Comparing alignment and de-novo approaches for gut microbiota metagenomic data analysis reveals differences in taxonomic resolution and novel functional insights.

Microbiome annotation based on metagenomic data is primarily conducted using two global approaches: alignment-based approach (AL) and de novo approach (DN). This study aimed to evaluate the limitations of each approach, explore correlations between their results, and assess the equivalence of findings derived from different methodologies when analyzing the same dataset. Shotgun metagenomic sequencing data from 346 fecal samples, collected longitudinally within individuals in Arkhangelsk, Northwestern Russia, were analyzed. Each of the 173 participants provided two samples, one during 2015-2017 and another in 2022. The alterations in the microbiota associated with BMI served as a critical variable for facilitating the comparisons between the AL and DN. Exploratory analyses, including PERMANOVA, alpha diversity and beta diversity, revealed no significant differences between the two approaches. However, differential abundance analysis based on the AL yielded more statistically significant results, with the DN producing only a subset of these findings. An analysis of the metagenome-assembled genomes (MAGs) of bacteria that were differentially abundant revealed that one group of MAGs of Alistipes onderdonkii encodes the enzyme 2,5-diketo-D-gluconate reductase A. Using AL and DN together offers complementary functional insights, as the methods produce partially overlapping results. The novel enzyme finding suggests a potential role in metabolic pathways and underscores the value of integrative metagenomic analysis.