DNA Contamination Research Articles

Presence of p25alpha-Domain in Seed Plants (Spermatophyta): Microbial/Animal Contaminations and/or Orthologs.

Genome and transcriptome assembly data often contain DNA and RNA contaminations from external organisms, introduced during nucleotide extraction or sequencing. In this study, contamination of seed plant (Spermatophyta) transcriptomes/genomes with p25alpha domain encoding RNA/DNA was systematically investigated. This domain only occurs in organisms possessing a eukaryotic flagellum (cilium), which seed plants usually do not have. Nucleotide sequences available at the National Center for Biotechnology Information website, including transcriptome shotgun assemblies (TSAs), whole-genome shotgun contigs (WGSs), and expressed sequence tags (ESTs), were searched for sequences containing a p25alpha domain in Spermatophyta. Despite the lack of proteins containing the p25alpha domain, such fragments or complete mRNAs in some EST and TSA databases were found. A phylogenetic analysis showed that these were contaminations whose possible sources were microorganisms (flagellated fungi, protists) and arthropods/worms; however, there were cases where it cannot be excluded that the sequences found were genuine hits and not of external origin.

Reverse transcription-quantitative PCR (RT-qPCR) without the need for prior removal of DNA

The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA’s integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.

Mpox virus DNA contamination can still be detected by qPCR analysis after autoclaving

Mpox virus DNA contamination can still be detected by qPCR analysis after autoclaving

P-727 New methods reveal the true incidence of DNA contamination in PGT-A samples for the first time and avoid errors that could result in serious misdiagnoses

Abstract Study question What is the occurrence of contamination in embryo biopsy samples and could this lead to incorrect interpretation of preimplantation genetic testing for aneuploidy (PGT-A) results? Summary answer On average, contamination affects 0.4% of biopsy samples, but can be significantly more common in some clinics. Misdiagnosis can occur when contamination is not detected. What is known already Until recently, most commercially available platforms for PGT-A have utilised whole genome amplification followed by sequencing of a random selection of DNA fragments scattered across the genome using next generation sequencing (NGS). However, the simple quantitative measurements of DNA fragments derived from each chromosome, provided by such methods, cannot reveal when a biopsy sample is contaminated with non-embryonic DNA. Negative controls are seldom used during PGT-A and are inadequate as they do not evaluate contamination in the actual tube containing the biopsy specimen. The extent to which failure to detect DNA contamination is a problem for PGT-A is unknown. Study design, size, duration This was a retrospective study involving analysis of 49,287 trophectoderm biopsy samples that underwent PGT-A over a three-year period. Embryos found to have a contaminated biopsy specimen typically underwent a second biopsy. In such cases, results from the two samples were compared to ascertain whether the contaminated sample would have been misdiagnosed if the analysis had been restricted to examining only the relative chromosome copy number, as is the case for most NGS-based PGT-A methods. Participants/materials, setting, methods All trophectoderm biopsies underwent targeted DNA amplification and next generation sequencing using a highly validated PGT-A method that evaluates the relative chromosomal copy number, similar to traditional PGT-A methods, but combines this with analysis of variations in DNA sequence (single nucleotide polymorphisms - SNPs). The genotype of each SNP, and the relative quantity of DNA fragments containing each of the different alleles, allows detection of otherwise invisible states, such as triploidy, haploidy, and contamination. Main results and the role of chance From the 49,287 TE biopsies analysed, contamination with non-embryonic DNA was detected in 218 (0.44%). There was variation in the rates of contamination between the 25 clinics that provided samples, ranging between 0% and 1.5%. Additionally, one clinic had a contamination rate of 7.7%, but the number of biopsies derived from that site was considered too small for reliable evaluation (n = 26). 156 of the embryos with a contaminated biopsy specimen underwent secondary biopsy (71.5%), allowing the relative chromosome copy number result from the contaminated specimen to be compared to that obtained from an uncontaminated sample. The results were split into three categories: 1) no change in interpretation between the first (contaminated) and second biopsy specimens; 2) false positive – the contaminated sample was euploid but would have been wrongly interpreted as triploid and would have been erroneously discarded, potentially impacting the patients chances of achieving a pregnancy; 3) false negative – the contaminated sample was fully aneuploid but would have been incorrectly classified mosaic or euploid and could have been eligible for transfer, potentially leading to implantation failure or abnormal pregnancy. 19% of contaminated samples gave a false negative result, while 24% gave a false positive, appearing to be triploid. Limitations, reasons for caution It is not possible to determine the origin of contaminants with certainty without having DNA from the contamination source for comparison. Additionally, we were unable to conclude whether contamination is more likely to occur in IVF or ICSI cycles as only 3% of samples were fertilized using IVF. Wider implications of the findings Contamination detection during PGT-A is important to prevent misdiagnosis of embryos. Misclassification due to undetected contamination can lead to discard of potentially viable embryos. It can also lead to the transfer aneuploid embryos, wrongly classified as mosaic, which could lead to increased rates of implantation failure, miscarriage and aneuploid pregnancy. Trial registration number Not applicable

P-731 The accuracy of truly non-invasive PGT using spent culture media is insufficient to justify routine clinical use

Abstract Study question Can non-invasive PGT (niPGT), using a protocol that avoids manipulation of the embryo before spent culture medium is collected, accurately determine embryo chromosomal status? Summary answer Accuracy using a truly non-invasive PGT method was low, suggesting this strategy is not appropriate for clinical use and should be restricted to research studies. What is known already PGT requires embryo biopsy to obtain small numbers of cells for genetic testing. The procedure requires skilled personnel and specialist equipment, significantly adding to the cost of PGT while also creating training and logistical bottlenecks that reduce patient access to PGT. Additionally, there have been concerns that biopsy could damage embryos. These considerations have stimulated interest in potential non-invasive PGT strategies. Thus far, niPGT methods have mostly focused on cell-free embryonic DNA found in spent culture media, but studies reporting highest accuracy have tended to involve embryo manipulations likely to promote DNA release and therefore cannot be considered truly non-invasive. Study design, size, duration Samples of spent culture medium (SCM) associated with 128 embryos were collected. Oocytes had been denuded of cumulus cells, fertilised using ICSI and the resulting embryos thoroughly washed on day-4 before transfer to a fresh drop of medium. SCM samples were collected on day-5 or day-6 of culture. Embryos had not previously been cryopreserved and underwent minimal manipulation prior to SCM collection. Results of SCM analysis were compared to those obtained following conventional ‘invasive’ PGT. Participants/materials, setting, methods Media samples were subjected to whole genome amplification using the PG-Seq Rapid Non-Invasive PGT kit. Concentrations of sequencing libraries were measured, and next-generation sequencing (NGS) was undertaken. The resulting NGS data was analysed with PG-Find Software to predict chromosomal status. In parallel, trophectoderm biopsies from the corresponding embryos were analysed using a well-established and highly validated PGT method, based upon amplification of thousands of sites across the genome, followed by NGS. Main results and the role of chance Despite the implementation of rigorous measures to prevent contamination of SCM samples, extraneous female DNA, likely of cumulus cell origin, was frequently observed. Depending on the clinic, 16.7% to 38.1% of media samples associated with male embryos gave a discordant (female) result. This highlights the difficulty of avoiding inadvertent sampling of maternal DNA. There were no instances of male DNA contamination affecting SCM samples from female embryos. We assessed whether clinic of origin, ploidy status of the embryo, and day of SCM collection influenced the quality of the sequencing libraries (ANOVA). Higher concentrations of libraries and superior sequencing quality scores were observed in SCM collected on day-6 compared to day-5 (p = 0.011 and p = 0.002, respectively). Chromosomal assessments obtained from SCM were compared to those from standard, trophectoderm-based PGT in a blinded fashion. 50% of SCM samples gave an identical predicted karyotype to their corresponding trophectoderm biopsy (concordant copy number for all chromosomes). When applying a simple aneuploid/euploid classification, 80% of embryos classified chromosomally abnormal using the validated PGT method were also aneuploid according to analysis of the associated SCM sample, but without necessarily having identical karyotypes, while 62% of embryos classified euploid by PGT received the same designation after SCM analysis. Limitations, reasons for caution SCM samples were designated ‘contaminated’ when embryos of one sex (determined using invasive PGT) were incorrectly classified as the opposite sex using niPGT. However, our study was unable to determine when SCM samples were contaminated with DNA of the same sex as the embryo. Consequently, contamination rates could be higher. Wider implications of the findings Most niPGT methods are not truly non-invasive, as they include embryo manipulations that increase likelihood of cell death and DNA release. Our results show that obtaining reliable niPGT results is difficult without deviating significantly from optimal embryological protocols. Currently, the accuracy of niPGT seems insufficient to justify routine clinical use. Trial registration number Not applicable

Avian sperm-borne RNAs: optimisation of a new isolation protocol

ABSTRACT 1. Sperm-borne RNAs are involved in sperm and embryonic protein translation, the regulation of early development and the epigenetic inheritance of the paternal phenotype. Sperm-borne RNA purification protocols generally include a cell purification stage to discard contamination by somatic cells. In avian species, no protocol is currently available to isolate all the populations composing sperm-borne RNAs. 2. This study evaluated the presence of somatic cells in semen samples of chickens and quails using visual examination after fluorescent nuclei staining. The efficiency of somatic cell lysis buffer (SCLB) on chicken liver cells and its impacts on chicken sperm cell integrity was explored. Three different approaches were tested to isolate RNA: two developed for mammalian sperm cells and a commercial kit for somatic cells. The efficiency and reliability of each approach was determined based on RNA quality and purity. Eventually, the presence of miRNA and mRNA in purified avian sperm-borne RNAs was investigated by RT-(q)PCR. 3. No somatic cells were found in chicken and quail semen. The SCLB totally lysed chicken liver cells but also induced sperm cell necrosis. Consequently, this treatment wasn’t performed on samples prior to RNA isolation. Among the tested RNA purification protocols, the commercial one was the least variable and isolated RNA with the highest purity levels. No DNA contamination was observed. Furthermore, the samples contained miRNA and mRNA already known as present in mammalian sperm cells (gga-miR-100-5p, gga-miR-191-5p, GAPDH and PLCZ1), but mRNAs associated with leucocytes (CD4) and Sertoli cells (SOX4, CLDN11) were not detected. This protocol was successfully applied to quail sperm cells. 4. Altogether, the study reveals that it is unnecessary to pre-treat samples to remove somatic cell contamination before RNA purification and successfully describes an isolation protocol for sperm-borne RNAs, including small non-coding and long coding RNAs, in two distinct avian species highly valuable as biological models.

Peptide analysis of tooth enamel - A sex estimation tool for archaeological, anthropological, or forensic research.

Proteomics has become an attractive method to study human and animal material, biological profile, and origin as an alternative to DNA analysis. It is limited by DNA amplification in ancient samples and its contamination, high cost, and limited preservation of nuclear DNA. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. Proteomics provides a new, seemingly simple, and relatively non-expensive way of sex estimation without the risk of contamination. Proteins can be preserved in hard teeth tissue (enamel) for tens of thousands of years. It uses two sexually distinct forms of the protein amelogenin in tooth enamel detectable by liquid chromatography-mass spectrometry; the protein amelogenin Y isoform is present in enamel dental tissue only in males, while amelogenin isoform X can be found in both sexes. From the point of view of archaeological, anthropological, and forensic research and applications, the reduced destruction of the methods used is essential, as well as the minimum requirements for sample size.

Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing.

Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the rep gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the rep-cap genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the rep/cap plasmid occurs under several events and rcAAVs spawn from diverse clones.

Shotgun sequencing of sonication fluid for the diagnosis of orthopaedic implant-associated infections with Cutibacterium acnes as suspected causative agent.

Orthopaedic implant-associated infections (OIAIs) due to Cutibacterium acnes can be difficult to diagnose. The aim of this pilot study was to determine if metagenomic next-generation sequencing (mNGS) can provide additional information to improve the diagnosis of C. acnes OIAIs. mNGS was performed on sonication fluid (SF) specimens derived from 24 implants. These were divided into three groups, based on culture results: group I, culture-negative (n = 4); group II, culture-positive for C. acnes (n = 10); and group III, culture-positive for other bacteria (n = 10). In group I, sequence reads from C. acnes were detected in only one SF sample, originating from a suspected case of OIAIs, which was SF and tissue culture-negative. In group II, C. acnes sequences were detected in 7/10 samples. In group III, C. acnes sequence reads were found in 5/10 samples, in addition to sequence reads that matched the bacterial species identified by culture. These samples could represent polymicrobial infections that were missed by culture. Taken together, mNGS was able to detect C. acnes DNA in more samples compared to culture and could be used to identify cases of suspected C. acnes OIAIs, in particular regarding possible polymicrobial infections, where the growth of C. acnes might be compromised due to a fast-growing bacterial species. However, since SF specimens are usually low-biomass samples, mNGS is prone to DNA contamination, possibly introduced during DNA extraction or sequencing procedures. Thus, it is advisable to set a sequence read count threshold, taking into account project- and NGS-specific criteria.

ChIP-Based Nuclear DNA Isolation for Genome Sequencing in Pyropia to Remove Cytosol and Bacterial DNA Contamination.

Contamination from cytosolic DNA (plastid and mitochondrion) and epiphytic bacteria is challenging the efficiency and accuracy of genome-wide analysis of nori-producing marine seaweed Pyropia yezoensis. Unlike bacteria and organellar DNA, Pyropia nuclear DNA is closely associated with histone proteins. In this study, we applied Chromatin Immunoprecipitation (ChIP) of histone H3 to isolate nuclear DNA, followed by high-throughput sequencing. More than 99.41% of ChIP-sequencing data were successfully aligned to the reference nuclear genome; this was remarkably higher than those from direct extraction and direct extraction data, in which 40.96% to 42.95% are from plastids. The proportion of data that were mapped to the bacterial database when using ChIP extraction was very low. Additionally, ChIP data can cover up to 89.00% of the nuclear genome, higher than direct extraction data at equal data size and comparable to the latter at equal sequencing depth. The uncovered regions from the three methods are mostly overlapping, suggesting that incomplete sequencing accounts for the missing data, rather than failed chromatin-antibody binding in the ChIP extraction method. This ChIP extraction method can successfully separate nuclear DNA from cytosolic DNA and bacterial DNA, thus overwhelmingly reducing the sequencing cost in a genome resequencing project and providing strictly purified reference data for genome assembly. The method's applicability to other macroalgae makes it a valuable contribution to the algal research community.

An optimized RNA extraction method for diverse leaves of Hawaiian Metrosideros, a hypervariable tree species complex.

The isolation of RNA from trees is challenging due to the interference of polyphenols and polysaccharides with downstream processes. Furthermore, many RNA extraction protocols are time consuming and involve hazardous chemicals. To address these issues, we aimed to develop a safe protocol for high-quality RNA extraction from diverse Metrosideros taxa representing a broad range of leaf toughness, pubescence, and secondary metabolites. We tested popular RNA isolation kits and protocols that were effective on other recalcitrant trees, including a broad range of optimization and purification steps. We optimized a protocol involving two silica-membrane column-based kits that yielded high-quantity RNA with an RNA integrity number >7 and without DNA contamination. All RNA samples were used successfully in a follow-on RNA-Seq experiment. We present an optimized high-throughput RNA extraction protocol that yielded high-quality and high-quantity RNA from three contrasting leaf phenotypes within a hyperdiverse woody species complex.

Benchmarking microbial DNA enrichment protocols from human intestinal biopsies.

Shotgun metagenomic sequencing is a powerful tool for studying bacterial communities in their natural habitats or sites of infection, without the need for cultivation. However, low microbial signals in metagenomic sequencing can be overwhelmed by host DNA contamination, resulting in decreased sensitivity for microbial read detection. Several commercial kits and other methods have been developed to enrich bacterial sequences; however, these assays have not been tested extensively for human intestinal tissues yet. Therefore, the objective of this study was to assess the effectiveness of various wet-lab and software-based approaches for depleting host DNA from microbiome samples. Four different microbiome DNA enrichment methods, namely the NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit, and Zymo HostZERO microbial DNA kit, were evaluated, along with a software-controlled adaptive sampling (AS) approach by Oxford Nanopore Technologies (ONT) providing microbial signal enrichment by aborting unwanted host DNA sequencing. The NEBNext and QIAamp kits proved to be effective in shotgun metagenomic sequencing studies, as they efficiently reduced host DNA contamination, resulting in 24% and 28% bacterial DNA sequences, respectively, compared to <1% in the AllPrep controls. Additional optimization steps using further detergents and bead-beating steps improved the efficacy of less efficient protocols but not of the QIAamp kit. In contrast, ONT AS increased the overall number of bacterial reads resulting in a better bacterial metagenomic assembly with more bacterial contigs with greater completeness compared to non-AS approaches. Additionally, AS also allowed for the recovery of antimicrobial resistance markers and the identification of plasmids, demonstrating the potential utility of AS for targeted sequencing of microbial signals in complex samples with high amounts of host DNA. However, ONT AS resulted in relevant shifts in the observed bacterial abundance, including 2 to 5 times more Escherichia coli reads. Furthermore, a modest enrichment of Bacteroides fragilis and Bacteroides thetaiotaomicron was also observed with AS. Overall, this study provides insight into the efficacy and limitations of various methods for reducing host DNA contamination in human intestinal samples to improve the utility of metagenomic sequencing.

Population genetics of freshwater stingray require investigation to confirm DNA contamination: Reply to Alshari et al. (2023)

Alshari et al. (2023; Endang Species Res 50:311-313) were able to collect 3 Fluvitrygon kittipongi specimens from the Pahang River, which were not available to us at the time of our study (Lim &amp; Then 2022; Endang Species Res 48:43-50). The cytochrome c oxidase I (COI) sequences (or haplotypes) of these 3 stingrays were identical to each other but differed from the haplotypes of our Perak River specimens. The result of 100% similarity between COI sequences of our Perak River rays and the eDNA water samples isolated from Pahang River in our study was suggested by Alshari et al. (2023) as DNA contamination in our study, which is plausible. However, further population genetics studies would be necessary to ascertain that the Perak River haplotype does not occur in stingrays of the Pahang River.

DNA contamination and taxonomic diversity hinder eDNA research on freshwater stingray species: Comment on Lim &amp; Then (2022)

Recently, Lim &amp; Then (2022; Endang Species Res 48:43-50) used an environmental DNA (eDNA) PCR-based method for detecting the presence of an endangered species of freshwater stingray, Fluvitrygon kittipongi (Dasyatidae), in the Pahang River basin, east Peninsular Malaysia. For that, they designed a species-specific pair of primers to amplify a 196 base pair (bp) fragment of the cytochrome c oxidase I (COI) gene. Lim &amp; Then (2022) reported positive detection of this species along an upstream stretch of the Pahang River during the inter-monsoon and the southwest monsoon seasons, with the PCR-amplified eDNA sequences identical to the reference COI sequences of F. kittipongi collected from the Perak River basin, west Peninsular Malaysia. Here, we argue that such positive results are likely the consequence of a DNA contamination because the 196 bp COI fragments of F. kittipongi from the Pahang River and the Perak River differ from each other by 4 nucleotide substitutions. The source of contamination in Lim &amp; Then (2022) could have been the Perak samples the authors handled to develop the primers. We also briefly discuss the potential impact of the presumed co-occurrence of freshwater stingrays of the genus Fluvitrygon in the Pahang River on the specificity of the F. kittipongi-specific primers designed by Lim &amp; Then (2022).

The analyzation of DNA and cross contamination inside the cyanoacrylate (superglue) fuming chamber.

The objectives of this experiment looked at were that the DNA, both skin cells and saliva, have the potential of binding with the superglue as it coats each object being fumed. If DNA has the potential of binding with the superglue, then it is very possible for DNA to be carried in the air by the humidifier, thus contaminating other objects. For the first experiment, two saliva covered microscope slides, one female and one male, were fumed along with 25 other sterile slides. Each sterile slide was swabbed and analyzed for DNA in an electrophoresis gel chamber. The second experiment fumed ten microscope slides presented with fingerprints. Each sterile slide was swabbed and analyzed for DNA in an electrophoresis gel chamber. The results showed that there was minimal cross contamination present on the slides fumed with the saliva based slides. The touch DNA experiment showed that there are potentially identifiable DNA markers present in fingerprints after fuming. The results concluded that it is possible to have DNA cross contamination between items that carry no DNA prior to the fumigation, and potential DNA can be found on fingerprints after fumigation.

Abstract 5930: Single day workflow for high-quality whole genome analysis of thousands of single cells

Abstract DNA sequencing technology provides the capability to tell the story of every cell in a heterogenous sample. ResolveDNA® generates uniform coverage of &amp;gt;95% of a single genome, enabling researchers to generate single-cell genomic data of unparalleled quality. Here, we report an enhancement to the ResolveDNA® workflow that increases the rate of data collection; the ability to reliably generate complete genomic information from up to 1536 cells in parallel from a single experiment. To achieve this, we have coupled a high-throughput automated process to a low volume reaction using the latest ResolveDNA® 2.5-hour chemistry. This establishes the potential for users to go from a single cell, or low-input DNA sample, to ready to sequence libraries in a single day for &amp;gt;3000 samples. As with earlier ResolveDNA® workflows, these reactions generate negligible amounts of contaminating and off target DNA during the reaction. A typical single-cell reaction yields ~ 85 ng of DNA. The single cell’s amplified DNA product is amenable to multiple post amplification library preparation processes. We utilized both BioSkryb ResolveDNA® and Watchmaker Library Preparation kits for Illumina sequencing. We did not identify significant differences in the performance of the two products. Libraries were assessed at a depth of ~ 2million reads and determined to be of high quality, with low rates of chimeric DNA formation and less than 1% mtDNA resulting in the reaction product. A subset of samples was probed at &amp;gt;200 million reads, with &amp;gt;85% of the genome covered at 1x. Consistent with earlier ResolveDNA® workflows, the libraries were characterized with &amp;gt;99% precision and &amp;gt;80% sensitivity. The story told from a single cell’s genome drives discovery and shines a light on the questions surrounding the causes of disease and cellular function. This workflow is designed to empower researchers to probe the genomic landscape of a heterogenous tissue sample. Citation Format: Jay A. West, Jeff G. Blackinton, Kyle Hukari, Joe Dahl. Single day workflow for high-quality whole genome analysis of thousands of single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5930.

External DNA contamination and efficiency of bleach decontamination for arthropod diet analysis

AbstractDNA metabarcoding is increasingly used to analyze the diet of arthropods, including spiders. However, high sensitivity to DNA contamination makes it difficult to apply to organisms obtained from mass‐sampling methods such as pitfall traps. An alternative is to hand‐sample spiders, but it is unclear how effectively this prevents external contamination, especially with new knowledge showing the wide spread of eDNA in the environment. Protocols using bleach to remove external DNA have been tested on several invertebrates, though testing with both mass‐sampling methods and spiders is lacking. Here, we used wolf spiders (Lycosidae) to assess the risk of external DNA contamination from pitfall trapping and hand sampling, and the efficacy of bleach decontamination. We first conducted a contamination experiment where we placed spiders in pitfall traps containing trapping medium and a nonprey insect species to simulate external DNA contamination. We also compared sampling methods by collecting spiders using pitfall traps and hand sampling. Spiders from the contamination experiment and sampling method comparison were either bleached or untreated, then metabarcoded using multiple primer pairs. The contamination experiment resulted in the contamination of almost all spiders from pitfall traps, which was successfully eliminated with bleaching. Interestingly, there was no difference in the number of amplicon sequence variants (ASVs) detected per spider between pitfall trapping and hand sampling but bleaching resulted in significantly fewer ASV detections for both methods. Additionally, bleaching, but not sampling method, affected the taxonomic diet composition for both hand‐sampled and pitfall‐trapped spiders, indicating similar levels of external contamination. Our results are the first to confirm that DNA metabarcoding can be used together with bleaching for spiders sampled from pitfall traps, and that hand sampling does not necessarily exclude external DNA contamination. Thus, diet studies using metabarcoding should address the risk of external contamination with field‐sampled arthropods, regardless of sampling method.

Application of PCR-based approaches for evaluation of cell-free DNA fragmentation in colorectal cancer.

Cell-free DNA (cfDNA) testing is the core of most liquid biopsy assays. In particular, cfDNA fragmentation features could facilitate non-invasive cancer detection due to their interconnection with tumor-specific epigenetic alterations. However, the final cfDNA fragmentation profile in a purified sample is the result of a complex interplay between informative biological and artificial technical factors. In this work, we use ddPCR to study cfDNA lengths in colorectal cancer patients and observe shorter and more variable cfDNA fragments in accessible chromatin loci compared to the densely packed pericentromeric region. We also report a convenient qPCR system suitable for screening cfDNA samples for artificial high molecular weight DNA contamination.

A quick and cost-effective method for DNA-free total RNA isolation using magnetic silica beads

Current RNA purification methods widely use silica-based columns that allow quick isolation of high-quality and good quantities of RNA. However, the major limitations include high cost, the requirement of different kits for small RNA isolation, genomic DNA contamination, and not being flexible. Here, we used the in-house RNA isolation reagent (RIR) for cell lysis, followed by RNA precipitation using isopropanol. RNA isolated using the in-house RIR resulted in a similar quantity and quality compared to the commercial TRIzol. Furthermore, the commercial RNA isolation kits with silica-based columns recommend genomic DNA digestion during or after RNA purification, adding time and cost to RNA purification. Here, we developed an optimized in-house protocol for isolating high-quality RNA free of genomic DNA contamination using magnetic silica beads without needing DNase digestion. Additionally, our method purifies total RNA along with the small RNA fraction, including miRNAs, which usually require a separate kit for extraction. Additionally, the RNA prepared with our method was equally suitable for mRNA and miRNA expression analysis using RT-qPCR. Together, the in-house method of RNA isolation using the magnetic silica beads has exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.

Is there a placental microbiota? A critical review and re-analysis of published placental microbiota datasets

The existence of a placental microbiota is debated. The human placenta has historically been considered sterile and microbial colonization was associated with adverse pregnancy outcomes. Yet, recent DNA sequencing investigations reported a microbiota in typical human term placentas. However, this detected microbiota could represent background DNA or delivery-associated contamination. Using fifteen publicly available 16S rRNA gene datasets, existing data were uniformly re-analyzed with DADA2 to maximize comparability. While Amplicon Sequence Variants (ASVs) identified as Lactobacillus, a typical vaginal bacterium, were highly abundant and prevalent across studies, this prevalence disappeared after applying likely DNA contaminant removal to placentas from term cesarean deliveries. A six-study sub-analysis targeting the 16S rRNA gene V4 hypervariable region demonstrated that bacterial profiles of placental samples and technical controls share principal bacterial ASVs and that placental samples clustered primarily by study origin and mode of delivery. Contemporary DNA-based evidence does not support the existence of a placental microbiota.ImportanceEarly-gestational microbial influences on human development are unclear. By applying DNA sequencing technologies to placental tissue, bacterial DNA signals were observed, leading some to conclude that a live bacterial placental microbiome exists in typical term pregnancy. However, the low-biomass nature of the proposed microbiome and high sensitivity of current DNA sequencing technologies indicate that the signal may alternatively derive from environmental or delivery-associated bacterial DNA contamination. Here we address these alternatives with a re-analysis of 16S rRNA gene sequencing data from 15 publicly available placental datasets. After identical DADA2 pipeline processing of the raw data, subanalyses were performed to control for mode of delivery and environmental DNA contamination. Both environment and mode of delivery profoundly influenced the bacterial DNA signal from term-delivered placentas. Aside from these contamination-associated signals, consistency was lacking across studies. Thus, placentas delivered at term are unlikely to be the original source of observed bacterial DNA signals.