Overview of microbial composition during decomposition
The average raw library size after paired-end assembly was 78,545 ± 10,670 (SD). After sequence trimming, quality filtering, and removal of chimeras, 17,696,903 high-quality sequences remained, with an average of 73,771 ± 9,688 (SD) reads per sample. Samples with library sizes close to, or larger than, 3000 reads showed convergence in their sampling effort curves. These high-quality sequences were assigned to a total of 5,099 operational taxonomic units (OTUs) by the UPARSE pipeline using a threshold of 97% identity. The OTUs belonged to 29 phyla, 67 classes,160 orders, 250 families, 473 genera, and 835 species. Among the 29 phyla, Firmicutes, Bacteroidetes, and Actinobacteria were dominant in all samples. (Fig. 1a). Lactobacillus was dominant in all groups at the genus level (Fig. 1b). The Lachnospiraceae NK4A136 group accounted for more than 15% of the total sequences before day 1. At the species level, Lactobacillus reuteri, Lactobacillus johnsonii, Enterococcus faecalis, Firmicutes bacterium M10-2, and Clostridium tetani E88 were the most abundant species (Fig. 1c). In general, the microbial community was dominated by OTUs with the Firmicutes (Fig. 1d) affiliated within the Lactobacillaceae.
Dynamics of microbial communities during decomposition
Although there appeared to be a significant amount of stochastic variation across the duration of this experiment, the overall reproducibility of these patterns between animals suggested that deterministic microbial community succession decreased with time (Fig. 1). As a result, only the following genera remained at day 15 postmortem: Gordonibacter, Bifidobacterium, Enterorhabdus, Lactococcus, Clostridium sensu stricto 18, Clostridium sensu stricto 15, Anaerosalibacter, Enterococcus, Dubosiella, and Lactobacillus (Fig. 1b). At the species level (Fig. 1c), the relative abundance of Lactobacillus reuteri, Clostridium tetani E88, Lactobacillus johnsonii, and Enterococcus faecalis showed notable variation during decomposition, which may aid in PMI estimation. Lactobacillus reuteri increased and exhibited peaks in abundances at both day 7 and day 15. Clostridium tetani E88 appeared on day 7 and decreased until day 15. Lactobacillus johnsonii showed a higher relative abundance one week after death than in the advanced stage of decomposition. Besides, Firmicutes bacterium M10-2 appeared on day 2 and showed an immediate increase from day 2 to day 4. Enterococcus faecalis appeared on day 2 and increased until day 10. Clostridium tetani E88 appeared on day 4 and increased and then decreased from then on. At the OTU level, OTU 1613 gradually increased in relative abundance during the 15-day decomposition and finally became the dominant OUT at day 15 (Fig. 1d). Among the Bacteroidetes and Actinobacteria, the OTUs had a similar change trend, increasing up to their peak abundances at day 2 and decreasing until day 4. However, OTU 2202, annotated to Helicobacter, gradually decreased during the 15-day decomposition (Fig. 1d). A heat map also showed discrepancies in microbial community abundance change trends during decomposition, with a color gradient from deep blue (low abundance) to deep red (high abundance) (Fig. 1e).
Classification of microbial succession patterns during decomposition
To analyze the microbial succession patterns, we applied hierarchical cluster analysis (HCA) based on the Manhattan distances between different taxa (Fig. 2a). Seven classes of taxon change patterns were obtained (Fig. 2b). Classes 1, 2, and 5showed a similar decreasing trend, which appeared with different PMIs. The taxa in lass 1 and class 5exhibited a fast decline before four days after death and a slow decline from then on. However, the declination range was greater in class 5 than in class 1. The taxa in Class 2 exhibited a continuous decrease during 15-day decomposition. In contrast, classes 3 and 4 showed an increase during the early PMI points. Class 3 showed a small increase two days after death, while class 4 showed a fast increase, a slow increase during 7-day decomposition, and a decrease from day 7 to day 13. Class 7 and class 6 assembled in clustering tree as both contained a lot of taxa with non-significant change trend during decomposition. Class 6 showed a relatively continuous profile, while class 7 showed a slow decline during the whole 15-day decomposition. After species annotation, class 1 mainly contained Lachnospiraceae, Ruminococcaceae, and Muribaculaceae at the family level and Lachnospiraceae NK4A136 group, Lactobacillus, Bacteroides, Lachnoclostridium, and Prevotellaceae UCG-001 at the genus level. Class 2 mainly contained Muribaculaceae, Lachnospiraceae, Ruminococcaceae, and Lachnospiraceae at the family level, Lactobacillus, Lachnospiraceae NK4A136 group, Ruminococcaceae UCG-014, Ruminiclostridium 5, Helicobacter, Ruminiclostridium 9, Alistipes, and Lachnoclostridium at the genus level. The microbial composition in class 1 and class 2 were similar. Class 5 also mainly contained Muribaculaceae, Lachnospiraceae, and Ruminococcaceae at the family level, and Lachnospiraceae NK4A136 group, Ruminococcaceae UCG-014, Lachnoclostridium, Bacteroides, Prevotellaceae UCG-001, Lactobacillus, Helicobacter, and Ruminiclostridium 9 at the genus level. Class 7 contained Enterorhabdus and Dubosiella. Dubosiella, Lactobacillus, Lactococcus, and Enterococcus were dominant genera in class 3 and class 4, increasing during early decomposition. We also counted the number of taxa in each class (Fig. 2b). Classes 6 and 7 contained 58.95% of the microbial taxa in all patterns. However, class 4 contained the fewest microbial taxa among all classes and was unimodal with an increasing profile. The blue lines in the plot represent relatively invariable taxa based on the Kruskal-Wallis test, which were mostly included in class 6. The other classes are mainly represented by the red lines, with significant variation during decomposition (P < 0.001, Kruskal-Wallis test). To display the changes in the patterns more obviously, the relative abundance changes of microbes in every class are shown in Fig. 3 (at the phylum level) and Supplementary Figure 1 (at the genus level). Specifically, in class 1, the relative abundance of Firmicutes gradually decreased, while Proteobacteria increased with PMI. The relative abundance of Bacteroidetes decreased during decomposition in class 2. Firmicutes in class 3 gradually increased. Latescibacteria in class 4 increased during 15-day decomposition. Bacteroidetes in class 5 gradually decreased during decomposition. The phyla in class 6 and class 7 did not show obvious variation during decomposition. In conclusion, the Lachnospiraceae NK4A136 group, Lactobacillus, Muribaculaceae, and Lachnospiraceae showed a declining profile, while Dubosiella, Enterococcus, Lactococcus, Clostridium_sensu_stricto_15, and Enterorhabdus were mainly increased during the 15-day decomposition. Then, we calculated the percentage of OTU annotation for the top 20 genera and every phylum. Firmicutes were the most dominant phylum in all classes (Fig. 4a). Class 6 contained the greatest number of phyla among the classes. Class 4 contained the fewest phyla, including Firmicutes, Actinobacteria, Bacteroidetes, Deferribacteres, Epsilonbacteraeota, and Proteobacteria. At the genus level (Fig. 4b), Lachnospiraceae NK4A136 group was the dominant genus in class 1. Lactobacillus was dominant in class 2. Dubosiella, Lactobacillus, and Enterococcus were dominant genera in class 3. Lactobacillus was the most dominant genus in class 4. Class 6 and class 7 contained many genera, while the distribution of the taxa in class 6 was discrete. Class 4 contained the fewest genera of the classes.
A PMI estimation model based on the microbial community succession trend data set
A random forests (RF) regression model was established to predict the PMI based on the microbial succession trend data set. The root mean square error (RMSE), the mean absolute error (MAE), and the squared correlation coefficient (R-squared, ) values in the validation set were 30.03 hours, 20.42 hours, 0.945, respectively. Those values in the testing set were 28.38 hours, 20.01 hours, and 0.947, respectively. Besides, the importance of variables was evaluated using the mean decrease accuracy (MDA) in the prediction illustrated in Fig. 5. In this model, the taxa with MDA values above 17.93, as 1.5 times the mean value of all MDA values, were regarded as significant strains, which was represented by the orange line in Fig. 5. The microbial taxa with MDA values over 17.93 were from all classes. According to the species annotation, significant taxa were mainly related to Lactobacillus, Dubosiella, Enterococcus, Lachnospiraceae NK4A136 group, Prevotellaceae UCG-001 and Clostridium sensu stricto 15 at the genus level.