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.     

A new set of 20 Multi-InDel markers for forensic application

Insertion/deletion markers (InDels) become an important marker for forensic medicine because of their compatible typing techniques with STRs and lower mutation rates. Recent years, a new kind of DNA marker named Multi-InDel was reported as characterized by two or more tightly linked InDel loci within a short length of physical position, usually 200–300 nucleotides. Many pieces of research showed that Multi-InDels had excellent application values in ancestry inference and forensic medicine. Since the identical number of insertion/deletion nucleotides of the InDel markers that composing the Multi-InDel marker, the genotypes of most reported Multi-InDels could not be directly typed by capillary electrophoresis (CE) due to the lack of length discrepancy among the composing InDel sequence. In this study, we applied a typing system of 20 Multi-InDels including 41 InDels, whose genotypes could be deduced by CE and assessed their potential applications in forensic medicine. A total of 200 unrelated Chinese Han individuals and five mother-child-father trios with proven paternity with one STR locus transmission incompatibilities from Shanxi province were genotyped by the multiplex system. The results showed that a total of 70 specific alleles were observed, more than three alleles were observed in 19 loci and seven alleles were observed in one locus. The combined probability of exclusion and the combined power of discrimination were 0.992 and 0.99999999993, respectively. This study demonstrates their potential usefulness for individual identification and paternity tests. The development of Multi-InDels provided another genetic tool inherent in higher polymorphic and lower mutation rates.     

Multiplex pyrosequencing of InDel markers for forensic DNA analysis

The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator^® DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator^® DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof‐of‐principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost‐effective alternative for some applications.     

Application of SNPs with low minor allele frequencies in missing person identification (MPI) through kinship analysis of DNA mixtures

The need to identify a missing person (MP) through kinship analysis of DNA samples found at a crime scene has become increasingly prevalent. DNA samples from MPs can be severely degraded, contain little DNA and mixed with other contributors, which often makes it difficult to apply conventional methods in practice. This study developed a massively parallel sequencing–based panel that contains 1661 single-nucleotide polymorphisms (SNPs) with low minor allele frequencies (MAFs) (averaged at 0.0613) in the Chinese Han population, and the strategy for relationship inference from DNA mixtures comprising different numbers of contributors (NOCs) and of varying allele dropout probabilities. Based on the simulated dataset and genotyping results of 42 artificial DNA mixtures (NOC = 2–4), it was observed that the present SNP panel was sufficient for balanced mixtures when referenced to the closest relatives (parents/offspring and full siblings). When the mixture profiles suffered from dropout, incorrect assignments were markedly associated with relatedness, NOC and the dropout level. We, therefore, indicate that SNPs with low MAFs could be reliably interpreted for MP identification through the kinship analysis of complex DNA mixtures. Further studies should be extended to more possible scenarios to test the feasibility of this present approach.     

Sample stacking capillary electrophoretic microdevice for highly sensitive mini Y short tandem repeat genotyping

Lab‐on‐a‐chip provides an ideal platform for short tandem repeat (STR) genotyping due to its intrinsic low sample consumption, rapid analysis, and high‐throughput capability. One of the challenges, however, in the forensic human identification on the microdevice is the detection sensitivity derived from the nanoliter volume sample handling. To overcome such a sensitivity issue, here we developed a sample stacking CE microdevice for mini Y STR genotyping. The mini Y STR includes redesigned primer sequences to generate smaller‐sized PCR amplicons to enhance the PCR efficiency and the success rate for a low copy number and degraded DNA. The mini Y STR amplicons occupied in the 5‐ and 10‐mm stacking microchannels are preconcentrated efficiently in a defined narrow region through the optimized sample stacking CE scheme, resulting in more than tenfold improved fluorescence peak intensities compared with that of a conventional cross‐injection microcapillary electrophoresis method. Such signal enhancement allows us to successfully analyze the Y STR typing with only 25 pg of male genomic DNA, with high background of female genomic DNA, and with highly degraded male genomic DNA. The combination of the mini Y STR system with the novel sample stacking CE microdevice provides the highly sensitive Y STR typing on a chip, making it promising to perform high‐performance on‐site forensic human identification.     

Massively parallel sequencing of STRs using a 29-plex panel reveals stutter sequence characteristics

Massively parallel sequencing of forensic STRs simultaneously provides length-based genotypes and core repeat sequences as well as flanking sequence variations. Here, we report primer sequences and concentrations of a next-generation sequencing (NGS)-based in-house panel covering 28 autosomal STR loci (CSF1PO, D1GATA113, D1S1627, D1S1656, D1S1677, D2S441, D2S1776, D3S3053, D5S818, D6S474, D6S1017, D6S1043, D8S1179, D9S2157, D10S1435, D11S4463, D13S317, D14S1434, D16S539, D18S51, D18S853, D20S482, D20S1082, D22S1045, FGA, TH01, TPOX, and vWA) and the sex determinant locus Amelogenin. Preliminary evaluation experiments showed that the panel yielded intralocus- and interlocus-balanced sequencing data with a sensitivity as low as 62.5 pg input DNA. A total of 203 individuals from Yunnan Bai population were sequenced with this panel. Comparative forensic genetic analyses showed that sequence-based matching probability of this 29-plex panel reached 2.37 × 10⁻²⁹, which was 23 times lower than the length-based data. Compound stutter sequences of eight STRs were compared with parental alleles. For seven loci, repeat motif insertions or deletions occurred in the longest uninterrupted repeat sequences (LUS). However, LUS and non-LUS stutters co-existed in the locus D6S474 with different sequencing depth ratios. These results supplemented our current knowledge of forensic STR stutters, and provided a sound basis for DNA mixture deconvolution.     

Development and validation of a new multiplex Y-STR panel designed to increase the power of discrimination

In an attempt to increase the discrimination capacity (DC) and reduce the adventitious match probability, a 6-dye multiplex Y-chromosomal short tandem repeat (Y-STR) panel named Y34plex was constructed that combined 25 Y-chromosomal markers (DYS456, DYS627, DYS390, DYS570, DYS635, DYS385a/b, DYS448, DYS437, DYS533, DYS449, DYS481, DYS392, DYS391, DYS389I, DYS460, YGATAH4, DYS438, DYS389II, DYS19, DYS458, DYF387S1a/b, DYS439, DYS393, DYS576, and DYS518) in widely used commercial kits, with nine highly polymorphic Y-STR loci (DYS557, DYS527a/b, DYS593, DYS444, DYS596, DYS643, DYS447, DYS549, and DYS645). The Y34plex is a promising type system to distinguish both unrelated and related male individuals due to the incorporation of rapidly mutated Y-STR loci. A validation study of the Y34plex was performed and followed the guidelines of the Scientific Working Group on DNA analysis methods. Results show that full Y-STR profiles were obtained from male/female DNA mixtures with 125 pg of male DNA in the presence of 50 ng of female DNA. The ability to tolerate polymerase chain reaction inhibitors commonly contained in forensic casework samples demonstrated the applicability and robustness of the Y34plex. Compared with the Yfiler Plus kit, the novel panel showed an increased power of discrimination in Chinese Wuxi Han population (n = 434). The overall haplotype diversity of the Y34plex was 0.999606, whereas DC value was 0.956221, which is suitable for use on forensic paternal investigation.     

Integrated massively parallel sequencing of 15 autosomal STRs and Amelogenin using a simplified library preparation approach

Massively parallel sequencing (MPS) technologies, also termed as next‐generation sequencing (NGS), are becoming increasingly popular in study of short tandem repeats (STR). However, current library preparation methods are usually based on ligation or two‐round PCR that requires more steps, making it time‐consuming (about 2 days), laborious and expensive. In this study, a 16‐plex STR typing system was designed with fusion primer strategy based on the Ion Torrent S5 XL platform which could effectively resolve the above challenges for forensic DNA database‐type samples (bloodstains, saliva stains, etc.). The efficiency of this system was tested in 253 Han Chinese participants. The libraries were prepared without DNA isolation and adapter ligation, and the whole process only required approximately 5 h. The proportion of thoroughly genotyped samples in which all the 16 loci were successfully genotyped was 86% (220/256). Of the samples, 99.7% showed 100% concordance between NGS‐based STR typing and capillary electrophoresis (CE)‐based STR typing. The inconsistency might have been caused by off‐ladder alleles and mutations in primer binding sites. Overall, this panel enabled the large‐scale genotyping of the DNA samples with controlled quality and quantity because it is a simple, operation‐friendly process flow that saves labor, time and costs.     

Developing and population analysis of a new multiplex panel of 18 microhaplotypes and compound markers using next generation sequencing and its application in the Shaanxi Han population

Compound marker consists of two different types of genetic markers, like deletion/insertion polymorphism and single nucleotide polymorphism in the genomic region of 200 bp, and microhaplotype consists of a series of closely linked single nucleotide polymorphisms in a small DNA segment (<300 bp), which show great potential for human identifications and mixture analyses. In this study, we initially selected 23 novel genetic markers comprising 10 microhaplotypes and 13 compound markers according to previously reported single nucleotide polymorphism or deletion/insertion polymorphism loci. Genetic distributions of these 23 loci in different continental populations showed that they could be used as valuable loci for forensic human identification purpose. Besides, high informativeness values (>0.1) were observed in six loci which could be further employed for forensic ancestry analyses. Finally, 18 loci were successfully developed into a multiplex panel and detected by the next generation sequencing (NGS) technology. Further analyses of these 18 loci in the studied Shaanxi Han population showed that 15 loci exhibited relatively high expected heterozygosities (>0.5). Cumulative power of discrimination (0.999 999 999 99 4835) of these 18 loci revealed that the multiplex panel could also be utilized for human identifications in the studied Shaanxi Han population.     

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.     

A new set of DIP‐SNP markers for detection of unbalanced and degraded DNA mixtures

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP‐SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP‐SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP‐SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP‐SNPs in a Chinese Han population. The DIP‐SNPs were capable of detecting the minor contributor's allele in home‐made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1 –10 ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP‐SNPs may serve as a valuable tool in detection of UDM in forensic medicine.     

Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies

We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.     

Development of a novel forensic STR multiplex for ancestry analysis and extended identity testing

There is growing interest in developing additional DNA typing techniques to provide better investigative leads in forensic analysis. These include inference of genetic ancestry and prediction of common physical characteristics of DNA donors. To date, forensic ancestry analysis has centered on population‐divergent SNPs but these binary loci cannot reliably detect DNA mixtures, common in forensic samples. Furthermore, STR genotypes, forming the principal DNA profiling system, are not routinely combined with forensic SNPs to strengthen frequency data available for ancestry inference. We report development of a 12‐STR multiplex composed of ancestry informative marker STRs (AIM‐STRs) selected from 434 tetranucleotide repeat loci. We adapted our online Bayesian classifier for AIM‐SNPs: Snipper, to handle multiallele STR data using frequency‐based training sets. We assessed the ability of the 12‐plex AIM‐STRs to differentiate CEPH Human Genome Diversity Panel populations, plus their informativeness combined with established forensic STRs and AIM‐SNPs. We found combining STRs and SNPs improves the success rate of ancestry assignments while providing a reliable mixture detection system lacking from SNP analysis alone. As the 12 STRs generally show a broad range of alleles in all populations, they provide highly informative supplementary STRs for extended relationship testing and identification of missing persons with incomplete reference pedigrees. Lastly, mixed marker approaches (combining STRs with binary loci) for simple ancestry inference tests beyond forensic analysis bring advantages and we discuss the genotyping options available.     

Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeq^TM DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub‐optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9–10.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as “low coverage” (< 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeq^TM DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1–44.8% and 10.9–58.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics.     

Mitochondrial DNA control region typing from highly degraded skeletal remains by single-multiplex next-generation sequencing

Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next-generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA-CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex-polymerase chain reaction (PCR) strategy to amplify and index all mtDNA-CR in a single reaction. Our study analyzes the success of mtDNA-CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in-house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA-CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in-house pipeline can provide variants concordant with the forensic software.     

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.     

Improved STR analysis of degraded DNA from human skeletal remains through in-house MPS-STR panel

DNA analysis of degraded samples and low-copy number DNA derived from skeletal remains, one of the most challenging forensic tasks, is common in disaster victim identification and genetic analysis of historical materials. Massively parallel sequencing (MPS) is a useful technique for STR analysis that enables the sequencing of smaller amplicons compared with conventional capillary electrophoresis (CE), which is valuable for the analysis of degraded DNA. In this study, 92 samples of human skeletal remains (70+ years postmortem) were tested using an in-house MPS-STR system designed for the analysis of degraded DNA. Multiple intrinsic factors of DNA from skeletal remains that affect STR typing were assessed. The recovery of STR alleles was influenced more by DNA input amount for amplification rather than DNA degradation, which may be attributed from the high quantity and quality of libraries prepared for MPS run. In addition, the higher success rate of STR typing was achieved using the MPS-STR system compared with a commercial CE-STR system by providing smaller sized fragments for amplification. The results can provide constructive information for the analysis of degraded sample, and this MPS-STR system will contribute in forensic application with regard to skeletal remain sample investigation.     

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.     

Slippage mutation rates in 15 autosomal short tandem repeat loci for forensic purposes in a Southeastern Brazilian population

Well‐defined estimates of mutation rates in highly polymorphic tetranucleotide STR loci are a prerequisite for human identification in genetics laboratory routines useful for civil and criminal investigations. Studying 15 autosomal STR loci of forensic interest (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, and vWA), we detected 193 slippage mutations (189 one‐step and four two‐step mutations) in 148 875 parent‐child allelic transfers from 5171 paternity cases with true biological relationship (15 096 individuals; 4754 trios and 417 duos; 9925 meiosis) from the state of São Paulo, a very representative population of Brazil. The overall mutation rate was 1.3 × 10^?3 and the highest rates were observed at loci vWA (2.8 × 10^?3), FGA and D18S51 (2.7 × 10^?3 for both), while loci TH01 and TPOX did not present any mutations. The mean slippage mutation rate of paternal origin (1.8 × 10^?3) was six times higher than that observed for maternal origin (0.3 × 10^?3).     

A new autosomal STR nineplex for canine identification and parentage testing

A single multiplex PCR assay capable of simultaneously amplifying nine canine‐specific autosomal STR markers (FH3210, FH3241, FH2004, FH2658, FH4012, REN214L11, FH2010, FH2361 and the newly described C38) was developed for individual identification and parentage testing in domestic dogs. In order to increase genotyping efficiency, amplicon sizes were optimized for a 90–350 bp range, with fluorescently labelled primers for use in Applied Biosystems, Inc., platforms. The performance of this new multiplex system was tested in 113 individuals from a case‐study population and 12 random dogs from mixed‐breed origin. Co‐dominant inheritance of STR alleles was investigated in 101 father, mother and son trios. Expected heterozygosity values vary between 0.5648 for REN214L11 and 0.9050 for C38. The high level of genetic diversity observed for most markers provides this multiplex with a very high discriminating power (matching probability=1.63/10¹⁰ and matching probability among siblings=4.9/10³). Allele sequences and a proposal for standardized nomenclature are also herein presented, aiming at implementing the use of this system in forensic DNA typing and population genetic studies. This approach resulted in an optimized and well‐characterized canine DNA genotyping system that is highly performing and straightforward to integrate and employ routinely. Although this STR multiplex was developed for use and tested in a case‐study population, the Portuguese breed Cão de Gado Transmontano, it proved to be useful for general identification purposes or parentage testing.