DNA Analysis Methods Research Articles

Molecular Genetic Methods and Computer Technologies in the System of Epidemiological Surveillance of Salmonella Infection

Introduction: Salmonella infection occupies a leading position in the structure of acute intestinal infections caused by bacterial pathogens. In recent years, with the development of molecular genetic methods and introduction of techniques of computerized data processing, the improvement of the epidemiological surveillance system in the light of the world scientific achievements has become of particular importance. This review is aimed at presenting the history of developing molecular genetic methods and computer technologies in the study of Salmonella infection, and the update on the issue in the Russian Federation based on recent findings of the Research Institute of Epidemiology and Microbiology named after G.P. Somov, one of the leading Russian centers for the study of Salmonella. Materials and methods: We used databases of the Laboratory of Molecular Epidemiology of Somov Research Institute of Epidemiology and Microbiology, and did a literature search in the Web of Science, PubMed, Scopus, Elsevier, Springer, and Google Scholar. Results: Since the second half of the 1980s, the method of plasmid DNA analysis of bacteria of the Salmonella genus has been put into practice. Since 1990, this method has been the basis for microbiological molecular genetic monitoring of the pathogen. The geography of the studied strains, restricted to Primorsky Krai in the 1990s, already in the 2000s encompassed the Far Eastern and Siberian Federal Districts of the Russian Federation. The studies conducted by the Laboratory of Molecular Epidemiology helped improve the system of epidemiological surveillance of the causative agent of salmonellosis in the Far East and revealed the structure of the Salmonella population based on the analysis of plasmids contained in Salmonella strains. Conclusion: Several issues related to microbiology, epidemiology, clinical picture, and prevention of Salmonella infection were resolved during the comprehensive research. We discuss prospects for the development of molecular genetic methods and computer technologies in the study of Salmonella infection in the epidemiological surveillance system in the Russian Federation and emphasize the importance of whole-genome sequencing as a new “gold standard” in molecular epidemiology.

Technical note: multi-alleles at the DYS385ab locus with high frequency in a Han Chinese population from southwestern China.

Y-chromosome short tandem repeat (Y-STR) markers have been widely used in forensic applications and usually show monoallelic or diallelic genotypic patterns at certain double-copied loci. In this study, we have found 13 samples among 703 males with multi-alleles at the DYS385ab locus, including one with five mutant alleles, nine with four, and three with three. The frequency of abnormal DYS385ab genotypes was 1.85% (13/703), which is very high in the Han Chinese population. The percentage of samples with diallelic patterns at DYS385ab was higher than that of monoallelic patterns (80.23% vs. 17.92%). Additionally, the percentage of samples with tetra-allelic patterns at DYS385ab was higher than that of tri-allelic patterns (1.28% vs. 0.43%), suggesting that there are possibly two copies with duplicated events happening frequently on the Y chromosome. Interestingly, the peak height of allele 13 was two to three-folds higher than that of other alleles. The allele 18 peak height was also two-fold higher than others, which could potentially be explained by a duplication event mechanism. We also found that tri-allelic genotypes for alleles 13, 17, and 20, tetra-allelic genotypes for alleles 13, 14, 19, and 20, and tetra-allelic genotypes for alleles 12, 13, 19 and 21 were more common than others. Furthermore, all 13 samples had multi-alleles containing allele 13, implying a founder effect in this particular Chinese-specific ethnic group. Taken together, this study provides new information for this population and will be useful for paternal lineage identification, kinship analysis, and family relationship reconstruction using Y-STR forensic DNA analysis methods.

FORENSIC EXPERT CHARACTERISTICS OF THE HUMAN GENOMEAND ITS IDENTIFICATION SIGNS

Purpose: introduction into expert practice of new methods of research of human biological traces to increase the level of identification capabilities. Research methods: comparative legal analysis, cognitive-analytical, etc. Results: it’s suggested that an innovative understanding of such basic legal properties of evidence as their significance, good quality and reliability and solution to the issue of criminal and other legal liability for violation of the procedure for obtaining evidence. Discussion: it’s emphasized that significant progress in obtaining identification evidence of a person’s involvement in a crime based on traces of biological origin has been achieved primarily through the use of molecular genetics. Molecular genetic analysis of DNA is only one of the stages of identification, and to obtain the final result requires statistical analysis of the data, especially important when the genotypes of the offender and the suspect: in this case it is sometimes about human life. Objects of biological origin that are removed as evidence from the scene include blood, semen, saliva, sweat, hair, vaginal secretions, urine, excrements, organs and tissues of the human body, bones and their fragments. The source of traces of biological origin is the human body, its organs. Under specific conditions, they, and especially blood, carry information about the mechanisms and circumstances of the crime. The shape of the traces of blood reflects the nature of the wounds, indicates some actions of the subject of the crime, the type of weapon used, and so on. Surveying the scene when looking for traces of biological origin, such as traces resembling blood, should be targeted, depending on the type of crime. The study of the shape of blood traces allows to model the mechanism of the crime. Such research is carried out at inspection of a scene. It allows you to establish the location and posture of the victim and the offender at the time of injury, the trajectory of the wounded, the special characteristics of the offender, the type of instrument of the crime and more. Trace detection should be done very carefully so as not to damage not only the tracks but also the media. Participants in the inspection should move only beyond the territory already inspected by the investigator. It is important to ensure that the inspection procedure is such that participants do not leave their own marks on the scene and in the areas not inspected. Thus, the search for and collection of traces of biological origin of man is based on the general principles of search activity. To date, the method of DNA analysis (genotyping) is one of the most promising areas of forensic expertise and its results are a fairly reliable proof of involvement in the crime of a particular person.

Degradation modeling of water environmental DNA: Experiments on multiple DNA sources in pond and seawater

AbstractEnvironmental DNA (eDNA) analysis methods have been developed to detect organism distribution and abundance/biomass in various environments. eDNA degradation is critical for eDNA evaluation. However, the dynamics and mechanisms of eDNA degradation are largely unknown, especially when considering different eDNA sources, for example, cells and fragmental DNA. We experimentally evaluated the degradation rates of eDNA derived from multiple sources, including fragmental DNA (internal PCR control [IPC]), free cells (from Oncorhynchus kisutch), and resident species. We conducted the experiment with pond and seawater to evaluate the differences between freshwater and marine habitats. We quantified the eDNA copies of free cells, fragmental DNA, and resident species (Cyprinus carpio in the pond and Trachurus japonicus in the sea). We found that eDNA derived from both cells and fragmental DNA decreased exponentially in both the sea and pond samples. The degradation of eDNA from resident species showed similar behavior to the cell‐derived eDNA. We evaluated three degradation models with different assumptions and degradation steps and found that a simple exponential model was effective in most cases. Our findings on cell‐ and fragmental DNA‐derived eDNA provide fundamental information about the eDNA degradation process and can be applied to quantify eDNA behavior in natural environments.

Developmental validation of VeriFiler™ Plus PCR Amplification Kit: A 6-dye multiplex assay designed for casework samples

The VeriFiler™ Plus PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 23 autosomal markers (D3S1358, vWA, D16S539, CSF1PO, D6S1043, D8S1179, D21S11, D18S51, D5S818, D2S441, D19S433, FGA, D10S1248, D22S1045, D1S1656, D13S317, D7S820, Penta E, Penta D, TH01, D12S391, D2S1338, and TPOX), a quality indicator system, and two sex-identification markers. Combined, the markers satisfy the requirements of the Chinese National autosomal DNA database as well as expanded CODIS (Combined DNA Index System). The VeriFiler Plus kit was developed with an improved Master Mix which incorporates the brighter TED™ dye, and accommodates a higher sample loading volume thus allowing for increased sensitivity and enabling maximum information recovery from challenging casework samples including touch, degraded, and inhibited samples. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

A Novel Panel of 43 Insertion/Deletion Loci for Human Identifications of Forensic Degraded DNA Samples: Development and Validation.

Insertion/deletion polymorphism is a promising genetic marker in the forensic genetic fields, especially in the forensic application of degraded sample at crime scene. In this research, a novel five-dye multiplex amplification panel containing 43 highly polymorphic Insertion/deletion (InDel) loci and one Amelogenin gene locus is designed and constructed in-house for the individual identification in East Asian populations. The amplicon sizes of 43 InDel loci are less than 200 bp, which help to ensure that full allele profiles can be obtained from degraded DNA sample. A series of optimizations and developmental validations including optimization of PCR conditions, detection efficiency of the degraded and casework samples, sensitivity, reproducibility, precision, tolerance for inhibitors, species specificity and DNA mixtures are performed according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) guideline. The results of the internal validation demonstrated that this novel InDel panel was a reliable, sensitive and accurate system with good tolerances to different inhibitors, and performed the considerable detection efficiency for the degraded or mixed samples, which could be used in the forensic applications.

A Feasibility Study on a Monitoring for Seagrass beds Using Environmental DNA

In order to protect marine resources listed in the SDGs, it is important to conduct surveys to understand the current status and transition of aquatic species such as fish and seagrass. However, the area and frequency of surveys are often limited due to the lack of manpower and cost. Environmental DNA (eDNA) analysis is a method to obtain information of aquatic species in the sea or rivers. The aquatic species are identified by analyzing DNA contained in the sampled water. The eDNA analysis can be utilized as a new and efficient method for investigating aquatic species. The author is developing a monitoring method for seagrass beds using eDNA. Seagrasses are sessile organisms and don’t move like a fish, the areas where eDNA is generated are fixed. Therefore, eDNA path in a seagrass beds area can be predicted by flow prediction simulation. The monitoring carries out in the path of eDNA released from seagrass. When there is a correlation between the amount of DNA and the amount of biomass, it is possible to gain knowledge of the amount of biomass from sampling water in the path of eDNA. For example, the changes in the amount of biomass due to growth and withering of seagrass beds can be studied by observing the changes in the amount of DNA. The purpose of this study is to examine the feasibility of the monitoring method using experiments, field surveys and numerical simulations for eDNA. The water tank experiments for 15 months, suggested that the amount of seagrass eDNA is related to the seasonal changes in the biomass of seagrass, which has been grown in the water tank. In the conducted four field surveys, the amount of eDNA tended to be high during times of spring with high amounts of seagrasses. In addition, the decomposition process of eDNA were examined by laboratory experiments using sea water. As a result, seagrass DNA contained in the sampling sea water was degraded and undetected within around 5 days. This result suggests that information obtained from the sampling water reflects biological information within a few days. These results suggest the possibility to estimate the changes in biomass by using eDNA in the sea. And, in the field surveys, it was shown that the amount of eDNA is small, so we have recognized that increasing the amount of sampling water and improvement of the DNA analysis method is an issue. This monitoring method is feasible when the collected eDNA is related to a specific seagrass bed. The feasibility of the method was considered using numerical simulation. In the numerical simulation, a particle tracking method using 10 types of simple bay shapes was used to trace the path of eDNA regarded as a particle. When the eDNA started from multiple positions, it was searched for position where the point where DNA starts and arrives is uniquely determined. As a result, at some observation points, the particles that departed from a specific position was observed without being mixed with particles departing from other starting positions, and this was true for all bay types. In addition, the same tendency could be obtained in calculations using the bathymetry of Ago bay, in Mie prefecture. These suggests that it may be possible to monitor seagrass beds by performing fixed-point observations according to the seagrass beds distribution and bay flow. Since few cases in the sea were conducted, the authors intend to continue the survey for the upcoming years and continue the study under various conditions including numerical analysis.

Heating Up Cold Cases: An Interview with Bruce Budowle on Human Identification

Heating Up Cold Cases: An Interview with Bruce Budowle on Human Identification

Developmental validation of the MGIEasy Signature Identification Library Prep Kit, an all-in-one multiplex system for forensic applications.

Analyzing genetic markers in nuclear and mitochondrial genomes is helpful in various forensic applications, such as individual identifications and kinship analyses. However, most commercial kits detect these markers separately, which is time-consuming, laborious, and more error-prone (mislabelling, contamination, ...). The MGIEasy Signature Identification Library Prep Kit (hereinafter "MGIEasy identification system"; MGI Tech, Shenzhen, China) has been designed to provide a simple, fast, and robust way to detect appropriate markers in one multiplex PCR reaction: 52 autosomal STRs, 27 X-chromosomal STRs, 48 Y-chromosomal STRs, 145 identity-informative SNPs, 53 ancestry-informative SNPs, 29 phenotype-informative SNPs, and the hypervariable regions of mitochondrial DNA (mtDNA). Here, we validated the performance of MGIEasy identification system following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), assessing species specificity, sensitivity, mixture identification, stability under non-optimal conditions (degraded samples, inhibitor contamination, and various substrates), repeatability, and concordance. Libraries prepared using MGIEasy identification system were sequenced on a MGISEQ-2000 instrument (MGI Tech). MGIEasy-derived STR, SNP, and mtDNA genotypes were highly concordant with CE-based STR genotypes (99.79%), MiSeq FGx-based SNP genotypes (99.78%), and Sanger-based mtDNA genotypes (100%), respectively. This system was strongly human-specific, resistant to four common PCR inhibitors, and reliably amplified both low quantities of DNA (as low as 0.125ng) and degraded DNA (~ 150nt). Most of the unique alleles from the minor contributor were detected in 1:10 male-female and male-male mixtures; some minor Y-STR alleles were even detected in 1:1000 male-female mixtures. MGIEasy also successfully directly amplified markers from blood stains on FTA cards, filter papers, and swabs. Thus, our results demonstrated that MGIEasy identification system was suitable for use in forensic analyses due to its robust and reliable performance on samples of varying quality and quantity.

Machine learning ATR-FTIR spectroscopy data for the screening of collagen for ZooMS analysis and mtDNA in archaeological bone

Faunal remains from archaeological sites allow for the identification of animal species that enables the better understanding of the relationships between humans and animals, not only from their morphological information, but also from the ancient biomolecules (lipids, proteins, and DNA) preserved in these remains for thousands and even millions of years. However, due to the costs and efforts required for ancient biomolecular analysis, there has been considerable research into development of accurate and efficient screening approaches for archaeological remains. FTIR spectroscopy is one such approach that has been considered for screening of proteins, but its widespread use has been hindered by the fact that its predictive accuracy can vary widely depending on the extent of sample preservation and the instrument used. Further, screening methods for ancient DNA (aDNA) analysis are scarce. Here we present a new approach to vastly improve upon FTIR-based screening methods prior to ZooMS (Zooarchaeology by Mass Spectrometry) and aDNA analysis through the use of random forest-based machine learning. To do so, we use ATR-FTIR to examine three sets of archaeological bone assemblages and analyse them by ZooMS (for taxonomic identification). Two of these are from Palaeolithic contexts, dominated by terrestrial fauna and include specimens with a variety of preservational conditions. The third set consists of Holocene faunal remains, with variable levels of preservation and is dominated by cetaceans. Using the Holocene faunal remains, we were able to more consistently evaluate ATR-FTIR-based screening for mtDNA as well as ZooMS success. We report on the potential of machine learning in ATR-FTIR-based screening for ancient mtDNA analysis, and our machine learning models conclusively improve the accuracy prior to usage of ATR-FTIR-based screening for ZooMS by 20–40%. The results also suggest this approach potentially allows for a universal screening system, applicable across multiple sites and largely independent of the spectrometers used.

Development and validation of a multiplex 19 X-chromosomal short tandem repeats typing system for forensic purposes

X-chromosome short tandem repeat (X-STR) markers are a powerful complementary system used for paternity and forensic casework. This study presents the development and validation of a new highly efficient multiplex-fluorescent-labeled 19 X-STR typing system, including DXS10079, DXS101, DXS10135, DXS10162, DXS6795, DXS6800, DXS6803, DXS6807, DXS6809, DXS6810, DXS7133, DXS7423, DXS981, DXS9902, DXS9907, GATA165B12, GATA172D05, GATA31E08 and HPRTB along with sex-typing locus, amelogenin. The system was validated according to guidelines issued by the Scientific Working Group on DNA Analysis Methods. Allele frequency and forensic parameters were investigated from 1085 (494 males and 591 females) unrelated Beijing Han individuals, the combined power of discrimination by the 19 X-STR loci in females and males, as well as the combined mean exclusion chance in trios and duos, were 0.999999999999999995, 0.99999999995, 0.9999999995, and 0.9999996, respectively. The results demonstrate that this multiplex system is robust and reliable, and considered to be a powerful tool for forensic application.

HUMAN BEING AS THE BEARER OF IDENTIFYING BIOLOGICAL INFORMATION

In forensic practice, biological traces of a person are very often used as material evidence - blood, hair, saliva, semen, urine, sweat, as well as parts of organs and tissues. Establishing the origin of these traces from a specific person is very important for the investigation of criminal offenses. The current level of development of molecular genetic research indicates the need to use DNA analysis in the detection and investigation of criminal offenses against a person. Today, molecular genetic identification reveal reliable prospects for solving identification problems in the criminal proceedings and developing the evidence base, and also has a number of advantages over traditional serological methods for studying human biological traces. It should be noted that along with the traditional method of nuclear DNA research, mitochondrial DNA research is also being carried out, which allows solving the problem of molecular genetic examination to establish biological affinity. The value of this method lies in its effectiveness in the study of a small amount of degraded DNA, secretions and heavily damaged objects, the study of which is impossible by traditional methods. When performing a forensic molecular genetic examination for the full identification of the detected traces when examining the places of committed criminal offenses, comparative samples are important, as well as the selection of appropriate biological samples to establish paternity and family ties. Molecular genetic analysis of DNA is only one of the stages of identification, and in order to arrive at the final result, a statistical analysis of the data obtained is necessary, which is especially important when the genotypes of the criminal and the suspect in mixed tracks coincide. For a probable-statistical assessment of the results of the identification significance of the set of established genetic traits, the frequencies of the distribution of the studied alleles in the population are required. Today, the DNA analysis method has become one of the most demanded directions in the development of forensic examinations, and its results are quite reliable evidence of the involvement of a specific person in a crime. Due to its unique capabilities, molecular genetic analysis of DNA is a powerful tool in the investigation of criminal proceedings.

Wykorzystanie potencjału mikrobiomu środowiskowego w kryminalistyce

Biometrics is one of the basic detection techniques used in law enforcement activities on a daily basis. Like other techniques, it is constantly changing. This article aims to provide an overview of what was in the past, what is now, and what will be in the near future. It presents the selected methods of collecting some of the data, as well as the systems used to rocess them. In addition, practical guidance is provided on how individual biometric data should be collected and, for specific cases, it explains why the data collection procedure is carried out in one way and not another, and what it means for its subsequent processing. As problems sometimes arise during recovery of the material they have been presented together with an overview of the reasons for that. In addition to information on the technical aspects, the article also includes references to legal acts regulatingissues related to biometrics.

DNA of centuries-old timber can reveal its origin

Oak wood was highly appreciated and widely used for construction in past centuries. As population sizes expanded in some regions of Europe, local forests were depleted of high-quality timber. Therefore, regions of soaring economies were importing timber initially from the European market and eventually from other continents. Origin of archaeological or historical timber is usually identified by means of dendroprovenancing, i.e. statistical matching of tree-ring-width (TRW) series of timber of unknown origin with TRW reference datasets. However, this method has pitfalls and limitations and therefore alternative techniques are needed. Here, we used three different DNA analysis methods to investigate the potential of using ancient (a)DNA, extracted from oak timber derived from historical buildings and shipwrecks from a variety of countries. All the material had also been analysed dendrochronologically, so its dating and provenance is demonstrated. We included heartwood samples in this analysis, for which DNA extraction is especially challenging as it contains chemicals that inhibit DNA amplification. We succeeded in amplifying DNA for at least one marker from 56% of samples (including heartwood samples), yielding crucial information that allowed us to identify the potential source area of centuries old timber buildings in Latvia and Denmark and of 750-year-old shipwreck material from Germany. Our results prove the strong potential of DNA analyses for identifying timber origin to the regional scale, but by combining these with the dendrochronological results, we can control the exactitude of the aDNA approach and demonstrate a more nuanced examination of the timber sources for these historic structures.

Developmental validation for Sanger sequencing of HV1 and HV2 in mitochondrial DNA

The method of Sanger sequencing of mitochondrial DNA (mtDNA) for forensic purposes has been validated previously. However, to our knowledge, there are few validation reports of Sanger sequencing of mtDNA that satisfy the latest SWGDAM Validation Guidelines for DNA Analysis Methods. Recently, we developed a new method that utilizes different chemistry, devices and protocols from those used in our current method of Sanger sequencing of mtDNA. In this study, our new protocol was developmentally validated according to the SWGDAM validation guidelines. This validated method can be used to obtain mtDNA sequences of forensic samples such as hair and bone.

Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel.

For the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology—Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge—has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra- and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.

A simple silica based DNA isolation method for cell-free DNA analysis from liquid biopsy

Abstract Objectives Cell-free DNA (cfDNA) is a promising biomarker for cancer diagnostics. Blood is used as a liquid biopsy material which transports many types of biological components such as protein, circulating tumor cells, cfDNA, and extracellular RNA molecules. In this study, the yield and purity of extracted DNA were compared of seven different in-house DNA isolation methods and two different commercial kits from human blood samples. Methods Different in-house methods, guanidine thiocyanate (GuSCN) with silica method, phenol-chloroform-isoamylalcohol method-1, phenol-chloroform-isoamylalcohol method-2, phenol-chloroform-isoamylalcohol with ammonium acetate method, sodium N-lauryl sarcosine with glycogen method, sodium acetate method, sodium chloride with silica method and two different commercial kits “Quick-cfDNA serum & plasma kit” and “plasma/serum cell-free circulating DNA purification mini kit” were used. After DNA isolation, methods were compared with real time PCR technique (qPCR) and spectrophotometric methods. Results The results showed that the newly modified GuSCN with silica method gave much higher purity and yield than any other analyzed in-house isolation methods and commercial kits that were used. Conclusions The GuSCN with silica method is simple to set up, easy to use, inexpensive and environmentally safe. The method would be an alternative method for cfDNA isolation from liquid biopsy samples.

DNA Profiling in Forensic Dentistry

In last few years, DNA analysis methods are applied to forensic cases. Forensic dental record comparison has been used for human identification in cases where destruction of bodily tissues or prolonged exposure to the environment has made other means of identification impractical, i.e., after fire exposure or mass disaster.Teeth play an important role in identification and criminology, due to their unique characteristics and relatively high degree of physical and chemical resistance. The use of DNA profile test in forensic dentistry offers a new perspective in human identification.DNA is responsible for storing all the genetic material and is unique to each individual. The currently available DNA tests have high reliability and are accepted as legal proofs in courts. This article gives an overview of the evolution of DNA technology in the last few years, highlighting its importance in cases of forensic investigation.

High-Throughput Amenable MALDI-MS Detection of RNA and DNA with On-Surface Analyte Enrichment Using Fluorous Partitioning.

High-throughput matrix-assisted laser desorption/ionization mass spectrometry (HT-MALDI-MS) has garnered considerable attention within the drug discovery industry as an information-rich alternative to assays using light-based detection methods. To date, these efforts have been primarily focused on assays using protein or peptide substrates. Methods for RNA or DNA analysis by HT-MALDI-MS have not been extensively reported due to the challenges associated with MALDI-MS of oligonucleotides, including the propensity to form multiple salt adducts, low ionization potential, and ease of fragmentation. The objective of this work was to develop a platform suitable for HT-MS analysis of RNA and DNA substrates that overcomes these hurdles by combining on-surface sample preparation with soft ionization. This has been accomplished through the selective immobilization of fluorous-tagged oligonucleotides on a fluorous-modified MS target plate, followed by on-surface enrichment, matrix addition, and direct laser desorption/ionization, a process dubbed fluorous HT-MS (F-HT-MS). The work has resulted in methods by which RNA and DNA substrates can be detected at nanomolar concentrations from a typical assay buffer system using procedures that are amenable to full automation. The protocols were applied to an miRNA biogenesis assay, demonstrating its potential for RNA processes and thereby filling a prominent gap in RNA drug discovery: the paucity of in vitro functional assays.

GeTallele: A Method for Analysis of DNA and RNA Allele Frequency Distributions.

Variant allele frequencies (VAF) are an important measure of genetic variation that can be estimated at single-nucleotide variant (SNV) sites. RNA and DNA VAFs are used as indicators of a wide-range of biological traits, including tumor purity and ploidy changes, allele-specific expression and gene-dosage transcriptional response. Here we present a novel methodology to assess gene and chromosomal allele asymmetries and to aid in identifying genomic alterations in RNA and DNA datasets. Our approach is based on analysis of the VAF distributions in chromosomal segments (continuous multi-SNV genomic regions). In each segment we estimate variant probability, a parameter of a random process that can generate synthetic VAF samples that closely resemble the observed data. We show that variant probability is a biologically interpretable quantitative descriptor of the VAF distribution in chromosomal segments which is consistent with other approaches. To this end, we apply the proposed methodology on data from 72 samples obtained from patients with breast invasive carcinoma (BRCA) from The Cancer Genome Atlas (TCGA). We compare DNA and RNA VAF distributions from matched RNA and whole exome sequencing (WES) datasets and find that both genomic signals give very similar segmentation and estimated variant probability profiles. We also find a correlation between variant probability with copy number alterations (CNA). Finally, to demonstrate a practical application of variant probabilities, we use them to estimate tumor purity. Tumor purity estimates based on variant probabilities demonstrate good concordance with other approaches (Pearson's correlation between 0.44 and 0.76). Our evaluation suggests that variant probabilities can serve as a dependable descriptor of VAF distribution, further enabling the statistical comparison of matched DNA and RNA datasets. Finally, they provide conceptual and mechanistic insights into relations between structure of VAF distributions and genetic events. The methodology is implemented in a Matlab toolbox that provides a suite of functions for analysis, statistical assessment and visualization of Genome and Transcriptome allele frequencies distributions. GeTallele is available at: https://github.com/SlowinskiPiotr/GeTallele.