The standardisation of the approach to metagenomic human gut analysis: from sample collection to microbiome profiling.

In recent years, the number of metagenomic studies increased significantly. Wide range of factors, including the tremendous community complexity and variability, is contributing to the challenge in reliable microbiome community profiling. Many approaches have been proposed to overcome these problems making hardly possible to compare results of different studies. The significant differences between procedures used in metagenomic research are reflected in a variation of the obtained results. This calls for the need for standardisation of the procedure, to reduce the confounding factors originating from DNA isolation, sequencing and bioinformatics analyses in order to ensure that the differences in microbiome composition are of a true biological origin. Although the best practices for metagenomics studies have been the topic of several publications and the main aim of the International Human Microbiome Standard (IHMS) project, standardisation of the procedure for generating and analysing metagenomic data is still far from being achieved. To highlight the difficulties in the standardisation of metagenomics methods, we thoroughly examined each step of the analysis of the human gut microbiome. We tested the DNA isolation procedure, preparation of NGS libraries for next-generation sequencing, and bioinformatics analysis, aimed at identifying microbial taxa. We showed that the homogenisation time is the leading factor impacting sample diversity, with the recommendation for a shorter homogenisation time (10 min). Ten minutes of homogenisation allows for better reflection of the bacteria gram-positive/gram-negative ratio, and the obtained results are the least heterogenous in terms of beta-diversity of samples microbial composition. Besides increasing the homogenisation time, we observed further potential impact of the library preparation kit on the gut microbiome profiling. Moreover, our analysis revealed that the choice of the library preparation kit influences the reproducibility of the results, which is an important factor that has to be taken into account in every experiment. In this study, a tagmentation-based kit allowed for obtaining the most reproducible results. We also considered the choice of the computational tool for determining the composition of intestinal microbiota, with Kraken2/Bracken pipeline outperforming MetaPhlAn2 in our in silico experiments. The design of an experiment and a detailed establishment of an experimental protocol may have a serious impact on determining the taxonomic profile of the intestinal microbiome community. Results of our experiment can be helpful for a wide range of studies that aim to better understand the role of the gut microbiome, as well as for clinical purposes.

Precision of scoring radiation-induced chromosomal aberrations and micronuclei by unexperienced scorers.

Purpose: Dose assessment plays an important role in case of radiological accidents and can be performed by scoring structural changes of chromosome morphology induced in cells by ionizing radiation. The results of such a test are biased by scorer experience, therefore, simple to learn assays are recommended to be used when fast analysis of a large amount of data is needed. The aim of this study was to compare the performance of two radiobiological assays - chromosomal aberrations and micronuclei - by unexperienced scorers with the reference values generated by an expert. Materials and methods: Each participant of an EU-funded two-week radiobiology course was asked to score Chinese hamster ovary cells exposed to gamma radiation up to 4 Gy. The congruence of students' and expert's scores at each dose and the coherence of the dose-response curve parameters between the students were investigated. Results: Micronucleus test tended to be faster and easier to learn than scoring chromosomal aberrations. However, both assays carried out by inexperienced students showed reasonable dose-response curves. Conclusions: In the case of a large radiological accident involving many casualties, the unexperienced scorers would support the process of biodosimetric triage by cytogenetic biological dosimetry.