Introduction to Molecular Diagnostics of Insects

Insects play an important role in biocenoses due to their abundance and wide (cosmopolitan) distribution. Many insects are crop pests. An effective pest control could be realized in case of proper species identification, which is usually managed by morphological analysis. Molecular methods allow to deep study of many issues of insect biology. In particular, traditional approach can not ordinary identify a species at all stages of their life cycle, whereas molecular methods can it. This review covers a wide range of issues related to the molecular genetic analysis of insects. In the first section we consider the methods of fixation and storage of insect specimens, as well as their impact on DNA quality. Further, we provide general information on population study design. Various schemes of DNA extraction, examples of both express techniques and more thorough protocols for DNA extraction and their purification are provided. In addition, methods of DNA isolation that allow to preserve a specimen integrity for further morphological studies are considered. The methods of DNA quality control are described in detail, that is important for PCR analysis. The last section provides various methods of PCR analysis, that we exemplify by studies aimed to elucidate both fundamental issues and practical problems.

Preserving avian blood and DNA sampled in the wild: a survey of personal experiences

Collecting and storing biological material from wild animals in a way that does not deteriorate data quality for analyses using DNA is instrumental for research in ecology and evolution. Our aims were to collect methods commonly used by researchers for the field collection and long-term storage of blood samples and DNA extracts from wild birds and gather reports on their effectiveness. Personal experiences were collected with an online survey targeted specifically at researchers sampling wild birds. Many researchers experienced problems with blood sample storage but not with DNA extract storage. Storage issues generated problems with obtaining adequate DNA quality and sufficient DNA quantity for the targeted molecular analyses, but were not related to season of blood sampling, access to equipment, transporting samples, temperature and method of blood storage. Final DNA quality and quantity were also not affected by storage time before DNA extraction or the methods used to extract DNA. We discuss practical aspects of field collection and storage and provide some general recommendations, with a list of pros and cons of different preservation methods of avian blood samples and DNA extracts.

Optimisation of an Automated DNA Extraction Method for Bone and Teeth Samples and Applicability to Two Forensic Cases

Bones and teeth are highly challenging sources of DNA in forensic science and human remains identification, requiring multiple laborious processing steps. In this study, we compared an organic phenol–chloroform method to the QIAamp® DNA Investigator and PrepFiler Express BTA™ methods in order to identify the most efficient automated DNA extraction method for bones and teeth. Results from individual tooth powder replicates showed that the PrepFiler Express BTA™ method extracted the highest yields of DNA per mg of tooth powder, returning a minimum of 20/21 PowerPlex® 21 loci. Samples extracted using the organic extraction or QIAamp® DNA Investigator methods produced PowerPlex® 21 profiles displaying a ski-slope morphology. The improved DNA quality and yield from the PrepFiler Express BTA™ method was verified using aged samples, where higher DNA yields per mg of powder and more informative profiles were obtained. Furthermore, the PrepFiler Express BTA™ method subsequently provided useful DNA profiles for two forensic cases involving degraded bone samples. Overall, this study showed that the PrepFiler Express BTA™ chemistry is a reliable and robust method for DNA extraction from bone and teeth samples, and will allow larger numbers of samples to be efficiently extracted in the event of a Disaster Victim Identification event.

Sperm DNA Fragmentation

Sperm DNA fragmentation refers to the accumulation of adducts as well as single- or double-strand DNA breaks and reflects the sperm DNA quality. Current data suggest that there are differences in sperm DNA quality among individuals with high or low fertility, and this observation has led to the idea that testing sperm DNA fragmentation could be a useful test of male fertility. However, sperm DNA fragmentation has become one of the most frequently debated topics in reproductive medicine, as there is no agreement about the optimal way to test for DNA fragmentation, the clinically significant level of sperm DNA fragmentation, as well as the best therapeutic options for infertile men. This article presents current evidence related to sperm DNA fragmentation and its role in managing male infertility.

Examining performance and likelihood ratios for two likelihood ratio systems using the PROVEDIt dataset

A likelihood ratio (LR) system is defined as the entire pipeline of the measurement and interpretation processes where probabilistic genotyping software (PGS) is a piece of the whole LR system. To gain understanding on how two LR systems perform, a total of 154 two-person, 147 three-person, and 127 four-person mixture profiles of varying DNA quality, DNA quantity, and mixture ratios were obtained from the filtered (.CSV) files of the GlobalFiler 29 cycles 15s PROVEDIt dataset and deconvolved in two independently developed fully continuous programs, STRmix v2.6 and EuroForMix v2.1.0. Various parameters were set in each software and LR computations obtained from the two software were based on same/fixed EPG features, same pair of propositions, number of contributors, theta, and population allele frequencies. The ability of each LR system to discriminate between contributor (H1-true) and non-contributor (H2-true) scenarios was evaluated qualitatively and quantitatively. Differences in the numeric LR values and their corresponding verbal classifications between the two LR systems were compared. The magnitude of the differences in the assigned LRs and the potential explanations for the observed differences greater than or equal to 3 on the log10 scale were described. Cases of LR < 1 for H1-true tests and LR > 1 for H2-true tests were also discussed. Our intent is to demonstrate the value of using a publicly available ground truth known mixture dataset to assess discrimination performance of any LR system and show the steps used to understand similarities and differences between different LR systems. We share our observations with the forensic community and describe how examining more than one PGS with similar discrimination power can be beneficial, help analysts compare interpretation especially with low-template profiles or minor contributor cases, and be a potential additional diagnostic check even if software in use does contain certain diagnostic statistics as part of the output.

P–121 Selection and separation of X- and Y- chromosome-bearing mammalian sperm using SPERMAN technology

Study question How can we select and separate X and Y-chromosome bearing sperm cells without causing cellular damage and reduced fertility? Summary answer AI powered SPERMAN technology can sort X and Y-chromosome bearing sperm populations without using harmful fluorescent dyes and lasers. What is known already Most common and reliable method to sex sorting (separation of X and Y- chromosome bearing sperm cells) is by using fluorescence activated cell sorting ( FACS) which takes advantage of the difference of amount of DNA in X and Y-chromosomes. Unfortunately this method causes reduced fertility and cellular damages due to the lasers and fluorescent dyes that are used. There are new and experimental developments in sex-sorting such as using immunological approaches to separate sperm cells. Current sorting method damages sperm cells. However, there is no automated and easy to use sperm sorting technology available. Study design, size, duration In this study, we have compared the quality parameters (motility, viability, AR status, DNA packaging) of sorted sperm cells to unsorted sperm cells. We have used epididymal sperm samples from 10 C57BL/6 mice (Charles River) and frozen semen samples from 6 Holstein bulls (Sexing Technologies) and frozen semen samples from 8 human sperm donors (Fairfax Cryobank). Samples were divided into two groups: sex-sorted and unsorted sperm. Participants/materials, setting, methods Sperm samples from 8-week-old C57BL/6 mice were collected from epididymal region, frozen bull semen samples and human semen samples were thawed at 37ºC then separated into two groups as sorted and unsorted sperm. Sorted samples in modified SP-TALP medium were loaded on the microfluidic chip of Sperman device, after the 30min long sorting process ended, all samples were centrifuged gently at 100g for 5 min, quality parameters were measured by using CASA and FACS. Main results and the role of chance Sperman device sorted X-bearing sperm cells at 81% purity and Y-bearing cells at 73% in mouse samples. Sperman device was able to sort X-bearing sperm cells at 78% purity and Y-bearing cells at 70% in bull samples.S perman device sorted X-bearing sperm cells at 85% purity and Y-bearing cells at 76% in human samples. Our study shows that in mouse, bull and human sperm samples, sperm DNA quality, sperm concentration, progressive motility and AR status results from the sorting with SPERMAN device are comparable with the unsorted samples. For sperm DNA quality, both the Spearman rank correlation coefficient and concordance correlation coefficient are above 0.97, indicating a high agreement between the unsorted samples and SPERMAN sorted samples. Limitations, reasons for caution In this study we only compared sperm quality parameters between unsorted sperm cells and Sperman-sorted sperm cells. Ideally a follow up study would be to look in detail at fertilization success rates and embryo growth. Also, genomic and proteomic profiles of unsorted and sorted sperm cells could be different. Wider implications of the findings: In livestock, sperm sex sorting has high value in terms of economic impact on the livestock management. Unfortunately, there is one expensive single technology dominates the sex-sorting market causing high fees poor outcomes. We believe that Sperman technology provides farmers more cheaper and better sex-sorting technology. Trial registration number Not applicable

Impact of SNP microarray analysis of compromised DNA on kinship classification success in the context of investigative genetic genealogy

Single nucleotide polymorphism (SNP) data generated with microarray technologies have been used to solve murder cases via investigative leads obtained from identifying relatives of the unknown perpetrator included in accessible genomic databases, referred to as investigative genetic genealogy (IGG). However, SNP microarrays were developed for relatively high input DNA quantity and quality, while SNP microarray data from compromised DNA typically obtainable from crime scene stains are largely missing. By applying the Illumina Global Screening Array (GSA) to 264 DNA samples with systematically altered quantity and quality, we empirically tested the impact of SNP microarray analysis of deprecated DNA on kinship classification success, as relevant in IGG. Reference data from manufacturer-recommended input DNA quality and quantity were used to estimate genotype accuracy in the compromised DNA samples and for simulating data of different degree relatives. Although stepwise decrease of input DNA amount from 200 nanogram to 6.25 picogram led to decreased SNP call rates and increased genotyping errors, kinship classification success did not decrease down to 250 picogram for siblings and 1st cousins, 1 nanogram for 2nd cousins, while at 25 picogram and below kinship classification success was zero. Stepwise decrease of input DNA quality via increased DNA fragmentation resulted in the decrease of genotyping accuracy as well as kinship classification success, which went down to zero at the average DNA fragment size of 150 base pairs. Combining decreased DNA quantity and quality in mock casework and skeletal samples further highlighted possibilities and limitations. Overall, GSA analysis achieved maximal kinship classification success from 800-200 times lower input DNA quantities than manufacturer-recommended, although DNA quality plays a key role too, while compromised DNA produced false negative kinship classifications rather than false positive ones.

EXAMINING DISCRIMINATION PERFORMANCE AND LIKELIHOOD RATIO VALUES FOR TWO DIFFERENT LIKELIHOOD RATIO SYSTEMS USING THE PROVEDIT DATASET

The conventional capillary electrophoresis (CE) genotyping workflow used in forensic DNA laboratories is composed of two processes: measurement and interpretation. The outcome of the measurement process is an electropherogram (EPG). The outcome of the interpretation process is a strength of evidence statement often reported in the form of a likelihood ratio (LR) which typically requires probabilistic genotyping software (PGS). An LR system is defined as the entire pipeline of the measurement and interpretation processes where PGS is a piece of the whole LR system. To gain understanding on how two LR systems perform, a total of 154 two-person mixture, 147 three-person mixture, and 127 four-person mixture profiles of varying DNA quality, DNA quantity, and mixture ratios were obtained from the filtered (.CSV) files of the GlobalFiler 29 cycles 15s PROVEDIt dataset and deconvolved in two independently developed fully continuous programs, STRmix v2.6 and EuroForMix v2.1.0. Various parameters were set in each software and LR computations obtained from the two software were based on same/fixed EPG features, same pair of propositions, number of contributors, theta, and population allele frequencies. The ability of each LR system to discriminate between contributor (H1-true) and non-contributor (H2-true) scenarios was evaluated qualitatively and quantitatively. Differences in the numeric LR values and their corresponding verbal classifications between the two LR systems were compared. The magnitude of the differences in the assigned LRs and the potential explanations for the observed differences greater than or equal to 3 on the log10 scale were described. Cases of LR < 1 for H1-true tests and LR > 1 for H2-true tests were also discussed. Our intent is to demonstrate the value of using a publicly available ground truth known mixture dataset to assess discrimination performance of any LR system and show the steps used to investigate and understand similarities and differences between different LR systems. We share our observations with the forensic community and describe how examining more than one PGS with similar discrimination power can be beneficial, help analysts compare interpretation especially with low-template profiles or minor contributor cases, and be a potential additional diagnostic check even if software in use does contain certain diagnostic statistics as part of the output.

Quality of metagenomic DNA extracted for molecular identification of microorganisms from CSF samples of patients with suspected cerebrospinal meningitis in northern Nigeria

Background: Following an increase in the practice of starting antimicrobial therapy prior to clinical sample collection, the ability to confirm pathogenic microorganisms of bacterial meningitis has decreased by approximately 30%. Culture results may be false negative when fastidious or culture-resistant bacteria are involved or when patient samples are obtained after antimicrobial therapy has started. Molecular diagnosis using PCR can be performed directly on clinical samples after metagenomic DNA (mDNA) extraction not requiring live organisms for a positive result. The specific objectives of this study are to perform mDNA extraction directly from cerebrospinal fluids (CSF) using appropriate spin column method, and to determine the quality of the mDNA elute.Methodology: Cerebrospinal fluid specimens were collected from 210 patients with suspected acute cerebrospinal meningitis (CSM) in the Federal Capital Territory and some States in Northern Nigeria during the 2017 and 2018 outbreak seasons. Metagenomic DNA was extracted from approximately 200µL of CSF specimens using the Qiagen QIAamp(R) DNA Mini kit specific for bacterial agents only. DNA quality check was performed on all DNA elutes using fluorometric, spectrophotometric and agarose gel electrophoresis methods.Results: Of the 210 CSF samples analyzed microbiologically, Gram reaction was positive in 94 cases (44.8 %) but only 17 (8.1 %) were culture positive for two of the three major bacterial causes of meningitis. One hundred and eighty (85.7%) samples had DNA concentrations ≥ 0.005 ng/µL, 55 (30.6 %) of these had DNA purity (A260/A280) of ≥ 1.7, 103 (57.2%) had purity value between 1.0 - 1.69, 14 (7.8%) had value of 0.57 - 0.99, and 8 (4.4%) failed purity evaluation with value of 0.00 at A260/A280.Conclusion: The essence of mDNA extraction is multipurpose. A multiplex PCR can be performed on the extracted mDNA to interrogate the presence of microbial pathogens of interest using specific primers and probes (when applicable). Quality mDNA from CSF samples will ensure successful qPCR results for rapid and accurate detection of bacterial pathogens in meningitis. This will eliminate the challenges associated with traditional culture methods. Keywords: Meningitis, CSF, DNA Quality Check, Fluorometry

Influence of degree of DNA degradation in formalin-fixed and paraffin-embedded tissue samples on accuracy of genome-wide DNA methylation analysis

Aim: Depending upon the degree of DNA degradation of formalin-fixed and paraffin-embedded tissue samples, accuracy of measurement by Infinium MethylationEPIC BeadChip assay (Illumina, CA, USA) was assessed. Materials & methods: DNA quality of six formalin-fixed and paraffin-embedded lung tissue samples with different formalin fixation periods was assessed by Illumina quality control, DNA copy number and DNA integrity number value. Infinium data from restored bisulfite treated DNA were compared with datum from a fresh-frozen sample. Results: The correlation coefficient decreased from 0.993 to 0.970 depending upon DNA degradation, even if the Illumina quality control was met. Exclusion of specific probes improved the correlation regardless of tissue. Conclusion: Poor DNA quality can be assessed as an amplifiable DNA copy number and DNA integrity number value. Probe filtering has the potential to improve assay accuracy.