Statistics for 16S rRNA data
Output data for microbiota sequencing included a total of 7.5 million raw reads with an average value of 2,57,096.85 ± 83008.43 reads per sample (Table S2). After eliminating both short and low-quality reads, adaptors, and other redundant sequences, a total of 5.1 million high-quality reads (~77.3% of total raw sequences) were obtained. To minimize the variation, the high quality reads were further rarefied to the minimum library size of 1,43,291 sequences. After quality filtering and rarefaction, the reads were classified into 1,54,790 unique OTUs by a 97% sequence similarity cutoff. For all samples, rarefaction curve showed an increase in OTU numbers as a function of the number of samples. The curve became asymptotically stable along with the OTU number being saturated, and an increasingly smaller number of new OTUs were added in each sample (Fig S1), indicating an adequate sequencing depth to obtain an accurate estimate of the OTU richness (with the Good's coverage > 99.7%). After eliminating low abundance and low variance features, a total of 3,847 OTUs were considered for downstream analysis.
Comparative microbiome analysis between K-Dwarf and Holstein samples
Alpha and Beta diversity analysis
Significant difference in the alpha diversity (Chao1 and Observed species) between the K-Dwarf and Holstein samples (Mann-Whitney, p< 0.0001) was observed that revealed a notable higher bacterial richness in K-Dwarf, compared to Holstein cattle (Fig 1a-b, Table 1). Further, the beta diversity projection on the PCoA plot revealed that the K-Dwarf and Holstein groups' fecal microbial communities were significantly different (ANOSIM, p-value < 0.001, Fig 2a-b).
The beta diversity analysis results were further supported by dendrogram clustering. Based on the individual samples taxon composition, the two cattle groups differentiated into two distinct clusters (Fig 3).
Metrics
|
|
K-Dwarf cattle
|
Holstein Cattle
|
p-value(using Mann-Whitney)
|
Observed Species
|
|
3433.6 ± 55.62
|
3273.3±53.11
|
0.0001
|
Chao1
|
|
3554.79 ±43.83
|
3417.26± 52.49
|
0.0002
|
Table 1 Comparison of alpha diversity metrics (Observed species and Chao 1) between K-Dwarf and Holstein cattle. The observed index was calculated as the actual number of unique taxa observed in each sample. While the Chao1 was estimated as the richness by inferring the number of rare organisms, that may have lost due to undersampling.
Taxonomic composition
According to the sequence similarity searches against the GREENGENES database, obtained OTUs were categorized into 22 phyla. Dominant phyla (>0.01) in both the K-Dwarf and Holstein groups were represented by Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, and Cyanobacteria and accounted for approximately 97% of the community (Fig S2a & b). Interestingly, a significant differential abundance of these four major phyla, including some minor contributing phyla, was observed across the cattle variety. For instance, Proteobacteria, Firmicutes, and Spirochaetes were significantly abundant in K-Dwarf, while Bacteroidetes were abundant in Holstein cattle. Also, TM7, Cyanobacteria, and Elusimicrobia lying under minor phyla were abundant in K-Dwarf, while phylum Actinobacteria and Tenericutes were significantly high in Holstein cattle. Dominant genera in K-Dwarf samples included Ruminobacter (20.9%), Oscillospira (10.3%),Clostridium (10.2%), Treponema (8.8%), and Succinivibrio (8.6%) (Fig 4a). Whereas, in the Holstein samples, the dominant genera were Oscillospira (20.7%), Clostridium (19.8%), Ruminococcus (10.8%), Phascolarctobacterium (9.8%), and Prevotella (7.5%) (Fig4b).
Differential taxonomic abundance and signature taxa analysis
To demonstrate the differential abundance analysis of microbial taxa and the degree to which the study groups differed at the phylum and genus level, the metagenomeSeq (fitZIG) approach was employed. Significant variations were observed at the phylum and genus level abundance between the K-Dwarf and Holstein groups (FDR adjusted p-value < 0.05).
At the phylum level, the relative abundance of nine phyla varied significantly between the study groups (MetagenomeSeq, FDR adjusted p-value < 0.001, Table S4). The phylum Proteobacteria, TM7, Cyanobacteria, Elusimicrobia, Firmicutes, and Spirochaetes were predominantly found in K-Dwarf, whereas in the Holstein group, Bacteroidetes, Actinobacteria, and Tenericutes were abundant.
At the genus level, 28 significant genera were differentially abundant between K-Dwarf and Holstein (MetagenomeSeq, FDR adjusted P < 0.05, Table S5) and highlighted through the heat map (Fig S3). The volcano plot displayed the existence of different community dynamics between K-Dwarf and Holstein samples with fold changes >2 and p values <0.05 (Fig 5, Table S3). Following Random Forest analysis, signature taxa were identified that potentially differentiate the K-Dwarf from Holstein groups (Fig 6). The signature genera investigated for K-Dwarf included Succinivibrio, Roseburia, Coprobacillus, Anaerovibrio, Anaerofustis, Paludibacter, Elusimicrobium, Candidatus-Azobacteroidetes, Sutterella, Ruminobacter, Oribacterium, and Coprococcus. On the other hand, Bifidobacterium, Prevotella, and L7A were found to be the marker taxa for the Holstein group. The mentioned features were ranked by their contribution to classification accuracy. The noticeable coherence observed in both the study groups showed the critical role of these genera in forming a distinct gut microbiome.
Association of beneficial bacterial genera with cattle type
Data mining results reveal 45 beneficial bacteria, out of which 27 were found differentially abundant between K-Dwarf and Holstein samples (MetagenomeSeq, FDR adjusted P < 0.05). Of 27 differentially abundant genera, 11 genera (9 in K-Dwarf and 2 in Holstein) were marked as signature taxa in Random Forest analysis. High beneficial bacterial genera were observed in K-Dwarf compared to Holstein samples. Bacteria that enhance the fermentation capacity, such as Ruminobacter, Sucinivibrio, Roseburia, Coprococcus, Dorea, Anaerovibrio, Blautia, Coprobacillus, Paludibacter, Sutterella, and Treponema were observed significantly abundant in the K-Dwarf group. However, in the Holstein samples, relative abundance of only five beneficial bacterial genera i.e., Oscillospira, Prevotella, Ruminococcus, Bifidobacterium, and Clostridium, was observed as predominant.
Correlation analysis of taxa between cattle type
Using the Spearman rank correlation coefficient (Rs), correlation analysis of highly abundant genera in K-Dwarf and Holstein cattle was performed (Fig 7). The results showed the taxa exhibiting a significant relationship with cattle type at the genus level. More than 10 beneficial bacterial genera such as Sutterella, Succinivibrio, Paludibacter, Coprobcillus, Anaerovibrio, Roseburia, Dorea, Oribacterium, Blautia, Anaerofustis, and Coprococcus was found to have a positive correlation with the K-Dwarf, (FDR adjusted p-value 0.05). Whereas, in the Holstein only three beneficial bacterial genera such as Ruminococcus, Oscillospira, and Prevotella were observed to be positively correlated (FDR adjusted p-value < 0.05).