Functionalized Ferroferric Oxide Nanomagnetic Beads for Extraction of Nucleic Acid and Its Application in Early Screening of Colorectal Cancer

Early screening is an important way to reduce the incidence and mortality of colorectal cancer (CRC). Fecal DNA testing stands out among many screening methods due to its high sensitivity. However, at this stage, researchers have not found a high-efficiency method for fecal DNA extraction. To this end, this work carried out a new round of exploration. Here, this experiment synthesized a kind of nanomagnetic beads (NH2-SiO2@Fe3O4) with good stability for nucleic acid extraction. A comparative study with the centrifugal adsorption column method revealed the significant advantages of the magnetic bead method in extracting fecal DNA. The DNA extracted by the magnetic bead method is of high purity, can also achieve high-throughput tests, and is more suitable for polymerase chain reaction detection, greatly simplifying the stool DNA detection process and providing a basis for the widespread promotion of early screening.

Identification of sample mix-ups and mixtures in microbiome data in Diversity Outbred mice

In a Diversity Outbred mouse project with genotype data on 500 mice, including 297 with microbiome data, we identified three sets of sample mix-ups (two pairs and one trio) as well as at least 15 microbiome samples that appear to be mixtures of pairs of mice. The microbiome data consisted of shotgun sequencing reads from fecal DNA, used to characterize the gut microbial communities present in these mice. These sequence reads included sufficient reads derived from the host mouse to identify the individual. A number of microbiome samples appeared to contain a mixture of DNA from two mice. We describe a method for identifying sample mix-ups in such microbiome data, as well as a method for evaluating sample mixtures in this context.

Determination of Butyrate Synthesis Capacity in Gut Microbiota: Quantification of but Gene Abundance by qPCR in Fecal Samples

Butyrate is formed in the gut during bacterial fermentation of dietary fiber and is attributed numerous beneficial effects on the host metabolism. We aimed to develop a method for the assessment of functional capacity of gut microbiota butyrate synthesis based on the qPCR quantification of bacterial gene coding butyryl-CoA:acetate CoA-transferase, the key enzyme of butyrate synthesis. In silico, we identified bacteria possessing but gene among human gut microbiota by searching but coding sequences in available databases. We designed and validated six sets of degenerate primers covering all selected bacteria, based on their phylogenetic nearness and sequence similarity, and developed a method for gene abundance normalization in human fecal DNA. We determined but gene abundance in fecal DNA of subjects with opposing dietary patterns and metabolic phenotypes—lean vegans (VG) and healthy obese omnivores (OB) with known fecal microbiota and metabolome composition. We found higher but gene copy number in VG compared with OB, in line with higher fecal butyrate content in VG group. We further found a positive correlation between the relative abundance of target bacterial genera identified by next-generation sequencing and groups of but genecontaining bacteria determined by specific primers. In conclusion, this approach represents a simple and feasible tool for estimation of microbial functional capacity.

The Effect of Common Viral Inactivation Techniques on 16S rRNA Amplicon-Based Analysis of the Gut Microbiota

Research investigating the gut microbiome (GM) during a viral infection may necessitate inactivation of the fecal viral load. Here, we assess how common viral inactivation techniques affect 16S rRNA-based analysis of the gut microbiome. Five common viral inactivation methods were applied to cross-matched fecal samples from sixteen female CD-1 mice of the same GM background prior to fecal DNA extraction. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA. Treatment-dependent effects on DNA yield, genus-level taxonomic abundance, and alpha and beta diversity metrics were assessed. A sodium dodecyl sulfate (SDS)-based inactivation method and Holder pasteurization had no effect on measures of microbial richness, while two Buffer AVL-based inactivation methods resulted in a decrease in detected richness. SDS inactivation, Holder pasteurization, and the AVL-based inactivation methods had no effect on measures of alpha diversity within samples or beta diversity between samples. Fecal DNA extracted with TRIzol-treated samples failed to amplify and sequence, making it unsuitable for microbiome analysis. These results provide guidance in the 16S rRNA microbiome analysis of fecal samples requiring viral inactivation.