A method of identifying the high-risk mutations of sudden cardiac death at KCNQ1 and KCNH2 genes.

Sudden Cardiac Death (SCD) often shows negative anatomy results after a systemic autopsy and the gene mutations of potassium channel play a key role in the etiology of SCD. We established a feasible system to detect SCD-related mutations and investigated the mutations at KCNQ1 and KCNH2 genes in the Chinese population. We established a mutation detection system combined with multiplex PCR, SNaPshot technique, and capillary electrophoresis. We genotyped 101 putative mutations at KCNQ1 and KCNH2 genes in 60 SCD of negative anatomy and 50 controls using the established assay and compared Odd Ratio (OR). Four coding variants were identified in the KCNQ1 gene: S546S, I145I, P448R, and G643S. The mutations of I145I and S546S did not differ significantly in the SCD compared with controls. 21 SCD individuals (35 %) and 1 control individual (2 %) showed a genotype of C/G at P448R (OR = 17.5, 95 % CI [2.40-127.82]). 24 SCD individuals (40 %) and 1 control individual (2 %) showed a genotype of C/G at G643S (OR = 20.0, 95 % CI [2.75-145.25]). We established a robust assay for rapid screening the putative SCD-related mutations in KCNQ1 and KCNH2 genes. The new assay in our study is easily amenable to the majority of laboratories without the need for new specialized equipment. Our method will meet the increasing requirement of mutation screening for SCD in regular DNA laboratories and will help screen mutations in those dead of SCD and their relatives.

Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing.

In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures. Traditional STR profiles face difficulties in such scenarios. Over recent years, RNA has emerged as a promising biomarker for body fluid identification, and mRNA polymorphism has shown excellent performance in identifying body fluid donors in previous studies. In this study, a massively parallel sequencing assay was developed, encompassing 202 coding region SNPs (cSNPs) from 45 body fluid/tissue-specific genes to identify both body fluid/tissue origin and the respective donors, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin. The specificity was evaluated by examining the single-source body fluids/tissue and revealed that the same body fluid exhibited similar expression profiles and the tissue origin could be identified. For laboratory-generated mixtures containing 2-6 different components and mock case mixtures, the donor of each component could be successfully identified, except for the skin donor. The discriminatory power for all body fluids ranged from 0.997176329 (menstrual blood) to 0.99999999827 (blood). The concordance of DNA typing and mRNA typing for the cSNPs in this system was also validated. This cSNP typing system exhibits excellent performance in mixture deconvolution.

A preliminary study characterizing temporal changes in soil bacterial communities after dismembered bones were buried.

Determining the burial time of skeletal remains is one of the most important issues of forensic medicine. We speculated that the microbiome of gravesoil may be a promising method to infer burial time by virtue of time-dependent. As we know, forensic scientists have established various models to predict the postmortem interval of a decedent based on the changes in body and soil microbiome communities. However, limited data are available on the burial time prediction for bones, especially dismembered bones. In this exploratory study, we initially conducted 16S rRNA amplicon high-throughput sequencing on the burial soil of 10 porcine femurs within a 120-day period and analyzed the changes in soil microbial communities. Compared with the control soil, a higher Shannon index in the microbial diversity of burial soil containing bones was observed. Correlation analysis identified 61 time-related bacterial families and the best subset selection method obtained best subset, containing Thermomonosporaceae, Clostridiaceae, 0319-A21, and Oxalobacteraceae, which were used to construct a simplified multiple linear regression model with a mean absolute error (MAE) of 56.69 accumulated degree day (ADD). An additional random forest model was established based on indicators for the minimum cross-validation error of Thermomonosporaceae, Clostridiaceae, 0319-A21, Oxalobacteraceae, and Syntrophobacteraceae, with an MAE of 55.65 ADD. The produced empirical data in this pilot study provided the evidence of feasibility that the microbial successional changes of burial soil will predict the burial time of dismembered bones and may also expand the current knowledge of the effects of bone burial on soil bacterial communities.

Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers.

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.

A human identification system for hair shaft using RNA polymorphism.

Hair is one of the common pieces of evidence at crime scenes, with abundant mitochondrial DNA but limited nuclear DNA in its shaft. It also helps to narrow the investigation scope to maternal lineage but fails to provide unique individual information. We assumed that RNA in hair shafts would be an alternative resource used to perform human identification based on the facts that (1) RNA retains the polymorphic information; (2) the multi-copy of RNA in a cell resists degradation as compared to the one-copy of nuclear DNA. In this study, we explored the potential of RNA polymorphism in hair shafts for forensic individual identification. A SNaPshot typing system was constructed using 18 SNPs located on 11 genes (ABCA13, AHNAK, EXPH5, KMT2D, KRT35, PPP1R15A, RBM33, S100A5, TBC1D4, TMC5, TRPV2). The RNA typing system was evaluated for sensitivity, species specificity, and feasibility for aged hair samples. Hair samples from a Shanxi population in China were used for the population study of the system. The detection limit of the assay was 2 ng RNA. The CDP of these 11 genes was 0.999969 in the Shanxi population. We also identified the concordance of the RNA and DNA typing results. In summary, we developed an RNA typing method to perform human identification from hair shafts, which performed as accurately as nuclear DNA typing. Our method provides a potential basis for solving the human identification problem from hair shafts, as well as other biological materials that lack nuclear DNA.

Exploration and Practice of the "One Combination, Two Highlights, Three Combinations, Four in One" Innovative Talents Training Mode in Forensic Medicine.

Talent is one of the basic and strategic supports for building a modern socialist country in all aspects. Since the 1980s, the establishment of forensic medicine major and the cultivation of innovative talents in forensic medicine have become hot topics in higher education in forensic medicine. Over the past 43 years, the forensic medicine team of Shanxi Medical University has adhered to the joint education of public security and colleges, and made collaborative innovation, forming a training mode of "One Combination, Two Highlights, Three Combinations, Four in One" for innovative talents in forensic medicine. It has carried out "5+3/X" integrated reform, and formed a relatively complete talent training innovation mode and management system in teaching, scientific research, identification, major, discipline, team, platform and cultural construction. It has made a historic contribution to China's higher forensic education, accumulated valuable experience for the construction of first-class major and first-class discipline of forensic medicine, and provided strong support for the construction of the national new forensic talent training system. The popularization of this training mode is conducive to the rapid and sustainable development of forensic science, and provides more excellent forensic talents for national building, regional social development and the discipline construction of forensic science.

A recombinase polymerase amplification (RPA) combined with strip visualization method for RNA-based presumptive tests of saliva and vaginal secretion.

Identifying the origin of body fluids is a critical step in a forensic investigation. One widely used method to identify human body fluids is based on the color visualization of immune antigen detection strips for detecting hemoglobin in blood and prostate-specific antigen in semen. It is highly imperative to construct an easy-to-perform, mRNA-based method for the point-of-care identification of other human body fluids, such as saliva and vaginal secretion. Here, we established specific strips with the mRNA markers STATH (for saliva) and SPINK5 (for vaginal secretion) via reverse transcription recombinase polymerase amplification (RT-RPA) and lateral flow dipstick (LFD) assays (RT-RPA-LFD). RT-RPA could be accomplished in a single tube at a wide temperature range of 30-42 â„ƒ within 10-25 min if we do not count time for RNA extraction. The diluted RPA products were added onto the LFD strip pad to visually observe the color change of the Control/Test line. The tissue specificity and detection limit of the assays were evaluated using the optimized reaction conditions of RPA at 37 â„ƒ for 15 min. The positive signals of STATH were observed both in saliva and nasal secretions. SPINK5 was positive in a template-dependent manner in 4 out of 30 female urine samples in addition to vaginal secretion and menstrual blood samples. Cross-reactions were not detected in semen, skin swabs, sweat, or male urine. Both assays were capable of detecting aged samples, which were stored for 180 days (saliva) or 300 days (vaginal secretion) at room temperature. Moreover, saliva or vaginal secretion was successfully detected in all kinds of mixtures made from various body fluids. Overall, the rapid strip test method by the RT-RPA-LFD assay is simple, time-saving and highly sensitive for estimating the tissue origin of saliva and vaginal secretion. This method for the rapid RNA-based presumptive tests of the tissue type of body fluids is easy to perform prior to a multiplex mRNA analysis, which can demonstrate more reliable saliva or vaginal secretion identification.

Estimating the time since deposition (TsD) in saliva stains using temporal changes in microbial markers.

Determining the time since deposition (TsD) of traces could be helpful in the investigation of criminal offenses. However, there are no reliable markers and models available for the inference of short-term TsD. The goal of this study was to investigate the potential of the succession pattern of human salivary microbial communities to serve as an efficiency TsD prediction tool in the resolution of the forensic cases. Saliva stains exposed to indoor conditions up to 20 days were collected and analyzed by 16S rRNA profiling using high-throughput sequencing technique. Noticeable differences in microbial composition were observed between different time points, and the indoor exposure time of saliva stains were inversely correlated with alpha diversity estimates across the measured time period. The sequencing results were used to identify TsD-dependent bacterial indicators to regress a generalized random forest model, resulting in a mean absolute deviation (MAD) of 1.41 days. Furthermore, a simplified TsD predictive model was also developed utilizing Enhydrobacter, Paenisporosarcina, and Janthinobacterium by quantitative PCR (qPCR) with a MAD of 1.32 days, and then forensic practice assessment were also performed by using mock samples with a MAD of 3.53 days. In conclusion, this study revealed significant changes in salivary microbial abundance as the prolongation of TsD. It demonstrated that the microbial biomarkers could be invoked as a "clock" for TsD estimation in human dried saliva stains.

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

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

Identification of the vaginal secretion donor in mixture stains using polymorphic cSNPs on mRNA biomarkers.

In recent years, RNA profiling has become an important technique in identifying the origin of human body fluids/tissues. Both perpetrators and victims can be identified from stains involving vaginal secretions (VS), such as sperm/VS mixtures left on condoms, bed sheets, or papers, etc. Body fluid specific RNA typing could link the source of body fluids/tissues and the identity of the donor. In this study, we aimed to trace the donor of VS in mixture stains using body fluid-specific mRNA markers and construct a coding single nucleotide polymorphism (cSNP) typing system for VS. We screened 8 VS-specific mRNA biomarkers (MUC4, SFTA2, CYP2A6, MYOZ1, FUT6, ESR1, SPINK5, and SERPINB13) encompassing 18 cSNPs. The RNA obtained from various body fluid/tissue samples was treated with reverse transcription polymerase chain reaction (RT-PCR) and then followed by a multiplex PCR and SNaPshot mini-sequencing assay. The detection limit of the assay was 0.08 ng RNA. For single-source body fluid, the positive cSNP typing was only shown in VS and void in non-VS body fluids/tissues. For laboratory-generated VS-containing mixtures, the minor VS contributor could be successfully detected at a ratio of 1:10-1:500. We also confirmed the concordance of DNA typing and mRNA typing for the cSNPs in this system. In summary, we established an 18-cSNP typing system for VS with high sensitivity and specificity, which could identify both the donor and the tissue origin simultaneously. This was shown to be a powerful tool for identifying the VS donor in those VS-containing mixture stains.

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.

Risk of sudden coronary death based on genetic background in Chinese Han population.

Associations between gene variations and sudden cardiac arrest or coronary artery disease have been reported by genome-wide association studies. However, the implication of the genetic status in cases of sudden coronary death (SCD) from the Chinese Han population has remained to be investigated. The present study established a mini-sequencing system to examine putative death-causing single nucleotide polymorphisms (SNPs) using multiplex PCR, single base extension reaction and capillary electrophoresis techniques. A total of 198 samples from the Chinese Han population (age range, 34-71 years; mean age, 53.86 years) were examined using this method. Samples were classified into three groups: Coronary heart disease (CHD, n=70), SCD (n=53) and control (n=75) group. Significant associations were identified for 10, 4 and 6 SNPs in CHD, SCD and sudden death from CHD, respectively, using the χ2 test. The SNPs obtained by binary logistic regression may be used to assess and predict the risk of disease. The predictive accuracy of the SNPs in each prediction model and their area under the receiver operating characteristic curve (AUC) values were determined. The AUC of the four SNPs (rs12429889, rs10829156, rs16942421 and rs12155623) to predict CHD was 0.928, the AUC of the six SNPs (rs2389202, rs2982694, rs10183640, rs597503, rs16942421 and rs12155623) to predict SCD was 0.922 and the AUC of the four SNPs (rs16866933, rs4621553, rs10829156 and rs12155623) to predict sudden death from CHD was 0.912. The multifactor dimensionality reduction values were as follows: 0.8690 (prediction model of CHD), 0.7601 (prediction model of SCD) and 0.7628 (prediction model of sudden death from CHD). Taken together, the results of the present study suggested that these SNPs have considerable potential for application in genetic tests to predict CHD or SCD. However, further studies are required to investigate the putative functions of these SNPs.

Development of a multiplex methylation-sensitive restriction enzyme-based SNP typing system for deconvolution of semen-containing mixtures.

The identification of mixed stains has always been a difficult problem in personal identification in the forensic field. In recent years, tissue-specific methylation sites have proven to be very stable biomarkers for distinguishing tissue origin. However, it is still challenging to perform tissue source identification and individual identification simultaneously. In this study, we developed a method that uses tissue-specific methylation markers combined with single-nucleotide polymorphism (SNP) markers to detect semen from mixed biofluids and to identify individuals simultaneously. Semen-specific CpG markers were chosen from the literature and further validated utilizing methylation-sensitive restriction endonuclease (MSRE) combined with PCR technology. The neighboring SNP markers were searched in the flanking sequence of the target CpG within 400 bp, and SNP typing was then carried out through a single-base extension reaction followed by capillary electrophoresis. Eventually, a method of MSRE combined with SNaPshot that could detect 12 compound CpG-SNP markers was developed. Using this system, 10 ng of total DNA and DNA mixture with semen content up to 25% could be typed successfully. Moreover, the cumulative discrimination power of the system in the northern Chinese Han population is 0.9998. This study provides a valuable strategy for forensic practice to perform tissue origin and individual identification from mixed stains simultaneously.

Predicting the postmortem interval of burial cadavers based on microbial community succession.

Previous studies have demonstrated that microbial community succession during the decomposition of cadavers could be used to estimate the postmortem interval (PMI). However, the vast majority of the existing studies focused on exposed cadavers. In fact, burial cadavers are common scenarios for forensic investigations. In this study, the microbial communities from gravesoil, rectum and skin of burial SD rat cadavers during decomposition were characterized using 16S rRNA gene high-throughput sequencing. We predicted PMI based on the microbial community succession. Obvious differences in microbial community structures were observed between different stages of decomposition. Later decay stages had a lower alpha diversity compared to earlier decay stages. Significant linear relationships between similarities of the microbial communities and postmortem intervals were observed, manifesting regular succession over the course of decomposition. Furthermore, we combined random forest models with postmortem microbial features to predict PMI. The model explained 86.83%, 84.55% and 81.67% of the variation in the microbial community, with a mean absolute error of 1.82, 2.06 and 2.13 days within 60 days of decomposition for gravesoil, rectum and skin of burial cadavers, respectively. Overall, our results suggested that postmortem microbial community data could serve as a potential forensic tool to estimate accurate PMI of burial cadavers.

Predicting human age by detecting DNA methylation status in hair.

Age prediction is of great importance for criminal investigation and judicial expertise. DNA methylation status is considered a promising method to infer tissue age by virtue of age-dependent changes on methylation sites. In recent years, forensic scientists have established various models to predict the chronological age of blood, saliva, and semen based on DNA methylation status. However, hair-inferred age has not been studied in the field of forensic science. In this study, we measured the methylation statuses of potential age-related CpG sites by using the multiplex methylation SNaPshot method. A total of 10 CpG sites from the LAG3, SCGN, ELOVL2, KLF14, C1orf132, SLC12A5, GRIA2, and PDE4C genes were found to be tightly associated with age in hair follicles. A correlation coefficient above 0.7 was found for four CpG sites (cg24724428 and Chr6:11044628 in ELOVL2, cg25148589 in GRIA2, and cg07547549 in SLC12A5). Among four age-prediction models, the multiple linear regression model consisting of 10 CpG sites provided the best-fitting results, with a median absolute deviation of 3.68 years. It is feasible to obtain both human identification and age information from a single scalp hair follicle. No significant differences in methylation degree were found between different sexes, hair types, or hair colors. In conclusion, we established a method to evaluate chronological age by assessing DNA methylation status in hair follicles.

Identification of coding region SNPs from specific and sensitive mRNA biomarkers for the deconvolution of the semen donor in a body fluid mixture.

mRNA markers provide a very promising method for the identification of human body fluids or tissues in the context of forensic investigations. Previous studies have shown that different body fluids can be distinguished from each other according to their specific mRNA biomarkers. In this study, we evaluated eight semen-specific mRNA markers (KLK3, NKX3-1, CKB, KLK2, PRAC1, SEMG1, TGM4, and SORD) that encompass 12 coding single nucleotide polymorphisms (cSNPs) to identify the semen contributor in a mixed stain. Five highly specific and sensitive mRNA markers for blood, menstrual blood, saliva, vaginal secretions, and skin were also incorporated into the PCR system as body fluid-positive controls. Reverse transcription polymerase chain reaction (RT-PCR), multiplex PCR and SNaPshot mini-sequencing assays were established for the identification of semen-specific mRNA. The amplicon size ranged from 133 to 337 bp. The semen-specific system was examined against blood, menstrual blood, saliva, vaginal secretions, and skin swabs. The eight mRNA biomarkers were semen-specific and could be successfully typed in laboratory-generated mixtures composed of different body fluids supplemented with 1 ng of semen cDNA. This system possessed a high sensitivity that ranged from 1:10-1:100 for detecting trace amounts of semen in semen-containing body fluid mixtures. Additionally, our results demonstrated that the cSNPs polymorphisms included in the mRNA markers were concordant with genomic DNA (gDNA). Despite the presence of other body fluids, the system exhibited high sensitivity and specificity to the semen in the mixture. In future studies, we will add other cSNPs from the semen-specific genes using massively parallel sequencing to further improve our system.

DIP-microhaplotypes: new markers for detection of unbalanced DNA mixtures.

The identification of a suspect in a degraded and unbalanced DNA mixture has been a challenge for the standard short tandem repeat polymorphisms (STR) typing. Several methods have been introduced to solve this problem, such as DIP-STR, DIP-SNP, and SNP-STR markers. In this study, we proposed DIP-microhaplotype (deletion/insertion linked a chain of SNPs) as a kind of new genetic marker to type the unbalanced and degraded DNA mixture. We established the detection method with ten DIP-microhaplotype markers including 26 SNPs using allele-specific multiplex PCR followed by SNaPshot assay. This novel compound marker allows us to detect the minor DNA with a sensitivity of 1:100 to 1:1000 in a DNA mixture of any gender. Most of the DIP-microhaplotype markers had a relatively high probability of informative alleles with an average informative value (I value) of 0.308. In all, we proposed DIP-microhaplotype as a novel type of DNA marker for the detection of minor contributor from unbalanced DNA mixtures. Due to their inherent shorter length, higher polymorphism, and sensitivity, DIP-microhaplotypes are promising markers for the examination of the degraded and unbalanced mixtures in forensic stains or clinical chimeras.

DNA typing from skeletal remains: a comparison between capillary electrophoresis and massively parallel sequencing platforms.

Skeletal remains encountered frequently in forensic applications are a challenging specimen, since their DNA is usually degraded due to harsh conditions, limiting the utilization of skeletal DNA. Forensic scientists have tried various methods to extract DNA from skeletal remains of low quantity and poor quality or improve detecting technology for more information from compromised DNA. Compared with traditional capillary electrophoresis (CE), massively parallel sequencing (MPS) is more sensitive to shorter fragments, able to detect allele sequences for variations from core motif or flanking regions, and able to detect more markers with a higher discrimination power. In this study, short tandem repeats (STR) and single nucleotide polymorphisms (SNP) from 35 human skeletons were genotyped by MPS platform, and CE method was also used to perform STR genotyping. The results indicated that the detection rates reached 100.00% in 16 of 35 samples with MPS method, while the same 100.00% was reached in only 9 samples with CE. The success rates of MPS were also higher than that of CE method in shared 21 loci (excluding Y-indel, DYS391, and SE33), especially in loci detected by MPS method only. Besides, all SNPs (124 and 90 SNPs in males and females) were detected in 18 samples of 35 samples by MPS method. Some intra-allelic sequence variants were observed in eight loci (D21S11, D8S1179, D5S2800, D3S1358, vWA, D2S1338, D1S1656, D12S391) using MPS technology. Interestingly, there is a sample showing genotyping disagreement in FGA locus. The clone sequencing verified that a "T" deletion discovered in flanking sequence of FGA led to wrong genotyping on Ampliseq Converge. Our results indicated that MPS could be adopted in qualified labs as a supplementary when the DNA of skeletal remains are hard to identify.

Correction to: DNA typing from skeletal remains: a comparison between capillary electrophoresis and massively parallel sequencing platforms.

The name of an author was misspelled. The correct spelling is "Linlin Gao" instead of Linin Gao.

A method of identifying the blood contributor in mixture stains through detecting blood-specific mRNA polymorphism.

In the past decades, messenger RNA (mRNA) biomarkers have been employed to identify the origin of body fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be applied to identify individuals in mixture samples composed of two body fluids. In this study, we selected five blood-specific mRNA biomarkers of venous blood (SPTB, CD3G, AMICA1, ANK1, and GYPA) that encompass 16 SNPs to identify the mixture contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva, and vaginal secretions were amplified and typed as body-fluid positive controls. We established the system of multiplex PCR and single base extension (SBE) reaction followed by CE. The amplicon size was between 90bp and 294bp. The peripheral blood specificity was examined against other human body fluids, including saliva, semen, skin, menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and could be successfully typed in homemade mixtures which are composed of different body fluids with 1 ng peripheral blood mRNA added. This system showed a supersensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese population. It was the first time to establish a method for identifying the blood donors and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot assay. This peripheral blood specific SNP typing system showed high sensitivity to the typing of blood source specific markers regardless of other body fluids in the mixture.