DNA analysis by capillary electrophoresis with fluorescent short tandem repeat (STR) detection plays a dominant role in DNA testing of various cases, but for difficult biological samples, such as highly corrosive and degraded bones, it is not effective due to the small number of motifs STR detected. This paper describes the application of second-generation sequencing technology in DNA testing of difficult biological samples such as highly degraded bones in forensic science practice. In a murder case that had remained unsolved for 30 years, the traditional STR testing method did not yield effective STR locus typing, but a complete autosomal locus typing was obtained by second-generation sequencing using the Precision ID GlobalFiler^TM NGS STR Panel v2 kit. However, through the second-generation sequencing method using Precision ID GlobalFiler^TM NGS STR Panel v2 kit, the genotypes of 31 autosomal loci were obtained, which helped the case unit to identify the source of the body and provided key technical support for the investigation of the case. By analyzing the advantages of NGS technology for such highly degradable samples and exploring the consistency between the STR locus typing results of CE electrophoresis detection and NGS typing results, it is shown that NGS technology can overcome the analysis limitations of CE for such highly degradable samples, supplement the shortcomings of CE technology, and provide new methods and technique for forensic DNA testing and analysis of difficult biological samples.

Presently, such an increasing demand emerges for the identification of complex genetic relationships in forensic practice that requires the combination of a variety of genetic markers, e.g., STRs, X/Y specific indicators, SNPs and mitochondrial DNA. Next generation sequencing (NGS) approach is able to integrate multiple genetic markers into one detection system, excelling the traditional capillary electrophoresis (CE) technology in overcoming the cumbersome operational procedures and experimental handling. Here, a typing panel, designated as DNATyper^TMNGSPanel v1.0, was developed with enclosure of human 29 autosomal STRs, 36 Y-STRs, 32 X-STRs, 71 Y-SNPs and the whole genome of mtDNA. Its validation was implemented under the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), having evaluated the items of repeatability, accuracy, consistency, sensitivity, mixture and species specificity. For the developed DNATyper^TMNGSPanel v1.0, a consistency of 99.72% was shown to the detection with CE, plus the complete identical results coming true between the genic loci shared with it and the Forenseq™ DNA Signature Prep Kit. No allelic loss occurred to the DNA template of 0.5 to 10 ng, yet the respective 2 and 9 loci being lost with the 0.25 ng and 0.125 ng of template. Regarding to the mixture of male and female substances, the mixed ratio of 2׃1 had the the female’s begun to appear allelic loss, contrasting that the mixed ratios of 9׃1, 4׃1, 2׃1 and 1׃1 brought the positive detections to 54.72%, 81.13%, 98.11% and 100%; and the mixed ratio of 1׃4 made the male’s happen to allelic loss, opposite the mixed ratios of 1׃1, 1׃2, 1׃4 and 1׃9 rendering the positive detections to 100%, 100%, 90.24% and 82.93%. The test of species specificity proved that the developed DNATyper^TMNGSPanel v1.0 hardly amplified the DNA from pig, cattle, mouse, either the cynomolgus monkey or the rhesus one. In conclusion, the developed DNATyper^TMNGSPanel v1.0 is of high detection throughput, high sensitivity and stability, accurate and repeatable typing and good ability for mixed sample to detect. Also, it can deliver the genetic information related to paternity and maternity at the same time in just a single run of test with any common biological samples, hence eligible for boosting the capability of individual identification and efficiency of consanguinity determination.

Targeting against small RNA (microRNA or miRNA) from five body fluids of forensic-relevant biological samples (venous blood, menstrual blood, saliva, semen and vaginal secretions), next generation sequencing (NGS) was adopted to sequence the expression profiles of miRNA from each of the above five body fluids. Partial least squares-discriminant analysis (PLS-DA) was operated to build the prediction model with the obtained data of expression profiles, successively having its applicability explored about tracing the source of forensic-relevant body fluids. Specifically, the small RNA library was optimized into its preparation process, then subjected to Ion Torrent S5 XL sequencing with the relevant above-mentioned five body fluids (10 samples each) collected from 20 unrelated healthy Han-ethnic individuals (10 ones of male and female, aging from 23 to 38 years old). The PLS-DA prediction model was constructed with the data from small RNAs of those five body fluids, and evaluated of its prediction accuracy for different subsets of combined miRNA markers. From the miRNA expression profiles and distribution characteristics of those five forensic-relevant body fluids, six were shown same among the top 10 miRNAs expressed in venous blood and the menstrual, and so were four in saliva and vaginal secretions. The prediction model achieved the respective accuracy of 0.95, 0.94 and 0.89 for the miRNA markers (full, 107 and 11 ones) to construct into their datasets. The miRNomes were here obtained from five forensic-relevant body fluids through NGS sequencing and other necessary processing. The resultant body fluid prediction model was tentatively established out of PLS-DA exploration, potential of value to provide a basis for miRNome to apply into forensic body fluid identification.

Microhaplotypes, an emerging type of forensic genetic marker, have been being used for individual identification, ancestry inference and mixture deconvolution as they were incessantly explored and developed. Nevertheless, the concise microhaplotype allelic names have not yet been suggested although there are standardized nomenclatures that were proposed for microhaplotype loci. Here, a proposal was put forward for discussion about microhaplotype alleles being designated with Arabic numerals. For a microhaplotype consisted of single nucleotide polymorphisms (SNPs), the SNPs are locally ordered with their positions in human genome, and have the relevant RefSNP alleles in the dbSNP database be accepted as their possible genotypes. Microhaplotype alleles are allowed to list in every possible combination of the RefSNP alleles before they are arranged in alphabetical order. The ordered alleles are subsequently named with consecutive positive integers starting from 1. Such a nomenclature would be convenient for forensic applications, especially the mixture deconvolution, capable of being enrolled into the software for forensic genetic calculations including PowerStats, Arlequin and STRUCTURE.