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The Infinium MethylationEPIC BeadChip (EPIC) is a reliable method for measuring the DNA methylation of more than 850,000 CpG positions. In clinical and forensic settings, it is critical to be able to work with low DNA amounts without risking reduced reproducibility. We evaluated the EPIC for a range of DNA amounts using two-fold serial dilutions investigated on two different days. While the β-value distributions were generally unaffected by decreasing DNA amounts, the median squared Pearson's correlation coefficient ( R 2 ) of between-days β-value comparisons decreased from 0.994 (500 ng DNA) to 0.957 (16 ng DNA). The median standard deviation of the β-values was 0.005 and up to 0.017 (median of medians: 0.014) for β-values around 0.6-0.7. With decreasing amounts of DNA from 500 ng to 16 ng, the percentage of probes with standard deviations ≤ 0.1 decreased from 99.9% to 99.4%. This study showed that high reproducibility results are obtained with DNA amounts in the range 125-500 ng DNA, while DNA amounts equal to 63 ng or below gave less reproducible results.

Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards with buccal swabs and compared the results with those obtained with DNA extracted using the EZ1 DNA Investigator Kit. Concordant profiles were obtained for all samples. Our protocol includes simple punch, wash, and PCR steps, reducing cost and hands-on time in the laboratory. Furthermore, it facilitates automation of DNA sequencing.

Archived formalin-fixed and paraffin-embedded (FFPE) heart tissue from autopsied individuals represents an important resource for investigating the DNA methylation of heart tissue of deceased individuals. The DNA quality of FFPE tissue from autopsies may be decreased, affecting the DNA methylation measurements. Therefore, inexpensive screening methods for estimating DNA quality are valuable. We investigated the correlation between the DNA quality of archived FFPE heart tissue examined with the Illumina Infinium HD FFPE QC assay (Infinium QC) and Thermo Fisher's Quantifiler Trio DNA Quantification kit (QuantifilerTrio), respectively, and the amount of usable DNA methylation data as measured by the probe detection rate (probe DR) obtained with the Illumina Infinium MethylationEPIC array. We observed a high correlation (r2 = 0.75; p < 10−11) between the QuantifilerTrio degradation index, DI, and the amount of usable DNA methylation data analysed with SeSAMe, whereas a much weaker correlation was observed between the Infinium QC and SeSAMe probe DR (r2 = 0.17; p < 0.05). Based on the results, QuantifilerTrio DI seems to predict the proportion of usable DNA methylation data analysed with the Illumina Infinium MethylationEPIC array and SeSAMe by a linear model: SeSAMe probe DR = 0.80–log10(DI) × 0.25. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) and invertebrate‐derived DNA (iDNA) are used to survey biodiversity non‐invasively to mitigate difficulties in obtaining wildlife samples, particularly in remote areas or for rare species. Recently, eDNA/iDNA were used to monitor known wildlife pathogens; however, most wildlife pathogens are unknown and often evolutionarily divergent.To detect and identify known and novel mammalian viruses from eDNA/iDNA, we used a curated set of RNA oligonucleotides as viral baits in a hybridization capture system coupled with high‐throughput sequencing.We detected multiple known and novel mammalian RNA and DNA viruses from multiple viral families from both waterhole eDNA and leech‐derived iDNA. Congruence was found between detected hosts and viruses identified in leeches and waterholes.Our results demonstrate that eDNA/iDNA samples represent an effective non‐invasive resource for studying wildlife viral diversity and for detecting novel potentially zoonotic viruses prior to their emergence. [ABSTRACT FROM AUTHOR]

Massively parallel sequencing (MPS) has revolutionised clinical genetics and research within human genetics by enabling the detection of variants in multiple genes in several samples at the same time. Today, multiple approaches for MPS of DNA are available, including targeted gene sequencing (TGS) panels, whole exome sequencing (WES), and whole genome sequencing (WGS). As MPS is becoming an integrated part of the work in genetic laboratories, it is important to investigate the variant detection performance of the various MPS methods. We compared the results of single nucleotide variant (SNV) detection of three MPS methods: WGS, WES, and HaloPlex target enrichment sequencing (HES) using matched DNA of 10 individuals. The detection performance was investigated in 100 genes associated with cardiomyopathies and channelopathies. The results showed that WGS overall performed better than those of WES and HES. WGS had a more uniform and widespread coverage of the investigated regions compared to WES and HES, which both had a right-skewed coverage distribution and difficulties in covering regions and genes with high GC-content. WGS and WES showed roughly the same high sensitivities for detection of SNVs, whereas HES showed a lower sensitivity due to a higher number of false negative results. [ABSTRACT FROM AUTHOR]

Sodium hypochlorite is an efficient reagent for removal of unwanted DNA from laboratory surfaces. Here, we tested two different chlorine wipes and compared their performance to a 0.9–1.8% hypochlorite solution. WipeClean Chlorine Disinfection wipes contain > 0.1 g sodium hypochlorite/kg, whereas WetWipe Chlorine Desinfection wipes contain > 1000 ppm active chlorine. Clean surfaces were contaminated with 10 µL 0.5 ng/µL of massively parallel sequencing libraries. The DNA was dried and left for 45 min before any treatment. The surfaces were cleaned using either 1) a 0.9–1.8% hypochlorite solution and clean wipes, 2) a WipeClean wipe, 3) a WetWipe, or 4) the surface was not cleaned. All experiments were repeated three times. Subsequently, the surfaces were swabbed using cotton swabs. DNA was extracted from the swabs and the DNA concentrations were determined in quadruplicates by real-time PCR. This protocol was repeated after the soft plastic wrapping around the wipes were left open or closed for several weeks. The results showed that the WipeClean wipes efficiently removed DNA for up to four weeks after the box with the wipes were opened, whereas the WetWipe wipes dried faster and gradually lost their cleaning effect. [ABSTRACT FROM AUTHOR]

In this study, we tested various approaches to remove DNA from hard laboratory surfaces. We contaminated clean surfaces with four different concentrations of massively parallel sequencing libraries. The DNA was dried and left for 45 min and for 24 h, respectively, before any treatment. The surfaces were cleaned with six different methods water, 96% ethanol, water followed by 96% ethanol, 3–6% hypochlorite solution, 0.9–1.8% hypochlorite solution, and no treatment. Subsequently, the surfaces were swabbed using cotton. DNA was extracted from the swabs and the DNA concentrations were determined by real-time PCR. The results showed that leaving the DNA traces for 24 h decreased the amount of amplifiable DNA approximately four times. The concentrations of amplifiable DNA were approximately five times lower after cleaning with 96% ethanol. Cleaning with water and water followed by 96% ethanol reduced the amount of amplifiable DNA 100–200 times, whereas cleaning with hypochlorite removed all traces of amplifiable DNA. This indicated that the ‘mechanical’ cleaning was efficient but cleaning with hypochlorite was superior. In conclusion, it is recommended to clean laboratory surfaces with 0.9–1.8% hypochlorite in order to eliminate laboratory contamination and simultaneously minimise any poisonous gases. [ABSTRACT FROM AUTHOR]