The geographical origin of forensic evidence provides important information for crime investigation and solving cases,and is one of the key elements of criminal cases.Previous studies have shown significant differences in the distribution of microorganisms in different regions.Detecting environmental DNA samples and inferring the geographical and spatial sources can provide clues and evidence for case handling.However,due to the diversity of criminal environments and the trace amount of frequently encountered exhibits,stable and reliable technical methods for inferring geographical origin from environmental DNA are not yet available.This article summarizes the sample collection and DNA extraction methods for four types of environmental samples:dust,soil,water,and air.It compares the differences between amplicon sequencing and metagenomic sequencing in studying environmental biological populations,outlines the full process of high-throughput sequencing-based data analysis,and focuses on reviewing the research progress in inferring geographical sources of environmental samples based on bacteria,fungi,and other eukaryotes,to provide references for establishing sequencing and analysis methods for environmental DNA in forensic DNA laboratories and exploring environmental DNA information for forensic applications.

Objective Sequencing depth is a key parameter in next generation sequencing,which is closely related to the accuracy of sequencing results.Forensic biological evidence examination requires extremely high accuracy.It is crucial to scientifically and reasonably set the sequencing depth analysis threshold for forensic next generation sequencing testing.Methods This study used targeted sequencing data of microhaplotypes from 50 samples with known genotypes.By calculating the accuracy,precision,recall,and F1 score of each locus under various threshold conditions,two types of analysis threshold setting methods,which were based on fixed read count and fixed sequencing depth ratio,were studied extensively.Results The results showed that false positives were observed when the analysis threshold was set at 50×or 100×.When the analysis threshold was set at 200×,false negatives were observed.When the analysis threshold was set at 1.5%,3.0%,or 4.5%,false positives were observed.This study further proposed a third type of analysis threshold setting method,which was based on sequencing depth ratio scatter plots.With this method,no false positive or false negative was observed in the results.This article then explored four factors that lead to significant differences in the sequencing depth of forensic next generation sequencing experiments,compared with the analysis threshold setting method for capillary electrophoresis technology,and discussed the correlation between analysis thresholds and the ability to distinguish mixed DNA.Conclusion Employing the sequencing depth ratio scatter plot method to set analysis threshold has significant application value in next generation sequencing-based forensic genetic marker genotyping.

To explore the expression of p62 protein in lung adenocarcinoma (LUAD). In this study, a cross-sectional study was adopted. From December 2011 to May 2013, 60 patients with lung adenocarcinoma who were diagnosed and treated in Tongji Hospital of Tongji University, Shanghai were selected for paraffin embedding and tissue chip preparation, and immunohistochemistry (IHC) technology was used to detect the expression of p62 in lung adenocarcinoma patients′ cancer tissues and adjacent tissues, and analyze the relationship between p62 expression and the clinicopathological characteristics and survival prognosis of patients with lung adenocarcinoma; at the same time, 6 cases of lung adenocarcinoma were selected by random sampling cancer tissues and adjacent tissues were detected by Western Blot (WB) to detect p62 protein and analyzed by gray value. Preoperative examination specimens of inpatients with lung adenocarcinoma diagnosed from April 2018 to early October 2019, and plasma specimens of healthy subjects were collected, and enzyme linked immunosorbent assay (ELISA) was used to detect lung adenocarcinoma patients and healthy patients. The expression of p62 in the plasma of the subjects was statistically analyzed using SPSS 22.0 software. The results of IHC showed that the positive expression rate of p62 in cancer tissues was significantly higher than that in adjacent tissues, and the difference was statistically significant ( t=5.593, P<0.001). Similarly, WB results showed that the expression of p62 protein in cancer tissues was significantly higher than that in adjacent tissues. It is statistically relevant ( t=2.238, P=0.049). The expression of p62 was statistically correlated with tumor size, clinicopathological stage and lymph node metastasis in patients with lung adenocarcinoma (all P<0.05). The overall survival of patients with lung adenocarcinoma with high p62 expression was worse than that of patients with low p62 expression (95 %CI was 0.238-0.870, P=0.028), suggesting that the high expression of p62 is related to the poor prognosis of patients with lung adenocarcinoma. The level of p62 protein in the plasma of patients with lung adenocarcinoma was significantly higher than that in the healthy control group. The difference was statistically significant ( t=8.533, P<0.001). The area under the receiver operating characteristic curve was 0.835 (95 %CI was 0.779-0.891, P<0.001), which is significantly higher than CEA, CA125, CA153 and other single traditional indicators, and the combined detection of four indicators has the highest diagnostic efficiency. p62 was strongly expressed in cancer tissues and serum, which is related to the poor prognosis and overall survival rate of LUAD patients.

To explore the expression of p62 protein in lung adenocarcinoma (LUAD). In this study, a cross-sectional study was adopted. From December 2011 to May 2013, 60 patients with lung adenocarcinoma who were diagnosed and treated in Tongji Hospital of Tongji University, Shanghai were selected for paraffin embedding and tissue chip preparation, and immunohistochemistry (IHC) technology was used to detect the expression of p62 in lung adenocarcinoma patients′ cancer tissues and adjacent tissues, and analyze the relationship between p62 expression and the clinicopathological characteristics and survival prognosis of patients with lung adenocarcinoma; at the same time, 6 cases of lung adenocarcinoma were selected by random sampling cancer tissues and adjacent tissues were detected by Western Blot (WB) to detect p62 protein and analyzed by gray value. Preoperative examination specimens of inpatients with lung adenocarcinoma diagnosed from April 2018 to early October 2019, and plasma specimens of healthy subjects were collected, and enzyme linked immunosorbent assay (ELISA) was used to detect lung adenocarcinoma patients and healthy patients. The expression of p62 in the plasma of the subjects was statistically analyzed using SPSS 22.0 software. The results of IHC showed that the positive expression rate of p62 in cancer tissues was significantly higher than that in adjacent tissues, and the difference was statistically significant ( t=5.593, P<0.001). Similarly, WB results showed that the expression of p62 protein in cancer tissues was significantly higher than that in adjacent tissues. It is statistically relevant ( t=2.238, P=0.049). The expression of p62 was statistically correlated with tumor size, clinicopathological stage and lymph node metastasis in patients with lung adenocarcinoma (all P<0.05). The overall survival of patients with lung adenocarcinoma with high p62 expression was worse than that of patients with low p62 expression (95 %CI was 0.238-0.870, P=0.028), suggesting that the high expression of p62 is related to the poor prognosis of patients with lung adenocarcinoma. The level of p62 protein in the plasma of patients with lung adenocarcinoma was significantly higher than that in the healthy control group. The difference was statistically significant ( t=8.533, P<0.001). The area under the receiver operating characteristic curve was 0.835 (95 %CI was 0.779-0.891, P<0.001), which is significantly higher than CEA, CA125, CA153 and other single traditional indicators, and the combined detection of four indicators has the highest diagnostic efficiency. p62 was strongly expressed in cancer tissues and serum, which is related to the poor prognosis and overall survival rate of LUAD patients.

Objective To establish a SYBR Green real-time PCR detection method with tissue-specific miRNAs and explore a novel approach for forensic body fluid identification. Methods The frequently reported 6 standard miRNAs were synthesized to establish a SYBR Green method, and verify with body fluid. The relative expression data for the 6 miRNAs were obtained using SYBR Green real-time PCR method in peripheral blood, menstrual blood, saliva and semen. Results The assays showed that miRNA205 permitted the unequivocal identification among different fluids. miRNA451 and miRNA144 could be used to distinguish blood from non-blood. Menstrual blood or peripheral blood could be identified through miRNA214. miRNA888 and miRNA891 was highly expressed in semen. Conclusion The results of this study indicate that miRNA SYBR Green profiling may provide a feasible and effective approach to body fluid identification for forensic casework.

Objective To genotype mixed samples with next generation sequencing and evaluate its prospects in forensic DNA application. Methods Three mixed biological samples from rapes cases and their reference samples were collected. DNA was extracted using the MagAttract M48 DNA Manual Kit(200). The ForenSeqTMDNA Signature Prep Kit was used for library preparation, and next generation sequencing was performed on the MiSeq FGx system. The ForenSeqTMUniversal Analysis v1.2.1 software was used for data analysis. NGS-based STR results were compared with CE-based genotypes. Results A single length polymorphic STR allele in the mixed profile could be recognized as two sequence polymorphic STR alleles from different donors, which would assist mixed profile analysis. Such phenomenon was observed in D3S1358, D9S1122 and D13S317 in this work. Conclusion Our results suggested that precision STR genotyping of mixed samples based on NGS can provide more information and hints for mixed STR profile separation.

Next generation sequencing (NGS),also known as massively parallel sequencing (MPS),with significant advantages such as high throughput,rapidness,integration and low cost,has been widely used in research and clinical diagnosis fields and promises bright future for forensic applications.Compared with traditional DNA sequencing methods,NGS technologies allow the simultaneous detection of various forensic genetic markers.The classical CE-based STR genotyping method only can differentiate alleles with length polymorphism.However,there are occasions like DNA base substitution and repeat sequence variations,which give different alleles with the same size of amplicons.These alleles are heritable from parents to off-springs,and could be of great importance for solving criminal cases.NGS-based STR genotyping can be compatible with current STR data output mode,and allows for the full resolution of STR loci by providing additional sequencing polymorphism information.In this article,the authors focused on the forensic STR genetic markers and next generation sequencing,and reviewed recent progresses on NGS-based STR genotyping.Perspectives of NGS-based full resolution STR genotyping were presented and possible challenges were discussed in the hope of providing a reference for related studies and applications.

Capillary electrophoresis-based STR genotyping is accepted as the gold standard for human individual identification. Next generation sequencing (NGS) allows for the full resolution of STR base composition, and has the potential to be widely used in the field of forensics. Compared with length polymorphism, STR sequencing could provide more information, and quantitatively calculating the forensic parameters is necessary. In this paper, we established simple models for length-based and sequence-based STRs, and calculated the forensic parameters for both models. The results showed that for a single STR locus, compared with length polymorphism, STR sequence polymorphism could provide higher power of discrimination and power of exclusion, indicating sequence-based STR marker have stronger ability for identifying unrelated individuals and exclude non biological father. By combining 15 non-linkage loci for forensic DNA analysis, the cumulative matching probability values for length-based and sequence-based STR models are at 10-18and 10-26levels, respectively. Only 10 non-linkage sequence-based STR is required to reach a cumulative matching probability of as high as 15 length-based STR loci. It is hoped that these simulated models and calculations can provide a reference for the forensic application of NGS-based STR genotyping.

DNA extracted from 100 unrelated Chinese were detected by using the Polymerase Chain Reaction (PCR), mini polyacryl gel vertical electrophorese and silver staining techniques at the p33. 4 locus. Among 100 unrelated individuals, 8 alleles were detected,Foster-Freeman BIOTRAC system indicated that the number of copies of the tandem repeats in different allele was 7, 10 to 15 respectively.There was a rare allele whose copy number was more than 13 but less than 14. The allele fragmehtlength varied from 603 to 1115bp,the allele freqency 0.5 % ~ 53. 5 %,heterozygosity 64 %,DP value84. 5 %. Pedigree analysis indicated that alleles of p33. 4 locus obey Mendel's Laws. Successful typingof various tissues and single hair folicle had confirmed that this technique is suitable for personal identification. In addition, by using Chelex we had established an alterntive method for extracting DNAfrom biological materials,whlch is rapid and easy to perform.

The polymorphisms of minisatellite p33.6 (D1S111) locus were typed rapidly and accurately us- ing polymerase chain reaction (PCR). The amplified fragments were analyzed by mini- polyacrylamide gel electrophoresis followed by silver stain. The distribution of allele frequencies among 100 unrelated Chinese is reported. 14 alleles, containing 9 to 22 repeat units, had been detected with a heterozygosity of 76%. The allele frenquencies were from 0.5 %to 35.5 %. The size of amplified fragments ranged from 435 bp to 925bp. The discrimination power of p33.6 locus was 0. 916. The Amp --FLP was inherited according to the Mendelian Law. The results showed that the polymorphisms of p33.6 locus can be used for individual identification and paternity test.