Total microbial DNA were extracted using the QIAamp PowerFecal Pro DNA Kit (Cat#51804, QIAGEN). DNA concentration was measured. 1 μg DNA per sample was used as input. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit (Cat# E7370L, NEB). DNA samples were fragmented by sonication to 350 bp, which were end-polished, A-tailed, and ligated. PCR products were purified. The clustering of the index-coded samples was performed on a cBot Cluster Generation System, and then sequenced on an Illumina Novaseq 6000 platform by Novogene (Novogene Tianjin, China).
QC process including trimming of low-quality bases, masking of human DNA contamination, and removal of duplicated reads were performed by using kneaddata (version v0.6.1). Human DNA contamination was identified by aligning all raw reads to the human reference genome (hg19) using bowtie2 (version 2.3.5.1). Taxonomic annotation of metagenome and the abundance quantification were performed by MetaPhlAn (version 2.0). Relative abundance of each clade was calculated at six levels (L2: phylum, L3: class, L4: order, L5: family, L6: genus, L7: species). Functional annotations were performed by using the data files from the HMP Unified Metabolic Analysis Network 3.0 (HUMAnN 3.0)74 (link). The clean paired-end sequencing data were merged into a single fastq file. The HUMAnN 3.0 toolkit was run by using the “humann–input myseq*.fq–output humann3/–threads 32–memory-use maximum -r -v” command, which calls Bowtie275 (link) to compare nucleic acid sequence and calls DIAMOND76 (link) to compare protein sequences to complete gene and protein function annotation to obtain KEGG pathway annotation. Differences in bacterial abundance and functional pathway were analyzed using MaAslin277 (link). Richness indices were calculated using the R Community Ecology Package vegan. Weighted Unifrac distance was calculated using Metaphlan3 R script “Unifrac_distance.r” and root-tree file “mpa_v30_CHOCOPhlAn_201901_species_tree.nwk”. The PCoA results were calculated and visualized using R build-in functions and the plot3D R package. The ANOSIM test was used to calculate the significance of dissimilarity using the R Community Ecology Package vegan. Pearson correlation and P values were evaluated using the rcorr function in the Hmisc R package.
QC process including trimming of low-quality bases, masking of human DNA contamination, and removal of duplicated reads were performed by using kneaddata (version v0.6.1). Human DNA contamination was identified by aligning all raw reads to the human reference genome (hg19) using bowtie2 (version 2.3.5.1). Taxonomic annotation of metagenome and the abundance quantification were performed by MetaPhlAn (version 2.0). Relative abundance of each clade was calculated at six levels (L2: phylum, L3: class, L4: order, L5: family, L6: genus, L7: species). Functional annotations were performed by using the data files from the HMP Unified Metabolic Analysis Network 3.0 (HUMAnN 3.0)74 (link). The clean paired-end sequencing data were merged into a single fastq file. The HUMAnN 3.0 toolkit was run by using the “humann–input myseq*.fq–output humann3/–threads 32–memory-use maximum -r -v” command, which calls Bowtie275 (link) to compare nucleic acid sequence and calls DIAMOND76 (link) to compare protein sequences to complete gene and protein function annotation to obtain KEGG pathway annotation. Differences in bacterial abundance and functional pathway were analyzed using MaAslin277 (link). Richness indices were calculated using the R Community Ecology Package vegan. Weighted Unifrac distance was calculated using Metaphlan3 R script “Unifrac_distance.r” and root-tree file “mpa_v30_CHOCOPhlAn_201901_species_tree.nwk”. The PCoA results were calculated and visualized using R build-in functions and the plot3D R package. The ANOSIM test was used to calculate the significance of dissimilarity using the R Community Ecology Package vegan. Pearson correlation and P values were evaluated using the rcorr function in the Hmisc R package.
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