DNA Analysis Research Articles

Current research status and clinical applications of noninvasive preimplantation genetic testing: A review

Noninvasive preimplantation genetic testing (ni-PGT) is conducted by obtaining genetic information from embryos through the analysis of free DNA released by embryos in spent embryo culture medium or blastocoel fluid. Compared to conventional preimplantation genetic testing relying on trophectoderm biopsy, ni-PGT is characterized by its noninvasiveness. It has demonstrated early advancements in the detection of embryonic chromosomal aneuploidies and the diagnosis of monogenic diseases, showcasing considerable potential for clinical application. However, there are substantial controversies in the literature concerning the reliability of ni-PGT, the source of cell-free DNA, and maternal contamination. This paper elaborates on the principles, research advancements, effectiveness, and limitations of ni-PGT to provide a basis for clinical applications.

Soibean: High-resolution Taxonomic Identification of Ancient Environmental DNA Using Mitochondrial Pangenome Graphs.

Ancient environmental DNA (aeDNA) is becoming a powerful tool to gain insights about past ecosystems, overcoming the limitations of conventional fossil records. However, several methodological challenges remain, particularly for classifying the DNA to species level and conducting phylogenetic analysis. Current methods, primarily tailored for modern datasets, fail to capture several idiosyncrasies of aeDNA, including species mixtures from closely related species and ancestral divergence. We introduce soibean, a novel tool that utilises mitochondrial pangenomic graphs for identifying species from aeDNA reads. It outperforms existing methods in accurately identifying species from multiple closely related sources within a sample, enhancing phylogenetic analysis for aeDNA. soibean employs a damage-aware likelihood model for precise identification at low coverage with a high damage rate. Additionally, we reconstructed ancestral sequences for soibean's database to handle aeDNA that is highly diverged from modern references. soibean demonstrates effectiveness through simulated data tests and empirical validation. Notably, our method uncovered new empirical results in published datasets, including using porpoise whales as food in a Mesolithic community in Sweden, demonstrating its potential to reveal previously unrecognised findings in aeDNA studies.

Multiplexed chromatin immunoprecipitation sequencing for quantitative study of histone modifications and chromatin factors.

ChIP-seq is a widely used technique for studying histone post-translational modifications and DNA-binding proteins. DNA fragments associated with a specific protein or histone modification epitope are captured by using antibodies, sequenced and mapped to a reference genome. Albeit versatile and popular, performing many parallel ChIP-seq experiments to compare different conditions, replicates and epitopes is laborious, is prone to experimental variation and does not allow quantitative comparisons unless adequate spike-in chromatin is included. We present a detailed protocol for performing and analyzing a multiplexed quantitative chromatin immunoprecipitation-sequencing experiment (MINUTE-ChIP), in which multiple samples are profiled against multiple epitopes in a single workflow. Multiplexing not only dramatically increases the throughput of ChIP-seq experiments (e.g., profiling 12 samples against multiple histone modifications or DNA-binding proteins in a single experiment), but also enables accurate quantitative comparisons. The protocol consists of four parts: sample preparation (i.e., lysis, chromatin fragmentation and barcoding of native or formaldehyde-fixed material), pooling and splitting of the barcoded chromatin into parallel immunoprecipitation reactions, preparation of next-generation sequencing libraries from input and immunoprecipitated DNA and data analysis using our dedicated analysis pipeline. This pipeline autonomously generates quantitatively scaled ChIP-seq tracks for downstream analysis and visualization, alongside necessary quality control indicators. The entire workflow requires basic knowledge in molecular biology and bioinformatics and can be completed in 1 week. MINUTE-ChIP empowers biologists to perform every ChIP-seq experiment with an appropriate number of replicates and control conditions, delivering more statistically robust, exquisitely quantitative and biologically meaningful results.

Raman-enhanced sensor based on CRISPR-SERS technology for the rapid and hypersensitive detection of Mycobacterium tuberculosis.

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)-based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100pM. The equation describing the standard curve was y = 24.10x + 1594, with R2 = 0.9914. The limit of detection was as low as 4.42pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 5 copy/μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology.

Familial hypodontia in bronze age Northwest China (1046–771BC)

ObjectiveThis research aimed to report hypodontia cases in a Middle Bronze Age high-tier cemetery in China and test the possible hereditary behind hypodontia by performing kinship tests on those individuals. DesignIn this study, dental anomalies were observed on a human skeletal sample (n = 45) uncovered from Yaoheyuan, China. Ancient DNA analysis was subsequently employed on a subsample of the Yaoheyuan individuals (n = 15), including individuals observed hypodontia and individuals randomly sampled from the cemetery for preliminary investigation on the cemetery demography. Kinship estimation tests (READ, TKGWV2, KIN, and F3 test) were subsequently employed. ResultsThe Yaoheyuan elite population had a prevalence (n = 7, 15 %) of tooth agenesis in either the maxilla or mandible, with one to two teeth missing. All missing teeth were incisors, except for one individual missing maxillary second molar. Preliminary ancient DNA results indicate that several kinship groups existed among interred individuals, including those with hypodontia, indicating the hereditary origin of these cases. ConclusionsThe prevalence of hypodontia observed on site is high compared to that in both modern East Asian populations and archaeological samples in the Chinese population. The preliminary kinship analysis suggests a case of familial hypodontia. Ancient DNA analysis should be thoroughly conducted in future studies to understand the genetic markers contributing to those hypodontia cases among the Yaoheyuan individuals.

Demonstration of potential DNA contamination introduced by laboratory consumables using Fluorescein

The development of increasingly efficient DNA extraction and profiling kits has increased the amount of allelic information obtained from trace DNA samples, but also inadvertently, increased the detection of DNA contamination. This study aimed to evaluate the potential of DNA transfer using fluorescein, fluorescent under an alternate light source, in the use of a range of forensically relevant DNA profiling consumables. An evaluation of two pre-lysis methods adopting three different sample tubes, some with deliberate seal damage, showed the PrepFiler™ Automated Forensic DNA Extraction Kit caused leakage and crusting when the rim of the PrepFiler™ LySep column was compromised, but no leakage was observed under the same conditions using the Investigator STAR Lyse&Prep kit. The AutoLys tube showed minimal leakage using the PrepFiler™ chemistry. A DNA extract tube with an external thread, similar to the AutoLys tube, showed no leakage after fridge or freezer storage. However, it highlighted that a centrifugal spin does not guarantee all the DNA will pool at the base of the tube. A comparison of adhesive plate sealing films to 8-well strip caps for sealing 96-well PCR plates showed the adhesive plate sealing films presented a lower risk of DNA transfer, largely due to the adhesion of dispersed liquid on the sticky surface of the film. Overall, this study highlighted a number of variables that may be considered in the development of more refined contamination minimisation protocols in respect to increased sensitivities of DNA profiling.

Advances in Forensic Genetics: exploring the potential of long read sequencing

Advances in Forensic Genetics: exploring the potential of long read sequencing

Limit Detection of Short Tandem Repeats (STR) Analysis on Touch DNA Samples

Forensic short tandem repeats (STR) profiling on touch DNA samples has emerged as a primary method for human identification. The stability and uniqueness of STR combination from the targeted locus in each individual make it a precision marker for human identification. Touch DNA samples can be found in traces of biological material shed from a person. This work aimed to identify the lowest concentration limit required for generating an interpretable DNA profile and the sensitivity of the STR loci applied. Touch DNA samples were collected from donors who were asked to hold a rope for 5 min. A double swab technique was used to lift the touch samples from the rope. These samples are subjected to DNA extraction and quantification. Two STR amplification cycles, 29 and 34 cycles, were used. DNA concentration greatly influences the success of amplifying the target allele at each STR locus to be interpreted into a complete DNA profile, shown by its allele peak. Touch DNA concentration >0.25 ng can produce a complete DNA profile. LCN method successfully amplified touch DNA with a concentration 0.0625–0.25 ng/µL. Limit detection of touch DNA analysis is 0.25 ng/µL. Low-copy DNA can still be analyzed within 0.0625–0.25 ng/µL.

The phyllosphere of Nigerian medicinal plants, Euphorbia lateriflora and Ficus thonningii is inhabited by a specific microbiota

The microbiota of medicinal plants is known to be highly specific and can contribute to medicinal activity. However, the majority of plant species have not yet been studied. Here, we investigated the phyllosphere composition of two common Nigerian medicinal plants, Euphorbia lateriflora and Ficus thonningii, by a polyphasic approach combining analyses of metagenomic DNA and isolates. Microbial abundance estimated via qPCR using specific marker gene primers showed that all leaf samples were densely colonized, with up to 108 per gram of leaf, with higher bacterial and fungal abundance than Archaea. While no statistically significant differences between both plant species were found for abundance, amplicon sequencing of 16S rRNA and ITS genes revealed distinct microbiota compositions. Only seven of the 27 genera isolated were represented on both plants, e.g. dominant Sphingomonas spp., and numerous members of Xanthomonadaceae and Enterobacteriaceae. The most dominant fungal families on both plants were Cladosporiaceae, Mycosphaerellaceae and Trichosphaeriaceae. In addition, 225 plant-specific isolates were identified, with Pseudomonadota and Enterobacteriaceae being dominant. Interestingly, 29 isolates are likely species previously unknown, and 14 of these belong to Burkholderiales. However, a high proportion, 56% and 40% of the isolates from E. lateriflora and F. thonningii, respectively, were characterized as various Escherichia coli. The growth of most of the bacterial isolates was not influenced by extractable secondary metabolites of plants. Our results suggest that a specific and diverse microbial community inhabits the leaves of both E. lateriflora and F. thonningii, including potentially new species and producers of antimicrobials.

Numerical investigation of centrifugal passive mixing in Omega shaped microchannel and comparison with fixed platform

The extraction of DNA from isolated cells is crucial for diagnosing and treating various diseases, including cancer, which has become a leading cause of death in recent years. Circulating tumor cells (CTCs) in the blood can be separated from other blood cells, allowing for DNA extraction and analysis for further research. Microfluidic methods are more cost-effective than conventional DNA extraction techniques and have significant potential for high-throughput mixing in lab-on-a-chip (LOC) and lab-on-a-disk (LOD) devices without damaging DNA. Chemical methods for cell lysis, where the mixing of reagents is essential, are particularly efficient on these platforms due to the generation of secondary flows. This is especially true in microchannels with a serpentine design, which enhances mixing quality—a critical parameter for cell lysis efficiency. This study presents and compares the mixing capabilities of the two microchannel platforms (LOC and LOD). Additionally, we introduce a new Omega-shaped serpentine microchannel that combines features of both straight and curved serpentine channels. The mixing quality across all geometries and both platforms has been investigated. Our findings indicate that the centrifugal platform is the most effective for mixing at low inlet velocities. Furthermore, we examine the number of stages in the Omega channel and conclude that 7 Ω stages are sufficient for achieving high mixing quality (∼100 %).

Genetic diversity and structure of Tunisian and Indian date palm (Phoenix dactylifera and sylvestris) cultivars and genotypes revealed by AFLP markers

The date palm breeding programs need to discover valid genetic fingerprints to characterize cultivars and assess their genetic diversity. This study assessed the genetic diversity among thirty-nine date palm cultivars from Tunisia ( Phoenix dactylifera ) and India (Phoenix sylvestris) by using six AFLP (Amplified Fragment Length Polymorphism) markers. 360 loci were amplified, with 127 loci polymorphic (34.35%). The Jaccard's similarity coefficient ranged from 0.161 to 0.931, with the mean genetic distances of 0.568. AFLP's average marker index value was 7.28, with a resolving power of 10.91. The analysis of population structure showed three main clusters with a clear separation between Tunisian and Indian cultivars.Furthermore, the heatmap analysis allowed the identification of 10 bands specific to the Indian accessions, which were not detected in Tunisian genotypes. These loci could be linked to genes involved in adapting the species in Indian lands, which allowed the study of the genetic diversity of date palm resources of different origins, confirming the existence of at least two origins of domestication. Additionally, identifying AFLP loci specific to P. dactylifera and P. sylvestris will significantly contribute to breeding programs by exploiting species-specific polymorphisms.

Fish diversity assessment through conventional morphological identification and recent advances in Saudi Arabia: A review

Fish identification in the Red Sea, particularly in Saudi Arabia, has a long history. Because of the vast fish diversity in Saudi Arabia, proper species identification is required. Indeed, identifying fish species is critical for biodiversity conservation, food and drug safety, and sustainable fishery management. Numerous approaches have been used to identify fish species, including conventional morphological identification, next-generation sequencing (NGS), nanopore sequencing, DNA barcoding, and environmental DNA analysis. In this review, we collected as much scientific information as possible on species identification in Saudi Arabia. Our findings suggest that the identification process has advanced and spread rapidly and broadly, as evidenced by the discovery of new fish species in Saudi Arabia. The advantages and disadvantages of each method were discussed as part of a comprehensive comparison. This study aimed to provide further scientific knowledge to promote the growth of fish diversity worldwide. Keywords: fish diversity, identification process, Saudi Arabia.

Prevalence and distribution of trematodes hosted by Bithynia siamensis in Southern Thailand.

This study aimed to investigate the occurrence of larval trematode infections in bithyniid snails across five provinces in southern Thailand. A total of 1,413 Bithynia siamensis snails were collected between October 2021 and October 2022 using handpicking and scooping methods. Among these, 844 were identified as B. s. siamensis and 569 as B. s. goniomphalos. The snail samples were examined for parasitic infections in the laboratory using shedding and crushing methods. Among the 27 sampling sites, snails from 6 sites were infected with various trematode species (infection rate: 4.95%, 70/1,413). Evaluation of the morphological and internal organ characteristics of the cercariae allowed for the categorization of the trematodes into five species of trematodes (belonging to four families). The identified species included Echinochasmus pelecani (family: Echinostomatidae), Echinostoma revolutum (family: Echinostomatidae), Haematoloechus similis (family: Haematoloechidae), Loxogenoides bicolor (family Lecithodendriidae), and Stictodora tridactyla (family: Heterophyidae), and the infection rates for these species were 3.26% (46/1,413), 0.35% (5/1,413), 0.42% (6/1,413), 0.78% (11/1,413), and 0.14% (2/1,413), respectively. The cercariae from the identified trematode species were studied for DNA analysis. The phylogenetic lineage reveals relationships among the species, confirming the morphological distinctions.

Gram-Negative Colonization and Bacterial Translocation Drive Neonatal Sepsis in the Indian Setting.

The gut microbiota, comprising billions of microorganisms, plays a pivotal role in health and disease. This study aims to investigate the effect of sepsis on gut microbiome of neonates admitted to the Neonatal Intensive Care Unit. A prospective cohort study was carried out in the NICU of tertiary care hospital in Karnataka, India, from January 2021 to September 2023. Preterm neonates with birth weight < 1500g and gestational age < 37 weeks were recruited, excluding those with congenital gastrointestinal anomalies, necrotizing enterocolitis, or blood culture-negative infections. The study population was divided into three groups: healthy neonates (Group A), neonates with drug-sensitive GNB sepsis (Group B), and neonates with pan drug-resistant GNB sepsis (Group C). Stool samples were collected aseptically, snapped in liquid nitrogen, and stored at -80⁰C for extraction of DNA and microbiome analysis. The gut microbiota of healthy neonates (Group A) was dominated by Proteobacteria (24.04%), Actinobacteria (27.13%), Firmicutes (12.74%), and Bacteroidetes (3%). Predominant genera included Bifidobacterium (55.17%), Enterobacter (12.55%), Enterococcus (50.69%), Streptococcus (7.92%), and Bacteroides (3.58%).Groups B and C, the microbiota exhibited higher Proteobacteria abundance (57.16% and 66.58%, respectively) and reduced diversity of beneficial bacteria. Notably, the presence of sepsis was associated with an increase in pathogenic bacteria and a decrease in beneficial commensal bacteria. Neonates with sepsis exhibited significant gut microbiome dysbiosis, characterized by increased Proteobacteria and reduced beneficial bacteria diversity. These findings highlight the potential of microbiome profiling as a diagnostic tool and underscore the importance of gut microbiota modulation in managing neonatal sepsis.

Diagnosis of bacteria isolated from patients with cutaneous eczema by PCR technique at Azadi Hospital, Kirkuk, Iraq

Eczema is a common skin condition that can be worsened or caused by a bacterial infection. This study aimed to investigate the bacterial pathogens associated with skin eczema in patients admitted to Azadi Hospital in Kirkuk using polymerase chain reaction (PCR) technology. The goal was to enhance the accuracy and efficiency of bacteria identification compared to traditional methods. A total of [300] patients suffering from symptoms of skin eczema between the ages of 1-40 years, females and males, were included. Bacterial isolates were obtained from skin samples, and conventional microbiological techniques were initially used for identification. Subsequently, molecular analysis using PCR was performed to confirm and characterize the bacterial species. The study included 165 male patients represented (55%), 135 female (45%), of participants who had symptoms of skin eczema. The PCR test targeted specific genes associated with common bacteria implicated in skin infections, such as Staphylococcus. aureus (33 %), Staphylococcus epidermidis (28%), Klebsiella pneumoniae (20 %) , pseudomonas aeruginosa (13 %), respectively, and the remaining( 6 %). of the samples were natural flora. The genetic fingerprints of these bacterial species were identified, an image of them was taken on an agarose gel electrophoreses , and the results were compared with those obtained through traditional methods to evaluate the sensitivity and specificity of PCR in diagnosing bacterial infections in cases of skin eczema. Preliminary results indicated that PCR significantly improved the accuracy and speed of bacterial identification compared to traditional methods. It has allowed the detection of specific bacterial species that might be missed using conventional methods. The prevalence and diversity of bacterial species causing skin eczema in Azadi Hospital in Kirkuk were also explored. This study contributes valuable insights into the microbial etiology of cutaneous eczema, emphasizing the importance of molecular techniques in accurate and rapid diagnosis. The findings may guide more targeted and effective treatment strategies for patients with bacteria-associated skin eczema, ultimately improving patient outcomes and reducing the risk of complications.

Developmental Validation of the Microreader 23HS Plex ID System: A Novel Supplementary Non-CODIS STR Multiplex Assay for Forensic Application.

A novel supplementary non-CODIS STR multiplex assay designated as the "Microreader 23HS Plex ID System" was developed. The Microreader 23HS Plex ID System enables simultaneous profiling of 23 STR loci and the amelogenin locus. The majority of these loci are non-CODIS STRs (D4S2408, D9S2157, D20S161, D3S2459, D18S1364, D13S305, D1S2142, D19S400, D6S1017, D7S1517, D2S1776, D2S1360, D3S1744, D16S3391, D3S1545, D11S4463, D20S85, D1S549, D10S2325, D21S2055), with the exception of three CODIS STRs (D2S441, D12S391, and D22S1045). Followed the recommendations of Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese validation standards, a comprehensive set of validation studies were conducted, encompassing PCR conditions, stutter ratio and peak height balance, sensitivity, precision and accuracy, reproducibility, species specificity, inhibition, as well as mixture testing. The results demonstrated that the Microreader 23HS Plex ID System is a reliable and robust assay, with well-balanced peak heights, high precision and accuracy, species specificity, and resistance to common inhibitors. The sensitivity of the assay was determined to be 0.125ng of template DNA. In mixture study, all minor alleles were detected in two-sample mixtures across various ratios (1:19, 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 4:1, 9:1, and 19:1). In population study, a total of 500 unrelated individuals of Han ethnicity from East China were genotyped. The allele frequencies and forensic population genetic parameters were calculated, with a cumulative random match probability of 7.757×10-27, and a total power of discrimination exceeding 0.999,999,999,999,999,999,999,999,99. In conclusion, the Microreader 23HS Plex ID System shows promise as a valuable supplementary tool for forensic applications, particularly in addressing complex kinship testing and challenges posed by STR mutation.

Application of PCR-Based Techniques for the Identification of Genetic Fingerprint Diversity of Dominant Bacteria in Fecal Samples of Children with Diarrhea in Vietnam

In Vietnam, diarrhea, especially persistent diarrhea, is one of the most common diseases in children, while a significant proportion of cases are negative with pathogens; thus, there is an urgent need to understand gut bacterial dysbiosis. In this study, bacteria in the fecal samples of five healthy and ten diarrheal children were separated from other residues, then adopted to extract their metagenomic DNA for evaluating their diversity based on V3 and V6–V8 regions and the 16S rRNA gene by PCR-RFLP and PCR-DGGE. As a result, bacterial metagenomic DNAs with high quality, quantity and diversity were successfully extracted using a GeneJET kit and a chemical protocol. A sequence analysis of 73 representative DNA fragments from gels indicated a remarkable bacterial dysbiosis in all groups of diarrhea. Viral diarrhea was characterized by extremely reduced bacterial diversity with the blossom of Bifidobacterium and Streptococcus. Streptococcus was also the most abundant in persistent diarrhea. Beneficial bacteria that may play a role in the self- rebalance in intestinal bacterial communities, such as Bifidobacterium, Lactobacillus, and Enterococcus, were seen in all diarrheal groups, while Bacteroides and Akkermansia muciniphila were seen in the healthy group but absent in the diarrheal groups. This study provides additional evidence for a relationship between intestinal bacterial dysbiosis and diarrhea in children, emphasizing an increase in Streptococcus.

Protective role of antibodies in enteric virus infections: Lessons from primary and secondary immune deficiencies.

Enteric viruses are the main cause of acute gastroenteritis worldwide with a significant morbidity and mortality, especially among children and aged adults. Some enteric viruses also cause disseminated infections and severe neurological manifestations such as poliomyelitis. Protective immunity against these viruses is not well understood in humans, with most knowledge coming from animal models, although the development of poliovirus and rotavirus vaccines has extended our knowledge. In a classical view, innate immunity involves the recognition of foreign DNA or RNA by pathogen recognition receptors leading to the production of interferons and other inflammatory cytokines. Antigen uptake and presentation to T cells and B cells then activate adaptive immunity and, in the case of the mucosal immunity, induce the secretion of dimeric IgA, the more potent immunoglobulins in viral neutralization. The study of Inborn errors of immunity (IEIs) offers a natural opportunity to study nonredundant immunity toward pathogens. In the case of enteric viruses, patients with a defective production of antibodies are at risk of developing neurological complications. Moreover, a recent description of patients with low or absent antibody production with protracted enteric viral infections associated with hepatitis reinforces the prominent role of B cells and immunoglobulins in the control of enteric virus.

Bio-mimicking DNA fingerprint profiling for HLS watermarking to counter hardware IP piracy

The multifaceted, multivendor-based global design supply chain induces hardware threats of intellectual property (IP) piracy for modern computing and electronic systems. Current hardware watermarking techniques fall short either in terms of watermark strength (size of covert constraints generated) or number of security layers/variables involved in the security constraints generation process. This paper presents a novel approach for high level synthesis (HLS) watermarking by bio-mimicking DNA fingerprint profiling to counter hardware IP piracy. The proposed approach effectively captures the vital DNA fingerprint profiling phases such as DNA sequencing, DNA fragmentation, fragment replication, DNA ligase, etc. and bio-mimics them to generate a digital watermarking framework. The presented approach has been demonstrated on convolutional layer and JPEG compression-decompression (CODEC) algorithms that are widely used in several medical and machine learning applications. The proposed approach has been thoroughly compared with several state-of-the-art approaches. The proposed approach depicts superior security in the probability of coincidence of up to ~ 104 and tamper tolerance of up to ~ 10368 at 0% overhead as compared to the prior approaches.

IdentiFLY: The Development and Validation of a 15-Plex SNP Assay for Forensic Identification of UK Blowfly Species (Calliphoridae)

Members of the blowfly family (Calliphoridae) are usually the first insect species to arrive at a corpse, using the body as an oviposition site, and, as such, they are the most important group of insects used to estimate the post-mortem interval (PMI). PMI estimations are based on species-specific developmental timings; therefore, accurate species identification is crucial. Current identification methods are based on morphological characteristics, which are time-consuming and difficult to perform on damaged, immature specimens and closely related species. Advances have led to specimens being identified via a host of molecular techniques, mainly DNA sequencing. Although molecular identification is becoming increasingly more common, there is currently a lack of genetic data regarding UK Calliphoridae species. This study aimed to address this issue. We present the development and validation of an identification assay capable of differentiating six UK species (Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia illustris, Lucilia caesar, and Protophormia terranovae). The sequencing of six genes, including both nuclear (28S rRNA and Elongation factor 1 alpha) and mitochondrial markers (Cytochrome oxidase I and II, Cytochrome b and 16S rRNA) identified 298 species-specific single nucleotide polymorphisms (SNPs). Fifteen SNPs from six genes were chosen for inclusion in a SNaPshot™ multiplex assay. The developed assay is capable of differentiating the species based on between 4 and 12 SNPs. Validation following guidelines by the International Society of Forensic Genetics (ISFG) demonstrated the assay to be accurate, reproducible, sensitive, and specific.