Optimization of DNA extraction and sampling methods for successful forensic microbiome analyses of the skin and saliva.

Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can provide information, such as geolocation and ancestry information, when short tandem repeat (STR) profiling fails. In this study, methods for DNA extraction and sampling from the skin and saliva were optimized for the construction of a Korean Forensic Microbiome Database (KFMD). DNA yields were estimated using four DNA extraction kits, including two automated kits (Maxwell® FSC DNA IQ™ Casework Kit and PrepFiler™ Forensic DNA Extraction Kit, updated) and two manual kits (QIAamp DNA Mini Kit and QIAamp DNA Micro Kit) commonly used in forensic DNA profiling laboratories. Next-generation sequencing of the 16S rRNA V4 region was performed to analyze microbial communities in samples. The Bacterial Transport Swab with Liquid Media (NobleBio), two cotton swabs (PoongSung and Puritan), and nylon-flocked swabs (NobleBio and COPAN) were tested for DNA recovery. The PrepFiler and Maxwell kits showed the highest yields of 3.884 ng/µL and 23.767 ng/µL from the scalp and saliva, respectively. With respect to DNA recovery, nylon-flocked swabs performed better than cotton swabs. The relative abundances of taxa sorted by DNA extraction kits were similar contributions; however, with significant differences in community composition between scalp and saliva samples. Lawsonella and Veillonella were the most abundant genera in the two sample types. Thus, the Maxwell® FSC DNA IQ™ Casework Kit and nylon-flocked swab (NobleBio) were optimal for DNA extraction and collection in microbiome analyses.

Effects of solvent-based adhesive removal on the subsequent dual analysis of fingerprint and DNA.

The combined approach of classical fingerprinting and DNA profiling is a powerful tool in forensic investigations of latent "touch" traces. However, little attention has been paid to the organic solvents frequently used in dactyloscopic laboratories to facilitate the separation of adhesive evidence prior to fingerprint development and downstream effects on subsequent DNA profiling. In the present study, we tested a selection of adhesive removers (n = 9) and assessed their potential impact on DNA recovery and amplification by PCR. Thereby, we identified and characterized novel PCR inhibitors. All investigated chemicals contain volatile organic compounds that evaporate under normal indoor atmospheric conditions. Exposure to certain solvents resulted in increased DNA degradation, but only if evaporation was prevented. A series of adhesive-removal experiments were conducted with prepared mock evidence (self-adhesive postage stamps affixed to paper envelope) to investigate the impact of treatment time and the location of applied traces on DNA recovery and dactyloscopy, respectively. Due to the early onset of print decomposition, we found that only a short treatment time was compatible with the development of fingerprints on the adhesive side of a stamp. Solvents also removed DNA from the adhesive surface, thus resulting in a marked shift in the substrate distribution of recovered DNA from the stamp to the envelope, but not in the reverse direction. Furthermore, we observed that treatment with conventional fingerprint reagents lead to a significant reduction in the amounts of DNA recovered from stamps, while the additional use of adhesive removers did not significantly enhance this effect.

Collection techniques of touch DNA deposited on human skin following a strangulation scenario.

Trace DNA is a significant type of evidence for its ability to be collected from touched items or surfaces at crime scenes to link suspects to their crimes. In cases of violent crimes like assault, sexual offences, or even homicide, often touch DNA is collected from the victim's skin. However, the collection of touch DNA from the victim's skin can be complex because of the mixture of DNA present, as there is likely to be a small quantity of the offender's DNA compared to the victim's DNA. Validating different collection methods or techniques can improve touch DNA sampling; therefore, this study investigated three collection techniques involving cotton and nylon swabs to test their efficiency for the collection of touch DNA from the human neck. There was a significant difference between the three recovery techniques used to recover touch DNA with a cotton swab (CS) (p < 0.05) and nylon swab (NS) (p < 0.05), with more alleles observed when the neck skin was moistened with 100 µL of distilled water using a spray bottle before collection with both swabs.

Comparison of five membrane filters to collect bioaerosols for airborne microbiome analysis.

AIMS: Recovering DNA of airborne micro-organisms (AM) from air is a challenging task. We compared five membrane filters for bioaerosol sampling-mixed cellulose ester (MCE), polyethersulfone (PES), polyamide (PA), polytetrafluorethylene (PTFE) and polyvinylidene fluoride (PVDF)-based on their bacterial, fungal and eukaryotic DNA recoveries. METHODS AND RESULTS: Bacterial, fungal and eukaryotic populations were quantified using quantitative PCR. With a bacterial consortium, PTFE exhibited the best recovery efficiency (113%), followed by PA (92%), PES (86%), MCE (48%) and PVDF (1%). When filters were compared with air, PA was used as a control to normalize results from the others. The bacterial, fungal and eukaryotic DNA recovery ratios were markedly greater in PES (9·3, 11·5 and 10·3 respectively) than in the remaining. Eukaryotic MiSeq sequencing revealed that PES recovered a more diverse and considerably richer assemblage (richness ratios, 4·97 vs ≤ 1·16 for PES vs the others). Rank abundance distribution analysis showed that distribution tails were longer (>4 times) in PES, but these did not differ between the remaining and PA. Community comparison showed that PES exhibited a lower variation across trials than the PA, while the remaining did not. CONCLUSIONS: PES filter markedly outperformed the other filters in quantitative and qualitative recovery of AM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings demonstrated the importance of filter selection for sampling AM.

Comparison of Isohelix™ and Rayon swabbing systems for touch DNA recovery from metal surfaces.

A previous study evaluating two swabbing systems found that DNA was best recovered from sterile metal substrates using an Isohelix™ swab wetted with isopropyl alcohol rather than a Rayon swab with water as the wetting agent. We tested the same swabbing systems on metal (aluminum, brass, and stainless steel) and plastic substrates in a regularly touched environment to simulate the non-deliberate transfer of touch evidence likely seen in a casework scenario, to ascertain the performance of these swabs in an uncontrolled situation. Higher amounts of touch DNA were recovered with Isohelix™ swabs (0.5 - 3.3 ng) compared to Rayon swabs (0.13 - 1.2 ng). The Isohelix™ swabbing system was found to significantly recover more touch DNA (p = 0.04) from the metal substrates than the Rayon swabbing system, consistent with the findings of our previous work. The results contribute to our understanding of the impact of sample collection techniques on touch DNA recovery from problematic metal surfaces and suggest that supplemental cleaning of substrates as a precautionary step against the spread of infections may affect touch DNA persistence and the recovery efficiency of swabs.

Optimization of CUT&Tag product recovery and library construction method.

The emerging cleavage under target and tagment (CUT&Tag) technology uses Tn5 transposase to cleavage near the DNA binding site of target protein and study the generated DNA fragments by the next-generation sequencing. It can quickly identify protein-DNA interactions, which greatly simplifies the experimental process of ChIP-Seq. After CUT&Tag tagment reaction, DNA recovery or other post-processing is required to perform library construction PCR. Different recovery methods have significant impact respectively. By establishing Streptavidin beads recovery CUT&Tag(srCUT&Tag), we can quickly and conveniently complete the product recovery of CUT&Tag. We carried out CUT&Tag assay of H3K4me3, RNA Polymerase II (RNA polymerase II, RNAPII), transcription factor CTCF and HMGA1 in K562 cells with different recovery methods, including ethanol precipitation, fragment separation magnetic beads (SPRI) Magnetic bead recovery, direct PCR method, as well as our srCUT&Tag recovery method. The results show that among the CUT&Tag results of four different targets, the SPRI magnetic bead recovery and our srCUT&Tag methods have higher recovery efficiency than the direct PCR method and ethanol precipitation method. All CUT&Tag results showed that the recovery of SPRI magnetic beads would lose most of the product fragments less than 150 bp. In the recovery of CTCF and HMGA1, direct PCR lost most of the fragments larger than 300 bp and has significant difference from result of other recovery method. This enables srCUT&Tag to provide more real and higher-resolution information than other recovery method. In summary, the newly established srCUT&Tag recovery method can improve the efficiency of CUT&Tag library construction and obtain better data quality compared with the existing CUT&Tag product recovery method, providing a better technical choice for epigenetics research.

Fecal Recovery of Ingested Cellular DNA: Implications for Noninvasive Detection of Upper Gastrointestinal Neoplasms.

BACKGROUND: Stool DNA testing represents a potential noninvasive approach to detect upper gastrointestinal (UGI) neoplasms. However, little is known about fecal recovery efficiency of DNA exfoliated from UGI tumors. AIMS: The purpose of this study was to establish a human ingestion model that quantitatively approximates daily cellular shedding from UGI neoplasms and to estimate fecal DNA marker recovery rates. METHODS: Healthy volunteers (n = 10) ingested two scheduled doses of raw salmon, 0.3 and 30 g, simulating the mass exfoliated daily from 1 to 4.5 cm lesions. To approach a steady-state, each dose was ingested over three consecutive days in randomized order. Following defecation of an indicator dye ingested with test meals, stools were collected over 48 h. Ingested salmon DNA was captured from stools using probes targeting pathognomonic Salmonidae sequences (SlmII). Captured DNA was quantified using PCR primers to generate 178, 138, 88 and 55 bp amplicons. RESULTS: SlmII sequences were recovered from all stools following salmon ingestion; recovery was proportional to amount ingested (p = 0.004). Fecal recovery of ingested salmon varied inversely with amplicon size targeted; mean recovery rates of SlmII were 0.49, 0.91, 3.63, and 7.31 copies per 100,000 copies ingested for 178, 134, 88, and 55 bp amplicons, respectively (p < 0.0001). Longer oro-anal transit was associated with reduced recovery. CONCLUSIONS: While recovery efficiencies are low, ingested cellular DNA simulating daily amounts shed from UGI tumors can readily be detected in stool. Assay of shorter-fragment analyte increases recovery. This ingestion model has potential value in studying the effects of perturbations relevant to the fecal recovery of DNA exfoliated from UGI tumors.

Preliminary study on mitochondrial DNA analysis from different sports items.

Criminals often attempt to conceal blood-stained weapons used in violent crimes, making forensic evidence crucial in solving cases. This study explores the recovery and extraction of trace DNA from sports equipment, including cricket bats, table tennis racquets, and hockey sticks, which are frequently implicated in such incidents. Our research evaluates various double swab collection methods for retrieving trace DNA from these sports items, emphasizing those associated with blunt force trauma. We also compare presumptive and confirmatory tests to establish a direct correlation. This research consistently demonstrated robust DNA recovery, surpassing a 50 % threshold across all tests. Specifically, DNA recovery from buried samples reached an impressive 87 %, while washed samples still yielded a substantial 80 % efficiency. We conducted a comparative analysis between presumptive and confirmatory testing methods, establishing a direct correlation between the two. Variability in DNA recovery efficiency was observed and attributed to factors like the type of surface the items contacted, and ambient humidity levels. In addition to presenting robust DNA recovery rates, statistical analyses were employed to compare methods, establishing correlations and highlighting the influence of environmental factors on DNA recovery efficiency. These findings have significant implications for forensic investigations involving silent weapons crafted from sports equipment, emphasizing the need for standardized protocols and consideration of environmental factors in DNA analysis.

Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces.

Previously, we demonstrated unique insertion/deletion polymorphisms of equine histidine-rich glycoprotein (eHRG) with five genotypes composed of 45-bp or 90-bp deletions in the histidine-rich region of eHRG in Thoroughbred horses. Although leukocytes are typically used to collect DNA for genotyping, blood sampling from animals is sometimes difficult and invasive. Moreover, the method for extracting DNA from blood leukocytes involves complicated steps and must be performed soon after blood sampling for sensitive gene analysis. In the present study, we performed eHRG genotyping using DNA, isolated from oral mucosa swabs collected by rubbing the mucosa on the underside of the upper lip of horses and 100 mg of freshly excreted feces obtained by scraping their surface. In the present study, we performed eHRG genotyping using DNA isolated from oral mucosa swabs and feces of horses (18 Thoroughbreds, 17 mixed breeds, 2 warm bloods), and compared the accuracy of this method with that of the method using DNA from leukocytes. The DNA derived from oral mucosa swabs was sufficient in quantity and quality for eHRG genotyping. However, DNA derived from fecal samples requires a more sensitive detection system because of contamination with non-horse DNA, and the test quality is low. Collection of oral mucosa swabs is less invasive than blood sampling; further, oral swabs can be stored for a longer period in a specified high-quality solution. Therefore, collecting DNA samples from oral mucosa swabs is recommended for the genetic analysis of not only horses but also other animals that are not accustomed to humans.

Comparison of swabbing and cutting-out DNA collection methods from cotton, paper, and cardboard surfaces.

Choosing an inappropriate method of sample collection can often have a detrimental impact on DNA recovery. Multiple studies highlight the importance of selecting the recovery method based on the type of surface the DNA sample is located on. This study aimed to investigate the efficacy of sample collection via the single cotton swabbing method in comparison to recovery directly from the material cut from the surface. The three types of surfaces included cotton, paper, and cardboard. DNA sources comprised cell-free and cellular DNA, as well as blood and saliva as examples of body fluids commonly encountered at crime scenes. The data analysis revealed that the cutting-out method resulted in higher DNA recovery from all but cardboard surfaces, making it the more efficient collection method. Despite its limitations, the cutting-out method should be considered as the DNA recovery method of choice when suitable.

Salmon sperm DNA increases sample recovery from cotton swabs.

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001-1-10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

Evaluation of the microbial wet-vacuum system (M-Vac®) for DNA sampling from rough, porous substrates, and its compatibility with fully automated platforms.

DNA retrieval methods traditionally used during forensic evidence recovery including swabbing and tape lifting, can have limited effectiveness when used on porous, rough substrates such as bricks and carpet. This is possibly due to the DNA material being dispersed and unreachable for surface sampling techniques. In this evaluation we investigated the effectiveness of the Microbial Wet-Vacuum System (M-Vac®; M-Vac® Systems, Inc., Sandy, UT), as it has been reported to retrieve greater amounts of DNA material from challenging exhibits. A four-stage evaluation was conducted, starting with seeding carpet and brick substrates with a known donor's saliva in two dilutions and comparing the DNA recovery of tape lifting, swabbing, and the M-Vac®. A victim struggle scenario on carpet was then mimicked to compare trace DNA recovery by each method. Two mock scenarios were also conducted; a shirt was submerged in a creek bed for a period of five days to sample for the wearer's DNA, and a car boot was sampled to assess the possibility of recovering a victim's DNA amongst background DNA from the usual car occupants. Finally, the compatibility of the M-Vac® sampling process was optimised for the fully automated DNA lysis and extraction platforms used in the NSW (Australia) jurisdiction by comparing filter subsampling methods. The results from the study were mixed. For bricks, none of the collection methods were effective in retrieving DNA. On carpet, the M-Vac® retrieved the greatest quantities of DNA from the saliva-seeded samples, however, tape lifts outperformed all methods for 'touch' DNA recovery. The M-Vac® retrieved the greatest amount of DNA from the t-shirt recovered from a creek bed as it was able to retrieve the embedded DNA. The final mock case car boot scenario resulted in greater victim DNA recovery from tape lifts, with the M-Vac® more likely to recover mixtures too weak and/or complex to be interpreted. Finally, operational considerations regarding the compatibility of the M-Vac® system with fully automated DNA lysis and extraction are discussed. Considering the substantial time and cost to deploy the M-Vac®, it is recommended to be utilised in casework only after swabbing and tape lifting methods have failed to yield sufficient DNA material, where the substrate properties would likely benefit from the M-Vac's® niche capabilities for retrieving embedded DNA, and low levels of background DNA may be anticipated.

Recovery of DNA from acetaminophen exploring physical state and sampling methods.

Research into the recovery of DNA from illicit drug samples has shown it is possible to get forensically useful profiles from such substrates. However, it is not yet known if the different physical states that drugs can be found in influences the quantity and quality of DNA that can be recovered or what is the best sampling method to adopt for powdered samples. This research used acetaminophen in four different states - large crystalline, powder, in solution, or residue - to determine the efficacy of current DNA technology in recovery and analysis of the resulting sample. Five replicates of each were prepared. Human blood was deposited on or mixed with the drug and left for 1 hour. The surface of the drug was sampled by wet/dry swabbing (where appropriate), or the entire sample was deposited in a tube, and the DNA then extracted using DNA-IQ™. The amount of DNA recovered (ng), degradation index, number of PCR cycles (Ct) required for the IPC to reach threshold, number of alleles in the DNA profile and average peak height (APH) were assessed. All samples, irrespective of the physical state they were collected from, returned full DNA profiles that corresponded to the DNA profile of the blood donor, with no degradation or inhibition detected. It was also found the wet/dry swabbing method returned higher levels of DNA than inclusion of the entire sample into the tube for powdered acetaminophen and the appropriate method to use will be dependent on casework circumstances. The findings of this research further develops our understanding of the recovery of DNA from drugs, and supports the need for further investigation to understand under what conditions DNA can be recovered from illicit substances.

The impact of substrate characteristics on the collection and persistence of biological materials, and their implications for forensic casework.

This study assessed the level of nucleic acid persistence on the substrate pre-, and post-swabbing, in order to assess whether biological materials (touch, saliva, semen, and blood) are collected differently depending on the substrate characteristics. A total of 48 samples per deposit and substrate variety (n = 384) were assessed by tracking the persistence of nucleic acid using Diamond™ Nucleic Acid Dye (DD) staining and Polilight photography. The number of DD nucleic acid fluorescent complexes formed post-staining were counted (fluorescent count) and in conjunction with the fluorescence signal intensity (DD nucleic acid complex accumulation) used to estimate the level of nucleic acid persistence on substrates. Touch deposits have shown to be the most persistent deposit with strong adhesion capabilities on both substrate verities. Saliva displayed a higher persistence than semen and/or blood. Semen displayed a high collection efficiency as well as a high fluorescence signal intensity. Blood displayed a low persistence on both substrates with a superior collection efficiency that may also indicate a higher probability to become dislodged from surfaces given a particular activity. Our research has shown that the persistence and recovery of biological deposits is not only measurable but more importantly, may have the potential to be estimated, as such, may build an understanding that can provide valuable guidance for collection efficiency evaluations, and the assessing of the probability of particular profiles, given alternate propositions of means of transfer occurring.

A comparison of six adhesive tapes as tape lifts for efficient trace DNA recovery without the transfer of PCR inhibitors.

Tape-lifting is a non-destructive method employed in the laboratory to recover and collect trace DNA evidence from crime scene exhibits with porous surfaces. The success of tape-lifting is a balance between capturing the biological material and compatibility with downstream DNA extraction processes to ensure efficient release of the tape-lifted material during DNA extraction. In this study, six commercially available low-, regular- and high-tack adhesive tapes were evaluated. The low-tack S183 tape and the highly adhesive S-Hold tape were compared for DNA recovery efficiency from different materials commonly encountered in casework. All tape-lifts were processed using PrepFiler Express™ BTA and AutoMate Express™ Forensic DNA extraction systems, DNA samples quantitated by Quantifiler TRIO, amplified using Powerplex® 21 and VeriFiler™ PLUS (VFP), and analysed on a 3500xl genetic analyser to evaluate the quality of the resultant STR profiles obtained. The more adhesive S-Hold tape recovered comparable or more DNA than the low-tack S183 tape from the majority of materials tested. However, STR profiles obtained from S183 tape-lifts were of markedly higher quality compared to S-Hold tape-lifts. This was most evident for towel, denim and printed chiffon, where S-Hold samples exhibited severe PCR inhibition, with VFP internal quality markers confirming the presence of inhibitors. The findings suggest that strong adhesion is not necessarily beneficial for tape-lifting, as the low tack S183 tape was able to efficiently recover cellular material from the surface of porous substrates commonly encountered in casework, while avoiding the co-transfer of PCR-inhibitory substances from the sampled material.

The detection of blood, semen and saliva through fabrics: A pilot study.

This study aimed to identify if biological material could be detected on the opposite side to deposition on fabric by commonly used presumptive and/or secondary tests. Additionally, this study aimed to ascertain if there is a difference in the DNA quantity and quality from samples obtained from both sides of the same substrate: cotton, polyester, denim, or combined viscose and polyester swatches. Blood, semen, or saliva (25 µL) was deposited on one side of 5 replicates of each fabric type and left for 24 h. Blood swatches were tested using Hemastix® and the ABACard® HemaTrace® immunoassay, semen swatches were tested using acid phosphatase (AP) reagent, the ABACard® p30® immunoassay and hematoxylin and eosin staining, and saliva swatches were tested using Phadebas® paper and the RSID-Saliva™ immunoassay. Both sides of each swatch were separately wet/dry swabbed and subjected to DNA analysis. Blood was able to be detected on the underside of all fabrics using both tests. Semen was able to be detected on the underside of swatches using the presumptive AP test but not p30®, and sperm was rarely observed. Saliva was able to be detected by RSID-Saliva™ but not Phadebas® paper when the underside of swatches were tested. Across all biological materials, DNA was able to be recovered from the top side of all 60 swatches. For the underside, DNA was able to be recovered from 54 swatches. Of the 6 swatches that DNA was unable to be recovered from, one sample was from semen and the rest were from saliva. This study has demonstrated that DNA and components of interest in forensically relevant biological material can be recovered from the opposite side to where it was originally deposited, and that observing biological material and/or DNA on one side of fabric does not definitively indicate direct deposition on that side.

Prevalence of male DNA on female worn undergarments. How small datasets may support robust opinions in activity level evaluative reporting.

In cases of sexual assault, the interpretation of biological traces on clothing, and particularly undergarments, may be complex. This is especially so when the complainant and defendant interact socially, for instance as (ex-)partners or by co-habitation. Here we present the results from a study where latent male DNA on female worn undergarments is recovered in four groups with different levels of male-female social interaction. The results conform to prior expectation, in that less interaction tend to result in less male DNA on undergarments. We explore the use of these experimental data for evaluative reporting given activity level propositions in a mock case scenario. We show how the selection of different populations to represent the social interaction between complainant and defendant may affect the strength of the evidence. We further show how datasets of limited size can be used for robust activity level evaluative reporting.

Effect of swabbing technique and duration on forensic DNA recovery.

Various factors have been shown to affect performance of the conventional wet-dry double and single wet swabbing techniques to recover DNA, such as pressure and angle of application, volume and type of wetting agent, and swab type. However, casework laboratories in some jurisdictions have recently adopted different swabbing techniques that include wet-moist double swabbing and moist-dry single swabbing. Factors affecting the effectiveness of these recent techniques in maximising DNA recovery therefore need to be investigated. Here, the performance of traditional and recent swabbing techniques was compared and the impact of swabbing duration on DNA recovery was investigated. Ten µl aliquots of a known concentration of DNA extracted from human blood were deposited on pre-cleaned DNA-free cotton swatches (porous) and porcelain tiles (non-porous). Five swabbing techniques were used, of which three were double swabbing techniques: wet-moist, wet-wet and wet-dry, and two were single swabbing techniques: wet and moist-dry. For a 'wet' or 'moist' swab, 100 or 50 µL water was added, respectively. For a moist-dry swab, water was applied to one side of the swab, leaving the other side drier. Each swabbing technique was applied for two durations, 15 and 30 s per swab, with 5 reps of each combination (n = 100 plus controls). All samples were extracted and quantified, and a sub-set was profiled. The results showed that the wet-moist double swabbing technique with a swabbing duration of 30 s maximised DNA recovery from cotton. From tile, a single wet or moist-dry swab maximised DNA recovery, but increasing swabbing duration from 15 to 30 s had no impact. These data can be used to inform standardisation of DNA collection protocols across casework laboratories.

Preliminary investigation into isolation and extraction of DNA recovered from drug residues.

It is a commonly held belief that drug residues may affect the integrity of DNA and/or interfere with DNA analysis, and therefore DNA on drug paraphernalia and the associated drugs may be overlooked as a source of evidence. This study investigated whether DNA could be isolated from a drug residue-bearing surface to ascertain whether a forensically useful DNA profile could be obtained. Human blood and pre-extracted "naked" DNA were deposited on samples of acetaminophen, codeine, morphine, oxycodone, ketamine, and synthetic cannabinoids and left for an hour before DNA extraction using DNA-IQ™. To investigate DNA integrity, the absolute amount of DNA recovered, degradation index, and number of PCR cycles required for the IPC to reach threshold (Ct), number of reportable alleles and average peak height (APH) in the DNA profile, were examined. The samples were also qualitatively analysed using LC:MS to determine if any residual drugs were present in the samples post-DNA extraction. Overall, the drugs had no to minimal degradation or inhibitory effects on the DNA with sufficient DNA recovered to generate a partial or full DNA profile in 80% of naked DNA samples and 100 % of blood samples. The amount of DNA collected was sufficient for further analysis in 86% of naked DNA samples, and 100% of blood samples, with all median APH values being over the 175 RFU standard. Chemical analysis showed that traces of the drug were still present in the samples after DNA extraction was performed. Therefore, this study demonstrates forensically useful DNA can be recovered from surfaces bearing drug residues, even when sampling directly from the samples of drugs.

An improved rapid method for DNA recovery from cotton swabs.

We present a novel rapid method for the recovery of cellular and free DNA from cotton swabs based on a simple elution buffer containing a high molecular weight polymer and detergent combined with a short proteinase K digestion to release cellular DNA. This method shows increased yields approaching 80% recovery of the input DNA compared to the QIAamp DNA Mini kit standard extraction protocol for swabs which has a recovery of 20-30%. The buffer components in the described method are compatible with direct PCR analysis of the isolated DNA without further purification. Recovery efficiencies were estimated by qPCR.