repeat analysis

Microfluidic technology has been utilized in the development of a modular system for DNA identification through STR (short tandem repeat) analysis, reducing the total analysis time from the ∼6 h required with conventional approaches to less than 3 h. Results demonstrate the utilization of microfluidic devices for the purification, amplification, separation and detection of 9 loci associated with a commercially-available miniSTR amplification kit commonly used in the forensic community. First, DNA from buccal swabs purified in a microdevice was proven amplifiable for the 9 miniSTR loci via infrared (IR)-mediated PCR (polymerase chain reaction) on a microdevice. Microchip electrophoresis (ME) was then demonstrated as an effective method for the separation and detection of the chip-purified and chip-amplified DNA with results equivalent to those obtained using conventional separation methods on an ABI 310 Genetic Analyzer. The 3-chip system presented here demonstrates development of a modular, microfluidic system for STR analysis, allowing for user-discretion as to how to proceed after each process during the analysis of forensic casework samples.     

A framework for the development of STR genotyping in domestic animal species: Characterization and population study of 12 canine X‐chromosome loci

This study reports the methodology used to search, select and characterize STR loci on the canine X chromosome using publicly available genome resources and following the current guidelines for human and non‐human forensic testing. After several rounds of selection, 12 X‐STR markers were optimized for simultaneous co‐amplification in a single PCR, and genetic profiles were determined in a sample of 103 unrelated dogs. Mendelian inheritance was verified and mutation rates were assessed using family groups. Alleles that varied in size were sequenced to create a standardized nomenclature proposal based on the number of repeats. All loci conformed to Hardy–Weinberg expectations. The resulting panel showed high forensic efficiency, presenting high values of power of discrimination (in males and females) and mean exclusion chance, both in trios involving female offspring and in duos composed of dam and male offspring. Its use may complement the information obtained by autosomal STR analysis and contribute to the resolution of complex cases of kinship in dogs. The presented methodology for the de novo construction of an STR multiplex may also provide a helpful framework for analogous work in other animal species. As an increasing number of reference genomes become available, convenient tools for individual identification and parentage testing based on STR loci selected from autosomes or sex chromosomes' sequences may be created following this strategy.     

Use of multi‐InDels as novel markers to analyze 13 X‐chromosome haplotype loci for forensic purposes

Many studies have been proposed to identify insertion/deletion (InDel) polymorphisms in humans for forensic genetic studies. However, the discriminatory power of InDels is limited by the poor polymorphisms of diallelic markers. To improve their discriminatory power, we developed multi‐InDel, a novel autosomal marker comprising more than two InDel loci that are tightly linked by their physical position and combined into a specific marker by a pair of PCR primers. This strategy gives at least three haplotypes for each multi‐InDel marker. Such markers can be potentially very useful in forensic applications. In this study, we focused on multi‐InDel markers located on X chromosome (ChrX). A multiplex system with 13 multi‐InDel markers, including 28 InDel loci in ChrX, was developed. To validate the multi‐InDel panel, the haplotype distribution in a population sample and in a set of pedigrees was investigated. This study demonstrates usefulness of these markers for individual identification and relationship studies. We highlight the fact that the multi‐InDel markers located on ChrX can provide new supporting information for complex kinship testing.     

Development of a new 25plex STRs typing system for forensic application

We have developed a novel STR 25‐plex florescence multiplex‐STR kit (DNATyper25) to genotype 23 autosomal and two sex‐linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non‐CODIS. The sex‐linked markers include a Y‐STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non‐human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/μL and hematin concentration <400 μM. For forensic evaluation, the 23 autosomal STRs followed the Hardy–Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10^?22, TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.