Sequencing data
After sequencing, the raw reads for all samples ranged from 54,075 to 99,209. The Q20 was more than 98% and Q30 was more than 97% for all samples. After quality control, the effective tags ranged from 80.72 to 95.17%.
OTU annotation
The top 10 maximum abundance of species in each group in genus level is shown in Fig. 1a. Bacteroides had the highest relative abundances in V, AF, CP and NP groups, and Faecalibacterium had the highest relative abundance in C group. In order to compare the species content in different groups of samples, the top 35 genera were selected for clustering analysis based on the species annotation and abundance information of samples at genus level. As shown in Fig. 1b, in the C group, Faecalibacterium, Bacteroides and Subdoligranulum were the dominant genera; in the V group, Bacteroides, Faecalibacterium and unidentified Enterobacteriaceae were the primary genera; in the AF group, Bacteroides, unidentified Enterobacteriaceae and Faecalibacterium were the dominant genera; in the CP group, Bacteroides, Faecalibacterium and Blautia were of higher abundance; meanwhile, in the NP group, the most abundant sequences were related to Bacteroides, Blautia and Faecalibacterium.
Alpha diversity analysis
The alpha diversity indexes, including observed species, Chao1, Shannon, Simpson, ACE, Goods_coverage and PD_whole_tree, for five groups are shown in Table 1. Generally, the community richness, community diversity and phylogenetic diversity in AF and V groups were higher than those in the other three groups. The rarefaction curve showed that the curve of each group tended to flatten, indicating that increasing sequencing depths did not help to discover new OTUs, which was in accordance with the sequencing depth index (Goods_coverage) as shown in Table 1.
Beta diversity analysis
PCoA analysis (unweighted UniFrac distance) showed that the samples in the NP group tended to cluster together, and samples in AF and V groups tended to cluster together (PC1, which explained 36.64% of variation in the community) (Fig. 2a). PCA revealed that the community structure was distinct between NP and the other groups (PC1, which explained 10.33% of variation in the community). Furthermore, the UPGMA clustering tree in phylum level was generated (Fig. 2b). In accordance with PCoA results, AF and V groups were clustered together.
Differential analysis of intestinal flora at the genera level
The differential microorganisms in digestive diseases (AF, C and V) groups versus CP group, digestive diseases (AF, C and V) groups versus NP group and among digestive diseases groups (AF, C and V) were analyzed. The abundance of 13, 50 and 34 genera was increased in AF, V and C groups compared with CP group, respectively; inversely, a total of 5, 28 and 54 genera were reduced in AF, V and C groups compared with CP group. The heatmaps of three comparison groups are shown in Fig. 3a. In the three comparison groups, the abundance of five common genera, namely Coprobacillus, Acinetobacter, Enterococcus, Weissella and Lysinibacillus, was increased, while that of five common genera, namely Terrisporobacter, Dysgonomonas, Adlercreutzia, Fusicatenibacter and Blautia, was identified as decreasing (Fig. 3a, b).
There were 31, 47 and 48 genera which were increased among digestive diseases (AF, C and V) groups compared with NP group, respectively; in addition, 32, 45 and 56 genera were decreased in the above three comparison groups. The heatmaps of three comparison groups are shown in Fig. 4a. A total of 13 common genera such as Carnobacterium, Coprobacillus, Caproiciproducens and Psychrobacter were observed to be increasing, while 27 common genera such as Novosphingobium, Dorea, Brevundimonas and Terrisporobacter were identified to be decreasing (Fig. 4a, b).
Furthermore, in the AF group, the abundance of 38 genera was increased and 31 genera were decreased compared with C group. The relative abundances of Aerococcus and Senegalimassilia in AF group were reduced than V group. In the C group, an increased abundance of 23 genera and a decreased abundance of 40 genera were found than V group (Fig. 5).