A metagenomic DNA sequencing assay that is robust against environmental DNA contamination

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied Coffee-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of inflammatory bowel disease in blood.

P–171 Automated oocyte and zygote denudation using a novel microfluidic device supervised by a computer vision algorithm

Study question Is it possible to remove cumulus cells using a 16-well microfluidic device with automated flows to facilitate vitrification, ICSI, NI-PGT or non-invasive metabolomics analysis? Summary answer The designed automated system and protocol efficiently denude 16 samples simultaneously with a x10 lower shear stress than the manual process and without human intervention. What is known already Most processes involved in IVF such as insemination, washing, denudation, embryo culture and selection are still manually performed, labor-intensive and require highly skilled professionals. This leads to a significant variability in the clinical outcomes achieved by different embryologists and labs. The automation of these processes is a promising approach to reduce costs and improve the accessibility to assisted reproductive therapies. Although a simple procedure, standardization of cumulus oocyte complex (COCs) and zygotes denudation is key to facilitate ICSI, vitrification and to avoid DNA contamination for NI-embryo testing (PGT or metabolomics), while avoiding damage to the oocyte by excessive shear stress. Study design, size, duration A total of 160 cow COCs were used due to their size similarity with human COCs. Half were denuded 16–20 hours post-insemination and half pre-insemination for 5–10 minutes. COCs were classified as partially denuded if fertilization assessment, ICSI or vitrification was possible, and completely denuded if no cumulus cells remained. COCs controls were manually denuded (Stripper® capillary 145μm ID) to compare shear stress between procedures. This study was conducted during 2020 – 2021. Participants/materials, setting, methods We developed a customized microfluidic biochip that exerts a particular fluid motion while avoiding egg entrapment within microfluidic channels. The denudation efficacy was established by subjectively scoring images of bovine oocytes after generating a continuous “Push & Pull” fluid motion inside the biochip wells. A Computer Vision model was developed in parallel in order to optically assess denudation completion. The model used was a Pytorch implementation of Faster-RCNN with ImageNet pretrained weights Main results and the role of chance 96 bovine COCs were microfuidically handled post insemination achieving complete (56/96) or partial (40/96) removal of the cumulus cells on day 1, while for day 3 double denudation group, 89/96 (92.7%) were completely denuded while the rest remained partially denuded. In comparison, 80/80 (100%) of manually denuded cow COCs, achieved complete denudation (50% post-insemination group and 50% pre-insemination group). In addition, 48/64 (75%) cow COCs treated pre-insemination were partially denuded, enough to carry out ICSI after 5–10 min of treatment. The results here obtained indicate that media needs to flow through the device at a rate that can generate enough shear to strip off the cumulus-corona cells while avoiding emptying of the reservoirs containing the fertilization or culture medium. The shear stress of our design was calculated to be smaller than 4.4 Pa, about ten times lower than the one applied by the manual process (∼44Pa). The deep learning algorithm was tested on 20 unseen human oocytes on day 1, with 10 true positives 9 true negatives, and 1 false negative (95% accuracy). Limitations, reasons for caution The success of the denudation procedure was dependent on the design of the biochip wells and the microfluidic protocol used. The accuracy of our findings is still limited because of the difficulty in manufacturing prototype biochips. Wider implications of the findings: Complete denudation is key to avoid DNA contamination for NI-PGT or metabolomics analysis, while avoiding damage to the oocyte by excessive shear stress. Our device, which has the potential of scaling up and treat each oocyte individually, can improve automation and increase efficiency of current ART procedures Trial registration number NA

The Effect of Genomic DNA Contamination on the Detection of Circulating Long Non-Coding RNAs: The Paradigm of MALAT1

The presence of contaminating gDNA in RNA preparations is a frequent cause of false positives in RT-PCR-based analysis. However, in some cases, this cannot be avoided, especially when there are no exons–intron junctions in the lncRNA sequences. Due to the lack of exons in few of long noncoding RNAs (lncRNAs) and the lack of DNAse treatment step in most studies reported so far, serious questions are raised about the specificity of lncRNA detection and the potential of reporting false-positive results. We hypothesized that minute amounts of gDNA usually co-extracted with RNA could give false-positive signals since primers would specifically bind to gDNA due to the lack of junction. In the current study, we evaluated the effect of gDNA and other forms of DNA like extrachromosomal circular DNAs (eccDNAs) contamination and the importance of including a DNAse treatment step on lncRNAsexpression.As a model, we have chosen as one of the most widely studied lncRNAs in cancer namely MALAT1, which lacks exons. When we tested this hypothesis in plasma and primary tissue samples from NSCLC patients, our findings clearly indicated that results on MALAT1 expression are highly affected by the presence of DNA contamination and that the DNAse treatment step is absolutely necessary to avoid false positive results.

Managing Contamination and Diverse Bacterial Loads in 16S rRNA Deep Sequencing of Clinical Samples: Implications of the Law of Small Numbers

There has been a gradual increase in 16S deep sequencing studies on infectious disease materials. Management of bacterial DNA contamination is a major challenge in such diagnostics, particularly in low biomass samples.

Characterization of maize root microbiome in two different soils by minimizing plant DNA contamination in metabarcoding analysis

A micropore-filtration method was used to reduce the proportion of plant DNA in microbial DNA samples isolated from roots prior to sequencing. We tested the impact of this pre-sequencing filtration methodology and used it to characterize the root microbiome of maize grown on two soils with different fertility levels. The micropore filtration reduced plant DNA contamination and unveiled potential in the N-poor soil for N fixation in roots and phosphate uptake by roots in the phosphate-poor soil. Our methodology and findings allude to the potential capability of plants to initiate plant-microbe interactions under sub-optimal soil fertility.

Sensitive detection of DNA contamination in tumor samples via microhaplotypes

BackgroundLow levels of sample contamination can have disastrous effects on the accurate identification of somatic variation in tumor samples. Detection of sample contamination in DNA is generally based on observation of low frequency variants that suggest more than a single source of DNA is present. This strategy works with standard DNA samples but is especially problematic in solid tumor FFPE samples because there can be huge variations in allele frequency (AF) due to massive copy number changes arising from large gains and losses across the genome. The tremendously variable allele frequencies make detection of contamination challenging. A method not based on individual AF is needed for accurate determination of whether a sample is contaminated and to what degree.MethodsWe used microhaplotypes to determine whether sample contamination is present. Microhaplotypes are sets of variants on the same sequencing read that can be unambiguously phased. Instead of measuring AF, the number and frequency of microhaplotypes is determined. Contamination detection becomes based on fundamental genomic properties, linkage disequilibrium (LD) and the diploid nature of human DNA, rather than variant frequencies. We optimized microhaplotype content based on 164 single nucleotide variant sets located in genes already sequenced within a cancer panel. Thus, contamination detection uses existing sequence data and does not require sequencing of any extraneous regions. The content is chosen based on LD data from the 1000 Genomes Project to be ancestry agnostic, providing the same sensitivity for contamination detection with samples from individuals of African, East Asian, and European ancestry.ResultsDetection of contamination at 1% and below is possible using this design. The methods described here can also be extended to other DNA mixtures such as forensic and non-invasive prenatal testing samples where DNA mixes of 1% or less can be similarly detected.ConclusionsThe microhaplotype method allows sensitive detection of DNA contamination in FFPE tumor samples. These methods provide a foundation for examining DNA mixtures in a variety of contexts. With the appropriate panels and high sequencing depth, low levels of secondary DNA can be detected and this can be valuable in a variety of applications.

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

ABSTRACTCurrent RNA purification methods widely use silica-based columns that allow quick isolation of high quality and right 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 for cell lysis, followed by precipitation of RNA using isopropanol resulted in a similar quantity and quality of RNA compared to the commercial TRIzol. 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 the need for 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 a magnetic bead exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.

When DNA gets in the way in RNA-seq experiments, a sequel

Using a newly developed method dubbed SILVER-Seq—enabling extracellular RNA sequencing (exRNA-seq) directly from a small volume of human serum or plasma— Yan et al. recently reported in Current Biology a potential exRNA biomarker for the early diagnosis of Alzheimer’s disease [1]. After the publication of the initial paper describing the SILVER-Seq method [2], we reported our concern regarding potential DNA contamination in their datasets [3]. Although the authors replied they were able to successfully treat RNA samples with DNase to avoid such contamination, they did not address our observations of the majority of reads without evidence of being derived from RNA, nor documented verified absence of DNA after DNase treatment [4]. To assess whether the newly data generated may suffer from DNA contamination, we downloaded the publicly available sequencing data and evaluated two quality control metrics (i.e., fraction of exonic and splice reads), which were not reported in the paper. We found that both quality metrics were much lower than expected for RNA-seq data (6.28% exonic and 0.478% splice reads), in line with our previous findings on the first SILVER-Seq paper. These observations suggest the data and results presented by Yan et al. are affected by DNA contamination, an issue that may be inherent to the SILVER-Seq technology.