2.6 Bioinformatics and statistical analysis
The macronutrients and micronutrients intake from the BKFS were calculated by the NutritionQuest. Based on the recommended daily nutritional intake from 2015-2020 dietary guideline, the child’s nutritional intake was categorized into three levels: inadequate level (less than 90% of the recommended dietary allowance [RDA]); adequate level (within the range of 90%-110% of the RDA); and excessive level (more than 110% of the RDA).
Quantitative Insight Into Microbial Ecology 2 (QIIME 2) was used to analyze the taxonomic composition and diversity of the gut microbiome33,34. QIIME 2 default parameters were used for sequencing data, and sequence quality was filtered with DADA235 to infer exact sequence variants (ASVs). By removing the length of primers, the raw sequences were trimmed at 0 and 0 base pairs, and then truncated at 250 and 215 base pairs based on the Phred Quality score >30. Taxonomies were assigned by a Naive Bayes classifier trained on the Greengenes database with sequences adapted to the 16S rRNA V4 gene region. The α-diversity (richness and evenness of the gut microbiome within samples) was calculated using four different parameters: Shannon’s index, Chao1, Faith’s phylogenetic diversity (Faith’s_PD), and Pielou’s evenness (Pielou’s_e). Mann-Whitney U tests were used to compare dietary status between children with cancer and healthy controls. Spearman’s correlation was used to explore correlations between the relative abundance of gut microbiome taxa and the alpha diversity index with diet. Pairwise permutational multivariate analysis of variance (PermANOVA) 36 was used to test taxa dissimilarities between nutritional intake levels. The analysis of composition of microbiomes (ANCOM) 37 was used to analyze associations between diet and the abundance of the gut microbiome.