Suspected sources of microplastics and nanoplastics: Contamination from experimental reagents and solvents.

There is an increasing concern about the potential effects of microplastics (MPs) and nanoplastics on human health and other organisms. For the separation and detection of MPs, there are various approaches, and the distinct procedures led to different results. However, the presence of MPs in the reagents was not addressed, which could cause false and/or inaccurate results during MPs detection. In this study, the chemical reagents commonly used for the separation and detection of MPs were selected to ascertain whether these reagents introduce MPs. It was shown that a large number of MPs were detected in the reagent and solvent samples. The largest number of MPs (>1 µm) was detected in the KOH reagent, with the abundance of 3070 items/g. The order of MPs abundance in the selected reagents was: KOH > NaCl > CaCl2 > SDS > NaI > H2O2. The types of MPs were the same as the body and stopper of the reagent packaging bottles. MPs size detected in reagent bottles was primarily smaller than 10 µm. The abundance of MPs in the reagents were independent of their purity, however, there was a certain difference in MPs abundance in reagents from different manufacturers. Furthermore, the presence of nanoplastics (< 1 µm) was verified in the reagents through Py-GCMS, with the abundance (39.47-43.01 mg/kg) higher than that of MPs. The obtained results in this study raised specific requirements and cautions for MPs and nanoplastics related research in terms of quality control. Also, this work can facilitate a more accurate assessment of MPs concentrations in the environment.

Metagenomic Identification of Viral Sequences in Laboratory Reagents.

Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterise the complete 'infectome' (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing ("metatranscriptomics") we documented the presence of contaminant viral sequences in multiple 'blank' negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank control libraries, as well as contaminating sequences from the Totiviridae, Tombusviridae and Lentiviridae families of RNA virus. These data suggest that viral contamination of common laboratory reagents is likely commonplace and can comprise a wide variety of viruses.

Prerequisites for Control of Contamination in Fungal Diagnosis.

Nucleic acid amplification methods facilitate rapid and sensitive detection of clinically relevant fungal pathogens, and can be employed using a variety of patient specimens. However, contamination from various exogenous sources constitutes a serious threat to the validity of amplification-based fungal assays. In this chapter, common origins of fungal contaminants that compromise molecular fungal testing are described, and measures for preventing contamination are proposed. Detailed guidelines for sample handling, reagent selection, contamination screening, and decontamination procedures are provided.

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics.

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100-1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.

Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.

BACKGROUND: Recent studies have suggested that bacteria associated with the placenta-a "placental microbiome"-may be important in reproductive health and disease. However, a challenge in working with specimens with low bacterial biomass, such as placental samples, is that some or all of the bacterial DNA may derive from contamination in dust or commercial reagents. To investigate this, we compared placental samples from healthy deliveries to a matched set of contamination controls, as well as to oral and vaginal samples from the same women. RESULTS: We quantified total 16S rRNA gene copies using quantitative PCR and found that placental samples and negative controls contained low and indistinguishable copy numbers. Oral and vaginal swab samples, in contrast, showed higher copy numbers. We carried out 16S rRNA gene sequencing and community analysis and found no separation between communities from placental samples and contamination controls, though oral and vaginal samples showed characteristic, distinctive composition. Two different DNA purification methods were compared with similar conclusions, though the composition of the contamination background differed. Authentically present microbiota should yield mostly similar results regardless of the purification method used-this was seen for oral samples, but no placental bacterial lineages were (1) shared between extraction methods, (2) present at >1 % of the total, and (3) present at greater abundance in placental samples than contamination controls. CONCLUSIONS: We conclude that for this sample set, using the methods described, we could not distinguish between placental samples and contamination introduced during DNA purification.