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Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a 5‐year survival rate of 7.2% in China. However, effective approaches for diagnosis of PDAC are limited. Tumor‐originating genomic and epigenomic aberration in circulating free DNA (cfDNA) have potential as liquid biopsy biomarkers for cancer diagnosis. Our study aims to assess the feasibility of cfDNA‐based liquid biopsy assay for PDAC diagnosis. In this study, we performed parallel genomic and epigenomic profiling of plasma cfDNA from Chinese PDAC patients and healthy individuals. Diagnostic models were built to distinguish PDAC patients from healthy individuals. Cancer‐specific changes in cfDNA methylation landscape were identified, and a diagnostic model based on six methylation markers achieved high sensitivity (88.7% for overall cases and 78.0% for stage I patients) and specificity (96.8%), outperforming the mutation‐based model significantly. Moreover, the combination of the methylation‐based model with carbohydrate antigen 19‐9 (CA19‐9) levels further improved the performance (sensitivity: 95.7% for overall cases and 95.5% for stage I patients; specificity: 93.3%). In conclusion, our findings suggest that both methylation‐based and integrated liquid biopsy assays hold promise as non‐invasive tools for detection of PDAC. [ABSTRACT FROM AUTHOR]

Highlights • A novel nomenclature of microhaplotype marker is proposed. • A new Perl-based tool Forensic-Loci-finder (FLfinder) was designed to profile the microhaplotype markers. • The direct and accurate genotypes of microhaplotype markers were got using massively parallel sequencing (MPS). • 1000 Genomes Phase 3 data was used to give a point that microhaplotype markers have the potential of ancestry inference. Abstract 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. [ABSTRACT FROM AUTHOR]

A set of CpG sites were detected and evaluated to identify body fluids based on DNA methylation using the amplification refractory mutation system-PCR (ARMS-PCR). In this research, two multiplex DNA methylation reactions composed of four promising CpG sites were used for the identification of forensic regular body fluids. ARMS-specific primers were used to amplify the CpG sites and the methylation values of each CpG site were detected by capillary electrophoresis. The DNA methylation profiling of four highly informative candidate CpG sites was consistent with previously reported results. It is the first time to apply the ARMS-PCR to detect DNA methylation in forensic science, which provides a new method for forensic body fluid identification. We proved this method will have great potential in detection of the methylation profiling at molecular level. [ABSTRACT FROM AUTHOR]

Non-protein coding RNA, miRNAs (microRNAs), are a class of promising molecular biomarkers for forensic body fluid identification (BFI) as their small size and tissue-specific expression manners. A number of studies have shown that semen can be distinguished from forensic-related body fluids (such as menstrual blood, venous blood, vaginal fluid, saliva, etc.) using semen-specific miRNAs through microassay screening and RT-qRCR. Infertility is becoming a global health problem, affecting 10%-15% of couples worldwide, and half of the cases are the result of male factors (Lian et al., 2009 [ 1 ]). Forensic researchers have to consider the impact of semen infertility on semen identification with a high incidence of infertility. In the present study, normal semen (NS) and four other types of infertile semen samples, including asthenospermia (AS), oligospermia (OS), azoospermia (AZ), oligospermia and asthenospermia (OSAS) semen, were collected. The expression levels of a set of semen-specific miRNA markers (miR-10a, miR-10b, miR-135a, miR-135b, miR-888 and miR-891a) were evaluated using a real-time quantitative PCR technique with a specific fluorescence-labelled TaqMan probe. The results showed the significantly high expression of these miRNAs in normal semen, and the molecules have semen specificity. Nevertheless, a distinct down-regulation in the expression of infertile samples compared with normal semen samples was observed. Moreover, differences in the results of selected optimal biomarkers between the discriminant function and two-dimensional scatter plots were also detected. The goal of the present study was to identify a small set of semen-specific miRNAs that efficiently and accurately distinguish semen (fertile and infertile) from other forensic-related body fluids. The results of the present study suggest that attention should be paid to infertile semen samples when using miRNAs to identify semen samples, for which would have a far-reaching impact on forensic identification. [ABSTRACT FROM AUTHOR]

The resolution of DNA mixtures is still a difficult problem that is worthy of further study. A common method applied for analysing mixtures is the use of autosomal STR markers as well as related calculation software based on genotypes; however, these markers have a limitation in detecting minor DNA in unbalanced mixtures if major DNA constitutes over 95% of the stain. Novel biomarkers, such as Y-STR, DIP-STR and SNP-STR, have been shown to perform well in distinguishing DNA donors in this type of mixture. DIP-STR can successfully target minor DNA in 1000-fold background DNA using two separate allele-specific primers. However, whether this method can successfully detect minor DNA primarily depends on the distribution of the DIPs in a population. Until now, only Swiss population data have been reported; therefore in this study, we selected 10 DIP-STR markers that performed well in the Swiss population and investigated whether these markers were also useful among the southwest Chinese Han population. The allele frequencies were estimated based on 152 samples, and six of the ten DIP-STR makers had a relatively high probability of informative markers ( I value), which indicated their potential usefulness in the southwest Chinese Han population. A comparative study of DIP-STR markers and autosomal STR markers demonstrated that DIP-STR markers detected minor DNA at a ratio of 1:1000, while autosomal STR markers often failed to genotype minor DNA because of strong background noises caused by large amount of major DNA. However, the discrimination power was not high enough using these six DIPs alone. Therefore, we suggest that development of a panel with more loci is imperative and that a panel combined with DIP-STR and SNP-STR markers may be a possible way to achieve better discrimination power. [ABSTRACT FROM AUTHOR]

The identification of menstrual blood (MB) and peripheral blood (PB) left at a crime scene is crucial for crime reconstruction, especially in sexual assaults. MicroRNAs (miRNAs), a class of non-protein coding molecules, have been demonstrated to be a viable tool for body fluid identification in forensic casework. Several groups have searched for miRNAs that are specific to different body fluids. Blood has been studied the most extensively. However, menstrual blood was only involved in five studies, and the results confirming the presence of specific miRNAs could not be reproduced in other studies. In this study, we attempted to screen new markers that can differentiate between menstrual blood and peripheral blood by using Exiqon’s miRCURY™ LNA Array. Five miRNAs were selected based on the microarray results, namely, miR-141-3p, miR-373-3p, miR-497-5p, miR-143-5p, and miR-136-5p, whose expression levels exhibited 27.95-, 17.96-, 16.74-, 10.14-, and 9.21-fold changes, respectively, compared to the level in peripheral blood. Two classic quantitative methods, TaqMan hydrolysis probes (TaqMan for short) and SYBR Green fluorochrome (SYBR Green for short), were applied in the confirmation step to study the impact of different quantitative methods on the results. Three miRNAs (miR-141-3p, miR-497-5p, and miR-143-5p) were confirmed by TaqMan and one (miR-141-3p) by SYBR Green. Furthermore, bioinformatic methods were applied to interpret the candidate miRNAs. Our results established a multi-step procedure for body fluid identification and showed that the choice of quantitative method is important when miRNAs are used to identify the origin of blood samples. [ABSTRACT FROM AUTHOR]

In the past decades, mRNA marker has been well demonstrated the remarkable progress and wide application for its high sensitivity, specificity and robustness. However, according to previous studies, infertility has become a global health problem. Therefore given the increasing occurrence of male infertility, forensic researchers have to consider the impact of semen infertility on semen identification. In present study, normal semen (NS) asthenospermia (AS) and oligospermia (OS) semen samples were collected. The expression of semen-specific mRNA markers (KLK3, PRM1) was detected using end-point PCR. The results showed that KLK3 was almost not influenced by infertile semen samples. In contrast, PRM1 couldn't be detected in all the infertile semen samples, indicating that it may not be ideal mRNA marker to identify semen samples. The present study aimed to explore the effect of the infertile semen on body fluid identification and to identify semen-specific mRNAs. [ABSTRACT FROM AUTHOR]

Facial morphology is the most distinguishable appearance and represents a promising subfield of Forensic DNA phenotyping. Located in the center of the face, eyes are important for facial recognition. In this study, epicanthal fold and palpebral fissure were selected for a preliminary genotype-phenotype analysis. Two SNPs were genotyped with SNaPshot method in 156 Chinese Han adults. A significant increased incidence of high ratio of palpebral fissure width to height was observed in the populations with AG genotype for SNP rs2074612 on gene HBEGF: AG versus GG, OR = 2.36, 95%CI = 1.08–5.18, p <0.05; AG versus GG/AA, OR = 2.07, 95%CI = 1.03–4.16, p < 0.05. No significant genotype difference was detected between epicanthal fold positive/negative populations for both SNPs (p > 0.05). Further study including more samples should be conducted to discover SNPs associated with epicanthal fold and palpebral fissure traits. [ABSTRACT FROM AUTHOR]

RNA molecules, including mRNAs and microRNAs (miRNAs), have been used for forensic body fluid identification. Specific body fluids present unique mRNA expression patterns, while miRNAs identifying body fluids are mainly differentially expressed. miRNAs are thought to be more stable than mRNAs, although this lacks adequate supporting data. In this study, we addressed perceived concerns regarding the stability of miRNAs and mRNAs in blood samples. The samples used in this study involved three groups. First, environmentally-degraded blood stain samples were exposed to a range of environmental conditions over 1–360 days to degrade naturally. Second, simulated-degraded samples were prepared using RNase A or high temperature (80 °C). Furthermore, two authentic casework samples that were proven to be degraded from short tandem repeat (STR) profiles were analyzed. mRNAs and miRNAs present in the same blood samples were simultaneously detected through reverse transcriptase qPCR (RT-qPCR). Furthermore, mRNAs expression was determined by an mRNA multiplex PCR system. Our results showed that both mRNAs and miRNAs were stable in dry environments. The stability of miRNAs was relatively higher than that of mRNAs in humid environments or at high temperature. RNase A had the most serious impact on RNA stability, both mRNA profiles and miRNAs expression patterns were altered. The results of this study provide data and support to demonstrate that miRNAs represent more stable RNA molecules in body fluid identification compared to mRNAs. • This study evaluates mRNAs and microRNAs stability in blood samples. • Blood samples were degraded to various degrees in different ways. • Relative humidity and RNase A disrupt the stability of both microRNAs and mRNAs. • mRNA species are more vulnerable to humidity and heat than microRNAs. • Our study confirmed miRNAs could represent an important supplement to mRNAs for BFI. [ABSTRACT FROM AUTHOR]

• ARMS-PCR technique could effectively detect the methylation levels in a simple and cost-effective manner. • 22 body fluid-specific CpG sites were included in development of the multiplex assays. • The random forest model performed well in predicting single source body fluids with high prediction accuracy (99.66%). • Random forest model is a practical and promising prediction method for the mixed body fluid identification. A set of DNA methylation markers was detected and evaluated to identify body fluids using the amplification refractory mutation system-PCR (ARMS-PCR) and random forest algorithm. In this study, four multiplex DNA methylation reactions composed of 22 promising methylation markers were used to identify regular forensic body fluids, including venous blood, saliva, semen, menstrual blood, and vaginal fluid. The ARMS-specific primers were used to amplify the candidate markers, and then the methylation values of each CpG site were detected through capillary electrophoresis (CE). The DNA methylation patterns of 22 highly informative methylation markers were consistent with previously reported results to a certain extent. To our knowledge, our study is a new method to apply the ARMS-PCR technique and random forest model to detect DNA methylation patterns and identify the type of body fluids in forensic science, thus providing a new method for forensic body fluid identification. Moreover, we proved that this method is robust, applicable and effective for identifying body fluids using the random forest model. The accuracy to predict all body fluids reached up to 0.9966. We firmly believe that this method will have a great potential in the detection of methylation profiles at the molecular level. [ABSTRACT FROM AUTHOR]

Postmortem interval (PMI) determination, helping exclude or confirm suspects, plays a key role in forensic cases. Recent years, degradation of mRNA has shown potentiality to determine time of death and be allowed to obtain a considerable time since death. In our study, we aimed to find an mRNA marker to assess PMI with accuracy. We performed hypoxia associated factor (HAF) mRNA degradation in 48 h after death within 29 time points in mouse brain to seek a more precise time of PMI determination. Caspase-3 DNA was carried out as normalization of HAF mRNA degradation. Nucleic acids were extracted by commercial kits and relative quantitive PCR was used to detect expression level of HAF mRNA and Caspase-3 DNA. Through analyzed profiling of HAF mRNA degradation, we acquired a statistical model between 48 h PMI and mRNA degradation. Then we found that abundance of 105 bp HAF mRNA fragment was increased in 48 h. Interestingly, in the first 4 h after death, PMI was well correlated with HAF mRNA degradation in mouse brain. We believe these results indicate that HAF mRNA was a suitable marker for PMI determination in early time since death and the statistical model of it can become a useful tool in forensic practice. [ABSTRACT FROM AUTHOR]

Estimating time of death in forensic routines is particularly important, and a range of time can be determined by traditional methods Degradation of mRNA has been used to estimate postmortem interval (PMI) and many potential mRNA markers are found in previous studies. In this study, we aimed to find a new mRNA marker which can determine PMI with its degradation. Besides, our previous results proposed that DNA was suitable as normalization. So, in this study we determined apoptosis inducing factor (AIF) degradation against Caspase-3 DNA to see if AIF is an effective marker to estimate time of death. To do that, corpses of mice in 37 °C were simulating the environment of summer days in Chengdu and heart tissue was obtained in different time points. RNA and DNA were co-extracted with Bioteke co-extraction kits followed by expression level detection with RT-qPCR of MiScript SYBR Green PCR Kit. The results showed that AIF was slowly degraded in 48 h PMI. What’s more, AIF represented a faster degradation process and a good liner correlation within 14–20 h PMI. Therefore, we suggested AIF was an useful marker to estimate time of death in14–20 h PMI. [ABSTRACT FROM AUTHOR]

Three microRNAs (miR-141-3p, miR-497-5p, miR-143-5p) have been screened out and confirmed to be promising markers to distinguish between menstrual blood and peripheral blood by our previous study. However, studies reported the expression levels of miRNAs might be different in menstrual blood samples of different menstrual cycle collection days. To evaluate the dynamic changes of expression levels of these three miRNAs in menstrual blood samples from different menstrual cycle collection days, 51 menstrual blood (day 1–5 of menstrual cycle) samples were collected and detected. Total RNA was extracted then corresponding cDNA was prepared and three TaqMan Assays were run in triplicate using the TaqMan Universal PCR Master Mix II without UNG (Life Technologies, USA). Bioinformatics analysis was used to search target genes of three miRNAs attempting to explain the results. After comparing with the expression levels of previous peripheral blood samples, we found that miR-141-3p and miR-497-5p were still reliable markers to distinguish menstrual blood from peripheral blood after the sample size was enlarged. [ABSTRACT FROM AUTHOR]

Non-protein coding RNAs, miRNAs (microRNAs), are a class of promising molecular biomarkers for forensic body fluid identification as the very small size and their tissue-specific expression manners. A number of studies have showed that semen can be distinguished from forensic related body fluids by the semen-specific miRNAs through microassay screening and RT-RCR. Infertility is becoming a global health problem affecting 10%–15% of couples worldwide and half of the cases are due to male factors. So high incidence of infertility forced the forensic researchers to consider the impact of the semen infertility on the semen identification. Hence, several kinds of semen from man infertility were collected into our research such as asthenospermia (AS) and azoospermia (AZ). The expression level of semen samples (fertile and infertile) and other common forensic body fluids were evaluated using two miRNA markers (miR-10b and miR-135b) by RT-PCR with TaqMan probe. Results showed that the two miRNAs expressed significantly higher in normal semen (NS) than other body fluids. Nevertheless, there was a distinct down-regulation in the expression of AS samples. The goal of our study was to ascertain whether the two miRNAs could distinguish semen (fertile and infertile) efficiently and accurately. This study reminded forensic workers should pay attention to samples in pathology when identifying body fluids, which would have a far-reaching significance for forensic application. [ABSTRACT FROM AUTHOR]

Identifying the origin of the blood left at a crime scene is crucial for crime scene reconstruction, especially in sexual assault. Nowadays there are no reliable and confirmatory assays to differentiate menstrual blood from peripheral blood. MiRNAs (MicroRNAs) are non-protein coding molecules with an important regulatory role at the post-transcriptional level. Although three study groups have screened out miRNA markers for menstrual blood identification, only one of them was confirmed through qPCR. In our study, we attempt to screen new markers for menstrual blood using Exiqon’s miRCURY™ LNA Array. MiR-141-3p was selected according to the results of the microarray whose expression levels was 27.95 fold changes comparing with peripheral blood, then it was confirmed via qPCR. According to the results, a multi-step procedure of blood identification was set up. First, blood should be recognized by blood-specific makers like miR-451a and miR-16-5p, then menstrual blood could be identified from peripheral blood by new marker miR-141-3p. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) has attracted wide interest of the forensic community since Hanson attempted to search specific microRNAs for body fluid identification from microarray. Research groups have screened out some sets of miRNA markers that can be used in forensic body fluids identification up to now. Most of those miRNA markers could not be repeated in different labs. The markers confirmed by SYBRgreen always could not be confirmed by another group in Taqman. Different reverse-transcription quantitative PCR methods might be the major cause of that. To seek out the influences of different reverse-transcription quantitative PCR methods on the expression of miRNA, we chose three miRNAs which showed conflicted or consistent results in different RT-qPCR methods according to previous studies, including miR-412-3p, miR-124a-3p, miR-10b-5p, for menstrual blood, vaginal secretions and semen respectively. MiRNAs were analyzed through Taqman and SYBRgreen according to the protocols respectively. Different ΔCq values of the same miRNA marker were detected by SYBRgreen and Taqman. Different results can be attributed to the sensitivity and specificity of two reverse-transcription quantitative methods. The choice of appropriate reverse-transcription quantitative method should be based on the study targets and the conditions of samples. [ABSTRACT FROM AUTHOR]

DIPs (insertion/deletion polymorphisms) combining the advantages of both SNPs (single nucleotide polymorphisms) and STRs (short tandem repeats) show a great value in forensic case works. A high sensitive 21 AIM (ancestry informative markers)-DIPs panel developed by Daniel et al. is sufficient to distinguish between three major global population groups. However, Chinese population was not included in Daniel’s research. In this study, DNA samples from three Chinese population groups living in west of China (Han ( n = 60), Uighur ( n = 60) and Tibetan ( n = 60)) were extracted and analyzed following Daniel’s procedure. The difference in biogeographic ancestry between the three Chinese population groups was compared. The consequence shows no significant difference between Chinese Han, Uighur and Tibetan populations. In summary, this 21-plex DIP panel is unable to distinguish the three Chinese populations. To characterize three groups in China, more AIM-DIPs are needed. [ABSTRACT FROM AUTHOR]