Evaluation of the protective capabilities of nucleosome STRs obtained by large‐scale sequencing

Partial DNA profiles are often obtained from degraded forensic samples and are hard to analyze and interpret. With in‐depth studies on degraded DNA, an increasing number of forensic scientists have focused on the intrinsic structural properties of DNA. In theory, nucleosomes offer protection to the bound DNA by limiting access to enzymes. In our study, we performed large‐scale DNA sequencing on nucleosome core DNA of human leucocytes. Five nucleosome short tandem repeats (STRs) were selected (including three forensic common STRs (i.e. TPOX, TH01, and D10S1248) and two unpublished STRs (i.e. AC012568.7 and AC007160.3)). We performed a population genetic investigation and forensic genetic statistical analysis of these two unpublished loci on 108 healthy unrelated individuals of the HeBei Han population in China. We estimated the protective capabilities of five selected nucleosome loci and MiniFiler? loci with artificial degraded DNA and case samples. We also analyzed differences between sequencing results and software predicted results. Our findings showed that nucleosome STRs were more likely to be detected than MiniFiler? loci. They were well protected from degradation by nucleosomes and could be candidates for further nucleosome multiplex construction, which would increase the chances of obtaining a better balanced profile with fewer allelic drop‐outs.     

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.     

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.     

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.     

Improved STR typing of telogen hair root and hair shaft DNA

Today the STR typing of telogen hair and hair shafts is regarded as a challenge. The small DNA quantity in the hair is highly degraded. Another problem are PCR inhibitors in the hair. In particular hair pigments, the melanins, are known to inhibit PCR. Hairs are exposed to sunlight and partly to chemical oxidation processes, which make them even more difficult to analyze. To increase the chances of a correct typing of hair, the small amount of DNA must be successfully isolated and the inhibitors have to be removed or neutralized. Furthermore, miniSTR typing improves the analysis of stains with degraded DNA like it is the case with hair. We introduce a nonorganic extraction method and in addition a miniSTR concept which is promising in typing stains with little and degraded DNA, especially hairs. The miniSTR concept including five database STRs (SE33, VWA, TH01, FGA, D3S1358) and the gender typing system Amelogenin was optimized for the amplification of hair DNA. Compared to commercial STR kits, this approach resulted in considerably higher success rates.     

17 to 23: A novel complementary mini Y‐STR panel to extend the Y‐STR databases from 17 to 23 markers for forensic purposes

A Y‐STR multiplex system has been developed with the purpose of complementing the widely used 17 Y‐STR haplotyping (AmpFlSTR Y Filer® PCR Amplification kit) routinely employed in forensic and population genetic studies. This new multiplex system includes six additional STR loci (DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643) to reach the 23 Y‐STR of the PowerPlex® Y23 System. In addition, this kit includes the DYS456 and DYS385 loci for traceability purposes. Male samples from 625 individuals from ten worldwide populations were genotyped, including three sample sets from populations previously published with the 17 Y‐STR system to expand their current data. Validation studies demonstrated good performance of the panel set in terms of concordance, sensitivity, and stability in the presence of inhibitors and artificially degraded DNA. The results obtained for haplotype diversity and discrimination capacity with this multiplex system were considerably high, providing further evidences of the suitability of this novel Y‐STR system for forensic purposes. Thus, the use of this multiplex for samples previously genotyped with 17 Y‐STRs will be an efficient and low‐cost alternative to complete the set of 23 Y‐STRs and improve allele databases for population and forensic purposes.     

Single nucleotide polymorphisms of mitochondrial DNA HVS‐I and HVS‐II in Chinese Bai ethnic group

For forensic and population genetic purposes, a total of 125 unrelated volunteers’ blood samples were collected from Chinese Bai ethnic minority group to analyze sequence variation of two hypervariable segments (HVS‐I and HVS‐II) in the mitochondrial DNA control region. Comparing the HVS‐I and HVS‐II sequences of the 125 Chinese Bais to the Anderson reference sequence, we found 86 polymorphic loci in HVS‐I and 40 in HVS‐II in mitochondrial DNA sequences of the Chinese Bai ethnic minority group, which defined 93 and 53 different haplotypes, respectively. Haplotype diversity and the mean pairwise differences were 0.992 ± 0.003 and 6.553 in HVS‐I, and 0.877 ± 0.027 and 2.407 in HVS‐II, respectively. We defined four macrohaplogroups R, M, N and D with the proportions ranging from 9.6% to 40.0%. With the analysis of the hypervariable domain from nucleotide 16 180–16 193 in HVS‐I, our study revealed new haplotypes of sequence variations. In addition, the Fst metric, phylogenetic tree, and principal component analysis demonstrated a close genetic relationship between the Bai group and Chinese Han populations from South China, Changsha, and Guangdong. The results support that the Bai group is a multiorigin ethnic minority that has merged with the Chinese Han population.     

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.     

Forensic applicability of multi‐allelic InDels with mononucleotide homopolymer structures

Insertion/deletion polymorphisms (InDels), which possess the characteristics of low mutation rates and a short amplicon size, have been regarded as promising markers for forensic DNA analysis. InDels can be classified as bi‐allelic or multi‐allelic, depending on the number of alleles. Many studies have explored the use of bi‐allelic InDels in forensic applications, such as individual identification and ancestry inference. However, multi‐allelic InDels have received relatively little attention. In this study, InDels with 2–6 alleles and a minor allele frequency ≥0.01, in Chinese Southern Han (CHS), were retrieved from the 1000 Genomes Project Phase III. Based on the structural analysis of all retrieved InDels, 17 multi‐allelic markers with mononucleotide homopolymer structures were selected and combined in one multiplex PCR reaction system. Sensitivity, species specificity and applicability in forensic case work of the multiplex were analyzed. A total of 218 unrelated individuals from a Chinese Han population were genotyped. The combined discriminatory power (CDP), the combined match probability (CMP) and the cumulative probability of exclusion (CPE) were 0.9999999999609, 3.91E‐13 and 0.9956, respectively. The results demonstrated that this InDel multiplex panel was highly informative in the investigated population and most of the 26 populations of the 1000 Genomes Project. The data also suggested that multi‐allelic InDel markers with monomeric base pair expansions are useful for forensic applications.     

A novel method for the analysis of 20 multi‐Indel polymorphisms and its forensic application

Insertion/deletion polymorphisms (Indels) have been considered as potential markers for forensic DNA analysis. However, the discrimination power of Indels is relatively lower due to the poor polymorphisms of diallelic markers. Here, two to three Indel loci that were very tightly linked in physical position were combined into a specific multi‐Indel marker to improve the discrimination, as well as a multiplex that consisted of a set of multi‐Indel markers was developed for forensic purpose. Finally, a multiplex system with 20 multi‐Indel markers including 43 Indel loci from dbSNP database was constructed and DNA sample can be analyzed by this multiplex in one PCR reaction and one CE run. A total of 150 unrelated individuals from Hunan province in South‐central China were genotyped by the multiplex system. The result showed that a total of 63 specific amplicons were detected, three alleles were observed in multi‐Indel markers including two Indel loci and four alleles were observed in the markers including three Indel loci. The cumulative probability of exclusion and the accumulated discrimination power were 0.9989 and 0.9999999999994, respectively. Our result demonstrated that the strategy could be efficient to develop higher polymorphic multi‐Indel markers, and the new multiplex could provide Supporting Information for forensic application.     

East Asian mtDNA haplogroup determination in Koreans: haplogroup-level coding region SNP analysis and subhaplogroup-level control region sequence analysis

The present study analyzed 21 coding region SNP markers and one deletion motif for the determination of East Asian mitochondrial DNA (mtDNA) haplogroups by designing three multiplex systems which apply single base extension methods. Using two multiplex systems, all 593 Korean mtDNAs were allocated into 15 haplogroups: M, D, D4, D5, G, M7, M8, M9, M10, M11, R, R9, B, A, and N9. As the D4 haplotypes occurred most frequently in Koreans, the third multiplex system was used to further define D4 subhaplogroups: D4a, D4b, D4e, D4g, D4h, and D4j. This method allowed the complementation of coding region information with control region mutation motifs and the resultant findings also suggest reliable control region mutation motifs for the assignment of East Asian mtDNA haplogroups. These three multiplex systems produce good results in degraded samples as they contain small PCR products (101-154 bp) for single base extension reactions. SNP scoring was performed in 101 old skeletal remains using these three systems to prove their utility in degraded samples. The sequence analysis of mtDNA control region with high incidence of haplogroup-specific mutations and the selective scoring of highly informative coding region SNPs using the three multiplex systems are useful tools for most applications involving East Asian mtDNA haplogroup determination and haplogroup-directed stringent quality control.     

Development of the 16 X‐STR loci typing system and genetic analysis in a Shanghai Han population from China

This study developed a new multiplex PCR system that simultaneously amplifies 16 X‐STR loci in the same PCR reaction, and the polymorphism and mutation rates of these 16 X‐STR loci were explored in a Shanghai Han population from China. These loci included DXS10134, DXS10159, DXS6789, DXS6795, DXS6800, DXS6803, DXS6807, DXS6810, DXS7132, DXS7424, DXS8378, DXS9902, GATA165B12, GATA172D05, GATA31E08, and HPRTB. Samples from 591 unrelated individuals (293 males and 298 females) and 400 two‐generation families were successfully analyzed using this multiplex system. Allele frequencies and mutation rates of the 16 loci were investigated, with the comparison of allele frequency distributions among different populations performed. Polymorphism information contents of these loci were all >0.6440 except the locus DXS6800 (0.4706). Nine cases of mutations were detected in the 16 loci from the investigation of 9232 meioses. Pairwise comparisons of allele frequency distributions showed significant differences for most loci among populations from different countries and ethnic groups but not among the Han population living in other areas of China. These results suggest that the 16 X‐STR loci system provides highly informative polymorphic data for paternity testing and forensic identification in the Han population in Shanghai, China, as a complementary tool.     

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.     

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.     

Analysis and interpretation of mixture DNA using AS‐PCR of mtDNA

Semi‐nested PCR with allele‐specific (AS) primers and sequencing of mitochondrial DNA (mtDNA) were performed to analyze and interpret DNA mixtures, especially when biological materials were degraded or contained a limited amount of DNA. SNP‐STR markers were available to identify the minor DNA component using AS‐PCR; moreover, SNPs in mtDNA could be used when the degraded or limited amounts of DNA mixtures were not successful with SNP‐STR markers. Five pairs of allele‐specific primers were designed based on three SNPs (G15043A, T16362C, and T16519C). The sequence of mtDNA control region of minor components was obtained using AS‐PCR and sequencing. Sequences of the amplification fragments were aligned and compared with the sequences of known suspects or databases. When this assay was used with the T16362C and T16519C SNPs, we found it to be highly sensitive for detecting small amounts of DNA (～30 pg) and analyzing DNA mixtures of two contributors, even at an approximately 1‰ ratio of minor and major components. An exception was tests based on the SNP G15043A, which required approximately 300 pg of a 1% DNA mixture. In simulated three contributor DNA mixtures (at rate of 1:1:1), control region fragments from each contributor were detected and interpreted. AS‐PCR combined with semi‐nested PCR was successfully used to identify the mtDNA control region of each contributor, providing biological evidence for excluding suspects in forensic cases, especially when biological materials were degraded or had a limited amount of DNA.     

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.     

An 18 Multi-InDels panel for analysis of highly degraded forensic biological samples

DNA genotyping from trace and highly degraded biological samples is one of the most significant challenges of forensic DNA identification. There is a lack of simple and effective methods for genotyping highly degraded samples. In this study, a multiple loci insertion/deletion polymorphisms (Multi-InDels) panel was designed for detecting 18 autosomal Multi-InDels through capillary electrophoresis (CE) with amplicon sizes no longer than 125 bp. Studies of sensitivity, degradation, and species specificity were performed and a population study was carried out using 192 samples from Han populations in Hunan province in the south of China. The combined random match probability (CMP) of these 18 Multi-InDels was 3.23 × 10^–12 and the cumulative probability of exclusion (CPE) was 0.9989, suggesting this panel could be used independently for human identification and could provide efficient supporting information for parentage testing. Complete profiles were obtained from as low as 62.5 pg of total input DNA after increasing the number of PCR cycles. Moreover, all alleles were detected from artificially highly degraded DNA after 80 min of boiling water bath treatment. This 18 Multi-InDels panel is simple, fast, and effective for the forensic analysis of highly degraded DNA.     

Development of a body fluid identification multiplex via DNA methylation analysis

The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers – BCAS4, CG06379435, VE_8, and ZC3H12D – were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.     

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.     

Improving the analysis of Y‐SNP haplogroups by a single highly informative 16 SNP multiplex PCR‐minisequencing assay

The SNP haplogroups of the Y‐chromosome are nonrandomly distributed among human populations. They are used for tracing the phylogeographical history of paternal lineages of male individuals and can be a useful tool for approaching the patrilineal bio‐geographic ancestry of unknown forensic evidences. With the aim of facilitating the inference of the principal informative worldwide Y‐SNP haplogroups, we have selected the minimum possible number of key Y‐SNPs to be amplified in a sensitive single multiplex PCR and detected by minisequencing. This assay, that includes 16 Y‐SNPs, was tested for male human specificity, sensitivity, and reproducibility. Its effectiveness was assessed in a set of degraded DNA samples and in a panel of male individuals from different worldwide populations. All these tests demonstrated the convenience of this assay for assigning the major Y haplogroups to forensic evidences by one single PCR‐minisequencing reaction.