Different types of molecular and genetic markers are widely used in basic research of forensic medicine and forensic practices, mainly concentrated on individual identification and parentage testing in forensic genetics. In addition, these markers are also applied for postmortem interval estimation, cause of death inference, drug metabolism, forensic age estimation, research of mental diseases, involving multiple disciplines such as forensic pathology, forensic toxicology, forensic anthropology, and forensic psychiatry.[2–5]
The process of deoxyribonucleic acid (DNA) self-replication, DNA transcription into messenger RNA (mRNA), and translation into protein is the central dogma followed by human genetic information transmission. DNA has a high degree of fidelity in the replication process, all cells share consistent DNA genetic information, which are the key premises for individual identification of different biological samples in forensic practices. Cells with different origins, functions, or states are significantly different in gene expression, and gene expression is of time sequence.[3,7] These differential expressions can be detected at the ribonucleic acid (RNA) level, which is also a major theoretical support for the application of RNA molecular markers in forensic medicine. Both DNA and RNA are nucleic acid molecules which have similar molecular structures and physicochemical properties, meeting the basic conditions for integrated detection.
DNA markers commonly used in forensic medicine, such as short tandem repeats (STRs), single-nucleotide polymorphisms (SNPs), insertion/deletion variants, and microhaplotypes, play a very important role in individual identification, parentage testing, ancestry inference, and detection of mixed samples. With the elaboration of RNA molecular marker screening and the diversification of analytical methods, mRNA, microRNA (miRNA), coding region SNP (cSNP), and long noncoding RNA have great application prospects in body fluids and tissues identification, age estimation, and determination of the age of stains, and can break through some technical bottlenecks that cannot be solved by DNA genetic markers, bringing new ideas and evidence to investigation and prosecution. Certain cell-specific RNA markers have been selected for the identification of common stains (peripheral blood, semen, saliva, menstrual blood, and vaginal secretion), skin, nasal mucosa, and rectal mucosa.[8–13] Zhao etal. comprehensively reviewed the biological foundation, detection method, and reference genes in the application of forensic mRNA for body fluid identification.[14–15] In a recent review, Haas etal. summarized the next-generation sequencing (NGS)-based research process of RNA in body fluid identification. These review articles provided crucial information for body fluid identification in forensic casework.
Currently, DNA and RNA are often studied separately. Especially for biological samples used in public security practices, the core content of detection is still DNA genetic markers represented by STR, and less attention is paid to the investigation clues and evidence value contained in RNA. In order to fully reveal variant information of biological samples and realize the evidence correlation of various types of information, it is necessary to study and analyze biological evidence from various perspectives. The method based on traditional capillary electrophoresis (CE) technology can achieve the integrated detection of a small number of DNA and RNA markers. However, in the past 10 years or so, the NGS technology has been continuously developing, and its high-throughput and integrated technical characteristics provide a new perspective for the integrated detection of DNA and RNA. With the realization of integrated detection of the two markers on the NGS platform, it is expected that they will play a unique role in saving the detection time, reducing the detection costs, and comprehensively analyzing biological information. In this paper, we will review the current experimental strategies for co-extraction of DNA and RNA in forensic medicine and the integrated detection methods based on the CE technology, and mainly summarize and analyze the routes and characteristics of the integrated detection technology based on the NGS platform in other research fields, so as to provide a reference for the integrated detection of DNA and RNA markers in future forensic medicine.
STRATEGIES FOR CO-EXTRACTION OF DEOXYRIBONUCLEIC ACID AND RIBONUCLEIC ACID
Under the premise of sufficient biological samples, a sufficient nucleic acid can be obtained by extracting DNA or RNA separately. However, for a limited number of biological samples from crime scenes, nucleic acid co-extraction is very important. In the early 21st century, Bauer and Patzelt and Alvarez etal. based on different organic extraction methods, achieved the simultaneous isolation of DNA and RNA for downstream detection from the same body fluid stain, revealing the possibility of co-extraction of DNA and RNA in forensic evidence.[18,19]
In 2011, Bowden etal. collected RNA from the lysate after the magnetic beads adsorbed DNA using RNA extraction reagents while extracting DNA from body fluids with the IQ magnetic bead method. This method can not only ensure the extraction efficiency of DNA but also achieve the extraction of RNA, grasping the key points of detection while realizing the full utilization of biological samples. On the basis of this research idea, some teams have used the combination of different DNA and RNA extraction kits for co-extraction.[8,21,22] Wang etal. showed that the DNA genotyping results of different co-extraction methods are basically the same, but the RNA extraction effects are different. Not all combination methods are suitable for co-extraction of nucleic acid, and reasonable protocol adjustment is necessary.
The launch of a commercial dual nucleic acid co-extraction kit simplifies experimental operations. Schweighardt etal. evaluated four commercial co-extraction kits and believed that the DNA and RNA extracted using the ZR-Duet™ DNA/RNA MiniPrep kit had the best detection effect. However, the extraction efficiency of the co-extraction kit is sometimes lower than that of the DNA/RNA extraction method alone, which may affect the DNA detection results of trace biological evidence.
Studies have shown that the experimental methods used to extract DNA or RNA alone are all likely to detect another nucleic acid. Lewis etal. detected miRNA s in DNA solutions extracted with different methods. Sun etal. obtained effective STR typing results of residual DNA in RNA extraction solution, which indicates that the experimental process of co-extraction has the potential to be further optimized and integrated.
DEOXYRIBONUCLEIC ACID AND RIBONUCLEIC ACID CO-ANALYSIS METHOD BASED ON CAPILLARY ELECTROPHORESIS PLATFORM
The CE technology is the most commonly used detection method in forensic genetics laboratories, so it is of great significance to detect different molecular markers on the CE platform. The European DNA typing group has carried out several collaborative studies and obtained a combination of mRNA molecular markers suitable for differentiating suitable for differentiating common body fluid stains and tissues, which has been validated across laboratories. While studying the RNA molecular markers, they also jointly analyzed the STR of the same sample, which fully proved the applicability of the CE technology to simultaneously analyze the two types of molecular markers.[9–12] Professor Hou Yiping’s team used unique multiple linear primers to perform reverse transcription of four miRNAs, and realized the co-analysis of miRNAs and STRs on the CE platform. The length design of linear primers is more flexible than that of stem-loop primers, which facilitates multiplex detection of more miRNA markers. The nucleic acid extraction method in the above study mainly relies on the commercial AllPrep RNA/DNA Mini kit or AllPrep RNA/DNA Micro kit for nucleic acid co-extraction, physical separation of DNA and RNA, and subsequently, a separate workflow will be arranged for experimental procedures of the two types of molecular markers.
Williams’ research group extracted total nucleic acid with the QIAamp DNA mini kit based on the nonphysical separation of DNA and RNA, and reverse transcribed the total nucleic acid mixture. miR-451 and miR-205 were selected as specific RNA molecular markers of blood and saliva, respectively. By adding customized miRNA primers to the commercial STR detection kit, the integrated detection of miRNA and STR in a single process was realized for the first time on the CE platform. The experimental steps in this study were highly integrated and the operation steps were simplified, which is an ideal idea for the integrated detection of DNA and RNA.
Selected RNA markers in the above studies are mainly applied to body fluid and tissue identification. Both DNA and RNA profiles from the same sample could make the interconnection between donor and body fluid type in caseworks, especially in sexual assault investigations. However, other forensic applications based on the CE platform are yet to be studied. Since CE technology is hardly used to detect transcript abundance, some potential forensic utilities are difficult to implement, such as the determination of the stain age and the donor age. In addition, limited by the fluorescence and the length of amplicon, the CE detection technology is quite challenging to continue to incorporate much RNA molecular markers in the same system. Therefore, there are few studies on the integrated detection of multiple types of molecular markers on the CE platform.
INTEGRATED DETECTION BASED ON THE NEXT-GENERATION SEQUENCING PLATFORM
The integrated detection of DNA and RNA based on NGS technology is rarely reported in forensic medicine, but it is widely used in clinical tumor molecular diagnosis and virus detection. Integrated detection methods in different fields can provide a reference for the application of this technology in forensic medicine.
Integrated detection methods based on separated library preparations for deoxyribonucleic acid and ribonucleic acid
In 2015, Professor Kayser’s team carried out integrated detection of 9 autosomal STRs, 1 sex-determining locus, and 14 mRNA molecular markers on the Ion personal genome machine sequencing platform, which can be used for individual identification and forensic tissue identification of the same sample. Based on the targeted amplification technology, this study used the Ion AmpliSeq™ RNA Library Kit and the Ion Plus Fragment Library kit to prepare RNA and DNA libraries, respectively and then loaded the two libraries into the same sequencing chip for sequencing. For the first time, it successfully realized the integrated detection of forensic DNA and RNA molecular markers on the NGS platform, which proved that NGS technology has great potential for the detection of multiple types of nucleic acid molecular markers. The technical route used in the study is very similar to that used in some clinical molecular diagnostics.[28–30] The experimental steps are shown in Figure 1a. The integrated detection is mainly reflected in the co-sequencing part, which requires two different sets of library preparation reagents and procedures, and its integration level is not high enough. Therefore, there is a large space for technical optimization.
In 2017, the TruSight™ Tumor 170 kit launched by Illumina can simultaneously detect 170 target genes associated with solid tumors at the DNA and RNA levels on the NGS platform. This technology could independently achieve the synthesis of complementary DNA (cDNA) and the fragmentation of genomic DNA (gDNA), and use the same workflow for end repair, A-tailing, adapter ligation, hybridization capture, and on-machine sequencing for cDNA and gDNA. Compared with the two independent nucleic acid library preparation methods, it can save time by half, but the targeted enrichment method of hybridization capture is complicated to operate, and the overall time-consumption is still long, and it takes more than 1 day to complete preparation of a library. In 2019, Haynes’ research group used amplicon sequencing to detect 135 RNA and 55 DNA detection targets in non-small cell lung cancer and prepared the RNA library using a commercial RNA library preparation kit. For preparation of DNA library, except for the use of specific DNA primer pools and the omission of reverse transcription, other operating procedures, library preparation reagents, and experimental conditions are consistent with those for the preparation of RNA library. The above two experimental steps are shown in Figure 1b. Although some optimizations have been made in the experimental process and reagent selection, in essence, the DNA and RNA libraries are still constructed separately.
Integrated detection methods based on simultaneous library preparations for deoxyribonucleic acid and ribonucleic acid
Since both cDNA generated by RNA reverse transcription and gDNA are DNA, meeting the conditions for combined preparation of libraries, which can simplify experimental operations and shorten detection time. Van Boheemen etal. used the commercial RNA library preparation reagent for combined preparation of RNA and DNA libraries, omitting the steps of mRNA enrichment, ribosomal RNA removal, and DNA enzyme treatment in the standard operating procedures of the RNA library preparation kit, and performed metagenomic sequencing.
Song etal. from Zhejiang Cancer Hospital used parallel amplification numerically optimized sequencing (PANO-Seq) technology to detect single nucleotide variants or insertion/deletion variants on 10 genes and gene fusion events on 3 genes at the DNA level while analyzing gene fusion events on 14 genes at the RNA level. After extracting the total nucleic acid of the tissue, the researchers directly reverse-transcribed the total nucleic acid without removing the gDNA to obtain a mixed solution of cDNA and gDNA, and prepare the library at the same time, without the need for separate preparation of DNA and RNA libraries, which greatly improves the efficiency of the library preparation, and simplifies the experimental process. Experimental steps are shown in Figure 1c. The ratio of input DNA and RNA cannot be controlled accurately with the nucleic acid co-extraction method, but the concentrations of the two types of nucleic acid primers in the primer pool can be adjusted according to the quality assessment of DNA and RNA to obtain relatively uniform sequencing data. For the quantification of DNA and RNA in total nucleic acids, the team reported a new quantitative reverse transcription polymerase chain reaction (PCR) method, which designed Taqman probes with two types of fluorescence in the intron and exon regions of the same housekeeping gene, respectively, to obtain the quantitative results of the two nucleic acids more accurately, and used this quantitative method to control the quality of the input template.
Lin established a pathogen DNA/RNA metagenomic co-sequencing method, which also adopted the total nucleic acid extraction and co-reverse transcription strategy, used the MGIEasy enzyme digestion DNA library preparation kit to perform the combined preparation of cDNA and gDNA libraries, and conducted sequencing on the BGISEQ-500 sequencer, realizing the integrated detection of viral DNA and RNA.
Nucleic acid input and next-generation sequencing data analysis for integrated detection
There will be certain differences in the nucleic acid input when different library preparation methods and different sequencing platforms are used, as shown in Table 1. Unlike clinical samples with a high total nucleic acid, forensic biological samples are often contain trace amounts of nucleic acid, with the DNA concentration as low as picogram-level. Compared with hybridization capture, the library preparation method based on targeted amplification requires less nucleic acid input and is more suitable for the detection of forensic biological evidence.
For obtained DNA and RNA sequencing data, analysis methods and reference sequences are variant in different research areas. Some software, such as the QuantideX® NGS Reporter Software and the Ion Reporter Software, can be used to analyze integrated NGS data resulting from corresponding commercial kits.[29–30] In most customized panels, however, analyses of one sequencing data containing both gDNA and cDNA reads remain separate pipelines.[28,32,34] These analysis pipelines are similar to detecting DNA or RNA separately.
OUTLOOK AND CHALLENGES
The integrated detection of forensic DNA and RNA markers is a very promising detection technology. Although a small amount of RNA molecular markers and STRs can be co-detected on the CE platform, it is still challenging to realize the integrated detection for a large number of molecular markers, as well as the integrated detection of RNA and SNPs. With the NGS platform, hundreds of molecular markers of different types can be integrated and detected, and the range of co-detection includes but is not limited to the combination of STR and mRNA, STR and miRNA, SNP and cSNP, and it is even possible to integrate all types of molecular markers. In addition to the advantages of high throughput and high integration, NGS technology can provide the detailed sequence information that is hard for CE methods. Furthermore, NGS can overcome the limitation of the targeted fragment length, so degraded samples with short nucleic acid fragments are also suitable for NGS analyzing. By obtaining multiple molecular marker data through one sequencing, and generating correlations between different types of data, it is expected to answer questions such as whom the forensic biological evidence came from, what is the tissue origin, the time when the sample was separated from the body, what molecular pathological characteristics it has, etc.
Obtaining forensic information of the same biological evidence in a fast, simple, and material-saving way can better serve the practice of forensic medicine. PANO-seq is currently the most integrated technical method among a number of studies on DNA and RNA co-detection based on NGS. Its principles and ideas for integrated detection can be used as a key reference in forensic research. However, there are still some problems to be solved if we tend to apply the highly integrated detection method to forensic practices. First, in addition to answering the question about gene expression with “yes” or “no,” the relative expression levels of different RNA molecular markers are also critical in many cases in forensic RNA level studies.[36,37] Whether the use of the nucleic acid co-extraction method and multiplex amplification will affect the true ratio of different types of markers is directly related to the reliability of sequencing results of RNA markers. Second, the use of combined preparation of library to simultaneously perform multiplex amplification of cDNA and gDNA, needs to meet strict requirements on the specificity of amplification primers. Especially in the design of RNA primers, it should be avoided that the forward and reverse primers are located in the same exon region, thereby reducing the interference of amplification on the detection of gene expression. Besides, how to analyze the massive sequencing data generated by NGS and how to verify the accuracy of the sequencing results are also issues to be solved. In addition to the above problems, researchers also need to consider whether they can further simplify experimental steps, such as integrating cDNA generation and multiplex amplification into a one-step operation by introducing the reverse transcription PCR technology. With a reasonable co-extraction strategy and an effective library construction and parallel sequencing scheme, the integrated detection technology of next-generation sequencing of DNA genetic markers and RNA molecular markers in forensic medicine will be well applied in forensic practices.
Financial support and sponsorship
This work was supported by the Ministry of Public Security of China (2019GABJC15) and the Institute of Forensic Science, Ministry of Public Security of China (2018JB007).
Conflicts of interest
There are no conflicts of interest.
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