Environmental Microbiology (2020) 00(00), 00–00
15000
Predicting postmortem interval based on microbial
community sequences and machine learning algorithms
Ruina Liu,1† Yuexi Gu,2† Mingwang Shen,3† Huan Li,4
Kai Zhang,1 Qi Wang,5 Xin Wei,1 Haohui Zhang,1
Di Wu,1 Kai Yu,1 Wumin Cai,1 Gongji Wang,1
Siruo Zhang,4 Qinru Sun,1* Ping Huang6* and
Zhenyuan Wang 1*
1
College of Forensic Medicine, Xi’an Jiaotong University,
Xi’an, 710061, China.
2 the most informative species in the decomposition
School of Mathematics and Statistics, Xi’an Jiaotong
University, Xi’an, 710061, China.
3
Department of Epidemiology and Biostatistics, School
of Public Health, Xi’an Jiaotong University Health
Science Center, Xi’an, Shaanxi, 710061, China.
4
Department of Microbiology and immunology, School of
Basic Medical Sciences, Xi’an Jiaotong University,
Xi’an, China.
5
Chongqing Medical University, College of Basic
Medicine, Department of Forensic Medicine,
Chongqing, 400016, China.
6
Shanghai Key Laboratory of Forensic Medicine,
Shanghai Forensic Service Platform, Academy of
Forensic Science, Ministry of Justice, Shanghai,
200063, China.
Summary
Microbes play an essential role in the decomposition
process but were poorly understood in their succes-
sion and behaviour. Previous researches have shown
that microbes show predictable behaviour that starts
at death and changes during the decomposition pro-
cess. Research of such behaviour enhances the
understanding of decomposition and benefits esti-
mating the postmortem interval (PMI) in forensic
investigations, which is critical but faces multiple
challenges. In this study, we combined microbial
community characterization, microbiome sequencing
from different organs (i.e. brain, heart and cecum)
and machine learning algorithms [random forest
(RF), support vector machine (SVM) and artificial
Received 22 October, 2019; revised 18 March, 2020; accepted 22
March, 2020. *For correspondence. E-mail qinrusun@mail.xjtu.edu.cn.
huangp@ssfjd.cn. wzy218@xjtu.edu.cn. Tel. +86-029-8265-5472; Fax
+86-029-8265-5472. †These authors contributed equally to this work.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.
doi:10.1111/1462-2920.
neural network (ANN)] to investigate microbial suc-
cession pattern during corpse decomposition and
estimate PMI in a mouse corpse system. Microbial
communities exhibited significant differences
between the death point and advanced decay stages.
Enterococcus faecalis, Anaerosalibacter bizertensis,
Lactobacillus reuteri, and so forth were identified as
process. Furthermore, the ANN model combined with
the postmortem microbial data set from the cecum,
which was the best combination among all candi-
dates, yielded a mean absolute error of 1.5
0.8 h
within 24-h decomposition and 14.5
4.4 h within
15-day decomposition. This integrated model can
serve as a reliable and accurate technology in PMI
estimation.
Introduction
In forensics investigations, accurate estimation of time
since death (postmortem interval, PMI) is critical but com-
plicated. Postmortem phenomena, such as algor mortis,
rigour mortis, livor mortis, discolouration of carrion, putre-
factive networks and digestion of gastric contents, are
commonly employed to estimate PMI (Kaatsch et al.,
1993; Henßge and Madea, 1996). However, these types
of physical evidence are sensitive to various environmen-
tal conditions and rely deeply on individual states and
investigators’ empirical judgments. To address the limita-
tions of traditional methods, scientists have been
employing various new techniques in PMI estimation,
such as molecular biology (Young et al., 2013; Li et al.,
2014; Feng et al., 2015; Ferreira et al., 2018), entomol-
ogy (Wells et al., 2015), spectroscopy (Li et al., 2017;
Zhang et al., 2017; Wang et al., 2017a; Wang et al.,
2017b) and thanatochemistry (Poloz and O’Day, 2009;
Kikuchi et al., 2010; Sara et al., 2014) analysis. Each
method provides significant information in investigations;
however, their feasibility and accuracy are limited under
the conditions and timeframe (e.g. days, weeks and
months) of PMI in a given case (Metcalf, 2019). For
example, forensic entomology is utilized to estimate PMI
with an error ranging from days to months because the
time and distribution of insects laying eggs on the
2 R. Liu et al.
corpses are uncertain (Metcalf et al., 2013; Guo et al., technology (Wang et al., 2018), which generate large
2016). Because of these limitations, developing a quick amounts of data. Thus, analysing massive amounts of
and accurate method for estimating PMI by forensic genomic data based on machine learning methods (the
pathologists is essential. core technology of artificial intelligence) to build a PMI
Recently, studies found that postmortem microbial com- estimation model is imperative. Machine learning algo-
munities respond to PMI in a predictive manner (Hyde rithms (Esteva et al., 2017; Kermany et al., 2018) are
et al., 2013; Metcalf et al., 2013; Pechal et al., 2014). This powerful methods for addressing high-dimensional data
discovery has spurred vast amounts of research (Javan sets because they are commonly applied to microbiome
et al., 2016; Metcalf et al., 2016) performed on human and data sets with mega DNA sequences (Belk et al., 2018).
animal remains to study microbial community succession With the considerable advantages in computing ability
after death (Finley et al., 2015; Hyde et al., 2017; Pechal and large data processing, machine learning methods
et al., 2018). Metcalf and colleagues (2013, 2016) demon- have been employed in the PMI estimation field by sev-
strated that the relative abundances of anaerobic gut bac- eral independent research groups (Metcalf et al., 2016)
teria (such as Lactobacillaceae and Bacteroidaceae) (Johnson et al., 2016; Pechal et al., 2018).
increased at the bloat stage, and aerobic bacteria and fac- Our study aims to develop an accurate PMI estimation
ultative anaerobic bacteria (such as Enterobacteriaceae) model in conjunction with a postmortem microbial
were dominant after rupture. Pechal and colleagues sequencing data set of internal organs in murine remains
(2014) found that Proteobacteria was the most abundant during 15-day decomposition. To accomplish this goal,
phylum in the early stages of decomposition, while informative features, suitable models and data sets were
Firmicutes dominated in the late stage. Damann and col- filtered, combined and evaluated. The feature extraction
leagues (2015) demonstrated that 99.2% of the bacterial process aids the discovery of microbial markers in PMI
genomic sequences of decomposing skeletons were from estimation. As the application of machine learning tech-
six phyla, Bacteroidetes, Firmicutes, Proteobacteria, niques in forensic science has not been well explored, it
Actinobacteria, Acidobacteria, and Chloroflexi, which is a novel addition to previous studies, and the data sets
were similar to the gut bacteria. In these studies, high- and multi-comparisons concluded here may improve cur-
throughput sequencing was used to reliably quantify the rent PMI estimation techniques.
relative abundance of postmortem microbial communities
and led to significant gains in the knowledge of bacterial
communities in mammal decomposition. Results
However, massively parallel high-throughput sequenc- Postmortem bacterial communities in internal organs
ing technologies, such as Illumina MiSeq and the Ion
S5TM XL system, are based on sequence-by-synthesis A total of 240 organ samples, including the brains, hearts
(Benbow et al., 2015) and semiconductor sequencing and ceca, were collected from 80 mice remains over
10 time points that spanned 15 days. A total of 176 sam-
ples remained and were labelled by PMI and body site
Table 1. A visual body score estimate of the decomposition stage after quality control by agarose gel electrophoresis,
according to Megyesi et al. (2005). except for brain and heart samples before day 2. A total
of 144 samples (D2Brain, D4Brain, D7Brain, D10Brain,
Head and neck Torso D13Brain, D15Brain, D2Heart, D4Heart, D7Heart,
Fresh D10Heart, D13Heart, D15Heart, D2Cecum, D4Cecum,
No discoloration (0 point) No discoloration (0 point) D7Cecum, D10Cecum, D13Cecum and D15Cecum)
Excoriation and trichomadesis Excoriation and trichomadesis
(1 point) (1 point) were used for the comparison of microbial comparison
Active decay between different organs. Visual decomposition stages
Discoloration: (2 points) Discoloration: (2 points) were estimated according to the body score estimation
Purging of decomposition Bloating of abdominal cavity
fluids out of eyes, nose, (3 points) method in Table 1 (the result was shown as Key_head,
or mouth Key_torso in see Additional file 2).
(3 points)
Bloating of neck and/or Precedes rupture and/or
face purging Bacterial analysis among internal organs at multiple
(4 points) of fluids (4 points). levels. The relative abundance of dominant bacteria
Advanced decay
Sagging of flesh (5 points) Sagging of flesh (5 points)
exhibited significant differences between ceca and hearts/
Sinking of flesh (6 points) Sinking of flesh (6 points) brains at multiple taxa. At the phylum level throughout
Caving in of flesh (7 points) Caving in of flesh decomposition duration, the dominant phyla were
(7 points)
Mummification (8 points) Mummification (8 points)
Firmicutes, Proteobacteria, and Bacteroidetes in brain and
heart samples, while Firmicutes, Proteobacteria,
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology

Bacteroidetes, and Actinobacteria had a relative abun-
dance of more than 1% of the total abundance in cecum
samples (Fig. 2A). At the family level, Enterobacteriaceae
and Peptostreptococcaceae were dominant in the brain
samples. Enterobacteriaceae and Lactobacillaceae were
abundant in the heart samples. By contrast, Lac-
tobacillaceae, Enterococcaceae and Erysipelotrichaceae
were the main components in the cecum samples (Fig. 2A).
At the genus level, Lactobacillus, Enterococcus and
Dubosiella were important genera in the cecum samples.
Morganella and Proteus were dominant in brain and heart
samples (Fig. 2A). At the species level, Lactobacillus
reuteri, Enterococcus faecalis and Firmicutes bacterium
M10-2 were the most abundant species of bacteria
detected in cecum samples. Clostridium novyi, Proteus
vulgaris, Anaerosalibacter bizertensis, and Clostridium
butyricum were dominant in heart and brain samples
(Fig. 2A). Enterococcus faecalis, Clostridium cochlearium
and A. bizertensis were predominant in brains, hearts and
ceca in the advanced decomposition of corrupted tissues
(Fig. 2A). Shared and unique operational taxonomic units
(OTUs) among different organs are exhibited in Venn plots
(see Additional file 1, Fig. S1).
Comparison of alpha and beta diversity among different
organs. The number of OTUs, observed species, the esti-
mators of community richness (ACE and Chao 1) and the
diversity (Shannon and Simpson) indexes of groups are
shown in Table S1 (see Additional file 1). Shannon index
comparison within different organs was performed with
analysis of variance, which indicated that the abundance
of microbial communities was significantly higher in
cecum groups than brain and heart groups before day
15 (Fig. 2B). Other alpha diversity index comparisons
between brain, heart and cecum samples also showed
significant differences (see Additional file 1, Fig. S2A). In
contrast, there were no significant differences between
brain and heart samples at the same time points during
decomposition (Fig. 2B, see Additional file 1, Fig. S2A).
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
Microbiome and AI in death time estimation 3
Fig. 1. The MAE and R2 of prediction
values of the ANN model, RF model
and SVM model on the testing data in
the cecum, brain and heart.
(A) and (B) corresponding to the
mean MAE and mean R2 on testing
data. The number of input variables of
the ANN model in the intestine, brain
and heart was 891, 160 and
116 respectively. Each model was
repeated 15 times, and the values of
MAE and R2 in this figure are the
mean values of the corresponding
15 repetitions.
To visualize the similarity and dissimilarity in postmor-
tem bacterial composition among different organs, princi-
pal coordinate analysis (PCoA) was used to demonstrate
the decomposing pattern in two-dimensional space
based on the unweighted and weighted UniFrac dis-
tances. Principal coordinate 1 (PCo1) and PCo2 (40.96%
and 24.67% of variance explained respectively) axes
showed that the microbial communities of brains and
ceca were separated before day 10 and had a tendency
to cluster after day 10 (Fig. 2B). The separation between
samples across body sites before day 10 and the similar-
ity at day 10–15 were more notable on Bray–Curtis
based non-metric multidimensional scaling (NMDS) plot
(see Additional file 1, Fig. S2C). The change trend was
similar between hearts and ceca (Fig. 2B, see Additional
file 1, Fig. S2C). Surprisingly, the bacterial communities
of brains and hearts clustered at the same postmortem
time point (see Additional file 1, Fig. S2B, S2C). The
Unweighted pair group method with arithmetic mean
(UPGMA) (Li and Xu, 2007) plot (Fig. 2C) indicated that
groups clustered by different organs based on weighted
UniFrac distance.
A t-test was performed to identify specific microorgan-
isms at the genus level with significant differences
(P ≤ 0.05) in different organs at the same PMI. The com-
parison of bacterial community differences between
hearts and brains revealed no significant differences
(P > 0.05), while this between brain and cecum samples
showed significant differences at multiple-taxon (see
Additional file 1, Table S2). We also employed linear dis-
criminant analysis effect size (LEfSe) analyses to detect
significant biomarkers among the sample sites during
decomposition at different levels (Fig. 2D). Morganella
and C. butyricum were detected in D2Heart. Clostridium
novyi. was considered a significant microbiome in
D4Brain. Firmicutes bacterium M10_2 was identified in
D4Cecum. Proteus vulgaris. and Clostridioides man-
genotii were significant species in D7Brain. Clostridium
sporogenes and Proteus mirabilis were significant
4 R. Liu et al.

Fig. 2. The significant differences in postmortem bacterial community composition of different organs.
A. Taxonomic profiles of postmortem microbial communities in internal organs during decomposition (community composition was based on the
top 10 phyla, families, genera and species with a classification of Other; Others were those found in some but not all samples). B. Comparison of
the Shannon index (ANOVA, *P < 0.05, **P < 0.05, ***P < 0.001) in different organs and PCoA plot based on weighted UniFrac distance coloured
by PMI points, shaped by organ type and coloured by PMI. Other alpha and beta diversity comparison plots are shown in Fig. S2 (see Additional
file 1). C. UPGMA analysis showed groups clustered according to different organs. D. LDA plots of bacteria at different levels among different
organs.

species in D10Brain. Enterococcus faecalis. and A.
bizertensis were regarded as representative in
D10Cecum. Faecalibacterium was a significant genus in
D13Brain. Clostridium cochlearium., Clostridium tetani
and Clostridiales bacterium mt7 were significant species
in D13Heart. Lactobacillus reuteri. was considered as a
significant species in D15Cecum. Bacteria at multiple
levels with significant differences are also displayed in
the MetaStat analysis figures (see Additional file
1, Fig. S3). The similarity percentage (Simper) is a
decomposition of the Bray–Curtis distances index, which
can quantify the contribution of each species to the differ-
ence between two groups. Figure S4 (see Additional file
1) shows that C. butyricum, L. reuteri and E. faecalis con-
tributed much higher than other species to the differences
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
between brains and ceca, and the results were similar in
the comparison of heart and ceca.
Significant metabolic differences among different
organs. The decomposition process was associated with
certain signalling pathways. To explore the connection
between postmortem microbiota and metabolic changes
within decomposition, Phylogenetic Investigation of Com-
munities by Reconstruction of Unobserved States
(PICRUSt) analysis was performed. In total, 4927 Kyoto
Encyclopaedia of Genes and Genomes (KEGG)
orthologue groups (Kos) were defined from our data and
annotated to three levels of KEGG pathways. The sec-
ond level of KEGG pathways is shown in the clustering
heatmap (Fig. 3A). The top four abundant pathways in all
 Microbiome and AI in death time estimation 5

Fig. 3. Predicted functional profiles of postmortem bacteria of internal organs in postmortem interval duration.
A. The relative abundance of KEGG pathway at level 2 in different organs is shown in the heat map. Gene copy numbers of samples within the
same sample group were pooled. The value of each functional gene (row) was log10 transformed. The significance test of the gene distribution
between groups was performed using the bootstrap Mann–Whitney U test with a cut-off of P < 0.01, FDR < 0.1 and mean counts >10. B. The
comparison histograms of the top two different pathways on metabolism (LSD-t, *P < 0.05, **P < 0.05, ***P < 0.001). C. PCoA plot of the third
level of metabolic pathway expression differences in internal organs after death.

groups for metabolism were membrane transport, carbo-
hydrate metabolism, amino acid metabolism, and replica-
tion and repair. Groups were clustered according to
different sample sites based on the relative abundance
expression level of KEGG pathways. Brain and heart
samples were clustered at different time points and sepa-
rated from cecum samples. Certain pathways, including
metabolism of cofactors and vitamins, cellular processes
and signalling and membrane transport, were much lower
in ceca than those in the brains and hearts (P < 0.05,
Fig. 3B, see Additional file 1, Fig. S5). In contrast, carbo-
hydrate metabolism, nucleotide metabolism, replication
and repair, and translation were lower in the brain and
heart samples than the cecum samples (P < 0.05, Fig. 3B,
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
see Additional file 1, Fig. S5). The comparison of the rela-
tive abundance of KEGG pathways between different
sample sites was analysed using SPSS software (version
18.0, Chicago, IL, USA), and the statistically significant
results are shown in Fig. 3B.
Postmortem bacterial community change in cecum
samples
After comparing the microbial community differences
among sample positions, the changes in the microbial
kingdom along the PMI were traced by high-throughput
sequencing. Using high-throughput sequencing data of
6 R. Liu et al.
cecum samples as an example, successive bacterial
community changes are described below.
Overview of postmortem bacterial composition in ceca.
A total of 18 858 845 raw sequences were generated in
the current study. After sequence trimming, quality filter-
ing and removal of chimeras, 17 696 903 high-quality
sequences remained, with an average of 73 771
9912
(SD) reads per sample. The rarefaction curves of the
Shannon diversity index indicated that species represen-
tation in each sample approached the plateau phase,
and it was unlikely that more bacteria would be detected
with additional sequencing efforts (see Additional file
1, Fig. S6A). These high-quality sequences were clus-
tered into 5099 OTUs by the VSEARCH pipeline using a
threshold of 97% identity. The results showed bacteria
belonging to 29 phyla, 67 classes, 160 orders, 250 fami-
lies, 473 genera and 835 species. Among the 29 phyla,
three phyla, Firmicutes, Bacteroidetes, and Prote-
obacteria, were dominant in all samples.
Epsilonbacteraeota, Deferribacteres, Actinobacteria and
Patescibacteria had a relative abundance of more than
1% of the total abundance (Fig. 4A). The 10 most abun-
dant genera included Lactobacillus, Lachnospiraceae,
Prevotellaceae, Muribaculaceae, Helicobacter,
Lachnoclostridium, Ruminococcaceae, Dubosiella,
Bacteroides and Ruminiclostridium, which accounted for
more than 50% of the total sequences (Fig. 4A). At the
species level, the most abundant species were L. reuteri,
E. faecalis, F. bacterium M10-2, A. bizertensis, Clostrid-
ium tetani, Vagococcus lutrae, Lachnospiraceae, Lacto-
bacillus johnsonii, Escherichia shigella and Helicobacter
(Fig. 4A).
The cecum microbiome change flow during decomposi-
tion. Intestinal microorganisms provided robust abun-
dance community profiles (Fig. 4A, Table 2). Using
community analysis at the genus level as an example,
several genera exhibited regular change flow during
decomposition. Lactobacillus was abundant during the
decomposition period (hour 0 to day 15). Enterococcus,
Anaerosalibacter and Clostridium sensu stricto 15 had
similar change trend, increasing up to the peak abun-
dance at the advanced decay stage (1 week after death)
and decreasing until day 15. Anaerosalibacter, Clostrid-
ium sensu stricto 15 and C. cochlearium appeared at
day 4. However, the Lachnospiraceae NK4A136 group,
Muribaculaceae, Lachnospiraceae, Ruminococcaceae,
Prevotellaceae_UCG-001, Lachnospiraceae_UCG-006
and Helicobacter decreased from hour 0 to day 15, and
almost disappeared at day 15. At the species level, the
relative abundance of L. reuteri, Clostridium tetani E88,
L. johnsonii and E. faecalis showed notable variation dur-
ing decomposition, which may aid in PMI estimation.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
Firmicutes bacterium. M10-2 appeared at day 2 and
showed an immediate increase from day 2 to day
4. Enterococcus faecalis. appeared at day 2 and showed
an increase until day 10. Clostridium tetani E88 appeared
at day 4 and showed an increase and then a decrease
from then on.
The heat map also showed differences in microbial
community abundance during decomposition (Fig. 4B),
indicating that there might be discrepancies in genera-
relative abundance between different groups with a col-
our gradient from deep blue (low abundance) to deep red
(high abundance). The diversity and similarity of microbial
communities in each group were based on the alpha and
beta values at the OTU level. To explore alpha diversity
among groups, we chose the ACE, Chao 1, Shannon
diversity and Simpson diversity indexes, with the last two
indexes considering the richness and evenness of the
microbial community (Fig. 4C, see Additional file
1, Fig. S6B). With the development of postmortem degra-
dation time, the Shannon index gradually decreased, and
other alpha diversity indexes also exhibited similar trends
(Fig. 4C, see Additional file 1, Fig. S6B). Only the follow-
ing genera remained at day 15 postmortem:
Gordonibacter, Bifidobacterium, Enterorhabdus,
Lactococcus, Clostridium sensu stricto 18, Clostridium
sensu stricto 15, Anaerosalibacter, Enterococcus,
Dubosiella and Lactobacillus. UniFrac distance is
designed to describe the difference between the two
groups. The score shown above is represented as a
divergence indicator. PCoA based on UniFrac distance
revealed the decomposition pattern in two-dimensional
space for Bray–Curtis distances (Fig. 4C). Groups clus-
tered based on PMI points shown on the principal coordi-
nate 1 axis (PC1, 16.90% of variance explained). The
LEfSe (Segata et al., 2011) analysis of core bacteria at
different levels revealed features that most likely
explained differences between groups associated with
PMI. Nine OTUs were identified as representative of the
following five time points: 0 h, 8 h, 2 days, 7 days and
15 days (Fig. 4E). In conclusion, among OTUs from lower
taxonomic levels, Ruminococcaceae UCG 014 and
Prevotellaceae UCG 001 (for 0 h samples);
Lachnospiraceae NK4A136 group (for 8 h samples);
Allobaculum (for 2 days samples); F. bacterium M10_2
(for 4 days samples); Clostridium sensu stricto 18 and
Clostridium tetani E88 (for 7 days samples); E. faecalis
(for 10 days samples), and Firmicutes (for 15 days sam-
ples) were significant in explaining the differences in
decomposition (Fig. 4E).
The shifts in the probable functions of the gut flora of
mice during decomposition were analysed by predicting
the 16S rRNA genes using PICRUSt (Fig. 5A). Similari-
ties in the variation trends among different level 2 KEGG
pathways during decomposition were observed (Fig. 5C).
 Microbiome and AI in death time estimation 7

Fig. 4. Bacterial community composition in cecum samples changed significantly and consistently over the course of decomposition.
A. The relative abundance of microbial communities at multiple level from postmortem cecum samples over PMI (the relative abundance of other
<0.01). B. The mean Shannon diversity index value decreased over time in cecum samples and PCoA analysis based on Bray–Curtis dis-
tance (Student T-test, *P < 0.05, **P < 0.05, ***P < 0.001). C. Heat map of the microbial 16S rRNA gene sequences (OTUs) at the generic taxo-
nomic resolution. D. LDA plots of bacteria at different levels as a result of all time points.

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
8 R. Liu et al.
Table 2. Relative abundance proportion of bacteria of cecum samples during decomposition.

Relative content percentage (%)

Bacteria h0 h8 h12 d1 d2 d4 d7 d10 d13 d15

Genera
Lactobacillus 21.36 22.62 29.08 26.24 26.64 23.92 50.20 24.67 35.04 42.77
Dubosiella 2.55 0.11 0.02 0.07 17.56 28.95 0.68 5.32 14.83 25.04
Enterococcus 0.11 0.06 0.94 2.63 4.01 11.82 14.59 26.96 13.04 17.23
Lachnospiraceae NK4A136 group 13.22 17.68 16.57 7.24 1.29 0.23 0.17 0.06 0.12 0.03
Anaerosalibacter 0.01 0.00 0.00 0.06 0.00 0.14 0.46 12.72 18.27 5.10
Muribaculaceae 4.25 3.97 3.93 3.44 7.35 4.05 1.69 0.30 0.29 0.07
Lachnospiraceae 8.54 8.29 4.33 5.07 1.10 0.20 0.45 0.10 0.18 0.04
Clostridium sensu stricto15 0.00 0.00 0.00 0.00 0.02 1.33 9.42 8.33 4.56 3.83
Ruminococcaceae 3.12 4.55 4.33 2.85 2.34 1.61 0.58 0.26 0.51 0.16
Helicobacter 3.20 1.87 3.75 3.26 2.55 0.22 0.14 0.06 0.04 0.00
Species
Lactobacillus reuteri 4.46 4.22 8.32 7.05 7.28 7.49 25.10 11.67 7.38 30.56
Enterococcus faecalis 0.01 0.03 0.05 0.17 0.83 3.10 3.68 15.99 3.40 10.07
Firmicutes bacterium M10-2 0.02 0.10 0.02 0.01 10.16 23.99 0.07 0.15 0.02 0.98
Lactobacillus johnsonii 2.83 5.11 6.65 3.33 2.28 1.33 2.85 0.24 5.41 0.67
Clostridium tetani E88 0.00 0.00 0.00 0.00 0.02 1.30 9.33 8.05 4.46 3.69
Anaerosalibacter massiliensis 0.00 0.00 0.00 0.02 0.00 0.03 0.12 2.74 4.94 1.24
Proteus mirabilis 0.00 0.07 0.00 0.00 0.29 2.61 0.77 0.36 0.29 0.01

The top 10 relative abundances of level 2 KEGG path-
ways showed that increases occurred at hour 8 and day
7, and decreases occurred at day 4 and day 13, which
was similar to the heat map results (Fig. 5C). The expres-
sion level of genes involved the endocrine system, amino
acid metabolism, carbohydrate metabolism pathways
showed obvious reductions during decomposition in
cecum samples (Fig. 5B).
Computing results and PMI estimation model
Feature selection
In the feature ranking list, features with small rankings
are more critical. We selected the top 20, 30, 40, 45 and
50 features to generate four different biomarker sets. To
identify the most stable biomarker sets, we established
four different ANN models, taking into account these four
biomarker sets. Similarly, each model was run 15 times,
and the mean value of the mean absolute error (MAE)
and goodness-of-fit (R2) of each experience were calcu-
lated and compared (Table 3). Biomarker sets with
45 genes had a lower MAE and higher R2 than other set
sizes.
PMI prediction model
The biomarker set in predicting PMI consisted of 45 differ-
ent strains in cecum samples. Figure 6 exhibits the pre-
diction results by ANN on the testing data run 15 times.
The ANN model is applied to two data sets. One is the
cecum data set with 891 different taxa, and the other is
the cecum data set with 45 taxon biomarkers selected as
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
previously described. The evaluation indexes of the
model are the MAE and R2. These indexes are computed
using the testing data. As the training and testing of the
ANN model will be affected by the partition of data sets,
the model in each data set was been repeated 15 times,
and the evaluation indexes of the model were calculated
as the mean value of these 15 experiments. The mean
MAEs (SD) of the ANN models in the data set with all
891 features and data set with 45 features were 14.483
(
4.443) and 18.684 (
3.566) respectively. The mean
R2 (SD) of the models in these two data sets was 0.951
(
0.032) and 0.948 (
0.021) respectively. Furthermore,
we specifically considered the prediction of testing sam-
ples that decomposed within 24 h. The mean MAEs
(SD) of the original data set and the biomarker data set
were 1.528 (
0.814) and 7.425 (
1.949) respectively.
That is, for mouse remains that decomposed in less than
24 h, the biomarker data set can predict PMI with a mean
error at 7.43 h. Because the original set provided the
ANN model with more features than the biomarker set,
the MAE value of the biomarker set was slightly larger
than the original set.
Discussion
Forensic microbiology is a developing tool for estimating
PMI in forensic investigations (Metcalf, 2019). Currently,
there remains a paucity of knowledge and motivation for
an in-depth interpretation concerning postmortem micro-
bial communities and their transmigration within internal
body sites. This study utilized 16S rDNA sequencing
approach to trace changes in bacterial community struc-
ture with PMI in internal organs of murine remains. In
 Microbiome and AI in death time estimation 9

Fig. 5. Predicted function in the metabiotic field.

A. Relative abundance of metabiotic pathways at multiple levels. B. Heat map of the top 50 abundant pathways at level 2. C. Line charts exhibit
an obvious shift of the level 2 KEGG pathways.

addition, mega microbial sequencing data sets were cadaver-associated soils and abdomen of mice, swine,
analysed with machine learning methods to extract infor- and human remains (Hyde et al., 2013; Metcalf et al.,
mative microbial species and establish a PMI estimation 2013; Pechal et al., 2014; Damann et al., 2015), as the
model. microbial data in those studies fluctuated with the sur-
This study is different from previous studies, which rounding biological and environmental conditions (e.-
examined microbial communities in the skin, bone, g. weather, insect accessibility and cadaver position).
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
10 R. Liu et al.
Table 3. Evaluation test of biomarker set size with MAE and R2. that the quantity of microorganisms in brain and heart
samples was not sufficient for high-throughput sequenc-
Size of biomarker set Mean MAE ± SD Mean R2 ± SD
ing process until 2 days after death in this study. Thereaf-
20 23.575 ± 3.073 0.922 ± 0.022 ter, brains and hearts were invaded by Morganella at
30 20.535 ± 4.337 0.946 ± 0.033 an earlier PMI (day 2) and Clostridium at later decompo-
40 18.844 ± 3.301 0.948 ± 0.023
45 18.683 ± 3.566 0.948 ± 0.021 sition as organs begin to emulsify at day
50 20.822 ± 2.811 0.940 ± 0.018 4. Paeniclostridium and C. novyi were detected in brain
samples at day 4, and A. bizertensis appeared in brain
samples at day 13 (Fig. 2A), which may contribute to PMI
Considering the influence of environmental factors, inter- estimation as specific biomarkers. Clostridium novyi. is a
nal organs (brains, hearts and ceca) were sampled in this pathogenic (Watanabe et al., 2019) anaerobic bacterium.
study, and microorganisms of ceca served as a contrast It is found in soil and faeces and becomes abundant with
to the postmortem microorganisms in brain and heart the consumption of oxygen. Anaerosalibacter bizertensis.
samples. Furthermore, the decomposition of these sam- is a kind of salt-tolerant microorganism (Rezgui et al.,
ples can represent the decomposition of cranial, thoracic 2012). These dominant postmortem microorganisms
and abdominal cavities. Referring to previous work detected in brains and hearts are known to be pathogenic
(Burcham et al., 2019; Can, Javan, Pozhitkov, & Noble, microorganisms. We infer that the immune system breaks
2014), most internal organs such as brains or hearts are down after death (Alan & Sarah, 2012), followed by prolif-
devoid of microorganisms in a healthy condition in the eration of microorganisms from the digestive system to
mammalian body, which is in accordance with the fact the whole body through capillaries of the vascular and

Fig. 6. The prediction results of the ANN model based on 45 biomarkers in the intestine.
In detail, the main picture shows the prediction time of all testing data, and the subgraph in the upper left specifically shows the prediction
corresponding to mouse remains that decomposed within 24 h. The diagonal value is the actual PMI the remains. The closer the predicted value
is to the diagonal, the more accurate this prediction is.

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology

lymphatic system (Paczkowski & Schutz, 2011) and the
mucus membranes in the respiratory system (Gill
et al., 1976).
A significant difference was observed between the
microbial communities of the brain, heart and cecum dur-
ing 7-day decomposition, as reported previously on
human cadavers (Burcham et al., 2019). Interestingly, E.
faecalis, C. cochlearium and A. bizertensis all appeared
in the three organs in the advanced decomposition of
corrupted tissues (Fig. 2A). This demonstrates that the
decomposers of different organs were similar at
advanced decay stages. Previous studies (Brenner et al.,
2001) also showed that C. cochlearium, which was dis-
covered in soil, human oral cavity, faeces, wounds and
septic infections, is a crucial contributor to the decompo-
sition of remains. In addition, E. faecalis caused the
majority of human enterococcal infections and contrib-
uted to the decomposition of corpses in previous reports
(Metcalf et al., 2013). These three species may help
describe the characteristics of different decay stages in
the carrion decomposition process.
To discover more significant microbial features of inter-
nal organs during the decomposition period, cecum sam-
ples were taken as an example, as they had a more
abundant and robust profile of microorganisms than brain
and heart samples did. Firmicutes, Bacteroidetes, Prote-
obacteria and Actinobacteria were predominant in all
PMI groups at the phylum level, which was coincidence
with several reports based on the human corpse system
(Finley et al., 2015). Firmicutes were found in the oral
cavity during prebloat, while Proteobacteria were found
during postbloat (Hyde et al., 2013). Proteobacteria are
commonly associated with the spoiling of meat and have
been found on the hides of slaughtered animals (Gill and
Newton, 1978). Actinobacteria play a vital role in the
recycling of refractory biological components through
decomposition and humus formation (Gill and Newton,
1978), and a group of Actinobacteria with filamentous
form have been shown to be important decomposers of
polysaccharides, proteins and lipids through the genera-
tion of extracellular enzymes, and have been found in
grave soil and bones at an advanced decay stage in
dead bodies (Cobaugh et al., 2015; Damann et al.,
2015). We found that relative abundance of
Epsilonbacteraeota decreased immediately after death
and disappeared at day 7, which is consistent with previ-
ous reports of many Epsilonbacteraeota species coloniz-
ing in the digestive tracts of animals and serving as
symbionts or pathogens. In addition, they reported that
carbon, nitrogen and sulphur cycling are the primary
drivers of functional divergence in environmental
Epsilonbacteraeota (Waite et al., 2017). At the family
level, the relative abundances of Ruminococcaceae and
Lachnospiraceae decreased, while those of
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
Microbiome and AI in death time estimation 11
Enterococcaceae and Clostridiaceae increased during
the 15-day decomposition. The former two were inhered
in the intestinal tract, and the following two were reported
as important contributors to decomposition. Additionally,
at the genus level, facultative anaerobes such as Entero-
coccus, Dubosiella and Anaerosalibacter (Fig. 4A),
which are widely recognized as opportunistic pathogens
and are associated with sewage and animal matter,
became abundant after rupture. Anaerosalibacter is a
halotolerant bacterium isolated from sludge (Rezgui
et al., 2012) and stool (Dione et al., 2016). The
Lachnospiraceae NK4A136 group and Bacteroidetes,
which occur in the human and mammal gut microbiota
(Debruyn and Hauther, 2017), became less abundant
after rupture at day 4. Several species also showed indic-
ative changes in relative abundance during PMI in this
study. For instance, Anaerosalibacter massiliensis, Pro-
teus mirabilis, Clostridium tetani E88, and L. reuteri
increased, whereas, L. johnsonii decreased with PMI.
Enterococcus faecalis. and F. bacterium M10-2
increased, following a decrease during the decomposition
procedure. These species may provide important infor-
mation in the PMI estimation model. In conclusion, based
on the trends in bacterial composition changes in this
study, we found that the diversity and evenness of the
cecum microbiome gradually decreased within 15 days of
decomposition. Also, in the early stage of death, micro-
bial participants depend on bacteria inherent in ceca,
which were similar to those in the stool of healthy
humans, as reported by the Human Microbiome Project
(Huttenhower et al., 2012). These shifts in bacteria taxa
were related to the shifts in functional gene abundances,
as the functional prediction made from PICRUSt. We
detected markedly predicted increases in genes associ-
ated with energy metabolism including carbohydrate
metabolism and amino acid metabolism on day 7 and
day 15, which was decreased on day 4 (Fig. 5C). These
results suggested that the level of amino acid and carbo-
hydrate degradation inside murine corpses decreased
after the body rupture on day 3 because nutrient-rich
fluids permeated the surrounding environment of the
corpses. Consistently, the functional gene abundances
associated with amino acid metabolism were significantly
increased in grave soil samples around human corpses
(Metcalf et al., 2016). Based on the postmortem micro-
biome correlation between this mouse decomposing sys-
tem and the human decomposing system, the microbes
found related to decomposition in this work could provide
fruitful information for projecting to humans in forensics
investigations.
Next, we focused on establishing a mathematical
model to predict PMI more accurately. Some previous
science scientific research teams utilized class- or
phylum-level taxonomy data sets to predict PMI with
12 R. Liu et al.
machine learning methods (Johnson et al., 2016; Belk
et al., 2018). Johnson and colleagues (2016) developed
a k-nearest neighbour regressor, combined with the
microbial data set from all nasal and ear samples at the
phylum level, which predicted the PMI with an average
error of
55 accumulated degree days, approximately
3 days. Metcalf and colleagues (2013) established a RFs
model combined with cadaver-associated skin data set
for PMI prediction, which allowed them to estimate PMI
within 3.30
2.52 days (MAE
SD) during 34-day
decomposition. To the best of our knowledge, this is the
first study to use OTU level data sets combined with an
ANN model to estimate PMI. In this study, we removed
OTUs with low relative abundances and less relative
abundance variance during decomposition, which were
considered as noise and low contribution features in this
study, and finally, 891, 160 and 116 features were identi-
fied in the cadaver-associated cecum, brain and heart
bacterial data set respectively. Figure 1 indicates that the
cecum data set predicted PMI through three models with
lower MAE and higher R2 than the brain and heart data
sets. The results of this data mining indicate that the most
robust models in PMI estimation are the cadaver-
associated cecum data set combined with the ANN
model and 16S rRNA gene marker summarized at OTU
level taxonomies. Among the 891 features ranked by the
support vector machine recursive feature elimination
(SVM-RFE) and RF models, the top 45 biomarker sets
performed better than the other three sets with the mini-
mized MAE and maximized R2 (Table 3). Regarding spe-
cies annotation, Clostridium tetani E88, A. massiliensis,
Vagococcus fluvialis, Candidatus Arthromitus sp. SFB
mouse Japan and Lactobacillus animals were included
in the ANN model for PMI estimation. Because microbial
succession is more rapid and diverse before 24 h than in
later stages of decomposition (Metcalf et al., 2013; Cob-
augh et al., 2015; Metcalf et al., 2016), we utilized the
ANN model to predict PMI specifically within 24 h. Our
ANN model predicted PMI with an MAE of 14.483
(
4.443) h during 15-day decomposition and 1.528
(
0.814) h within 24-h decomposition.
This result demonstrated that the ANN model with pre-
dictable postmortem microbiome data sets is more accu-
rate for PMI estimation during 15-day decomposition.
Currently, the greatest methodological and technological
gap (Metcalf, 2019) in developing generalizable microbial
models for predicting PMI is the limitation of the size of
cadaver-associated data sets and low sampling fre-
quency. Our research team is currently trying to over-
come these barriers and establish lager data set
(240 cecum samples) to train, test and generate robust
models. However, we have a limited sampling time
frame, and the longer sampling span will allow for a more
thorough understanding of microbial behaviour in the
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
decomposition process. Therefore, the longer time frame
to study microbial community changes and the effect of
temperature and humidity on microbial succession need
to be improved and established in future work. In addi-
tion, the sample positions in this study only reflected the
internal postmortem microbial succession. The influence
of outside environment and complicated inter-reaction
with the hosts of microbiome should be focused in future
studies. This present work formed a preliminary basis for
future investigations with human remains. However, how
to leverage the results of this study in practical forensic
investigations with human corpses needs further studies.
In conclusion, the present study evaluated the post-
mortem microbiome community structure during the
15-day decomposition. Consistent with previous works,
the microbial decomposers of the brain, heart and cecum
became more similar as PMI progressed. We utilized
machine learning algorithms to evaluate the effectiveness
of different data sets, and the results indicate that the
most robust model of PMI estimation is postmortem bac-
teria data in cecum samples associated with the ANN
model at the OTU level. We also detected several spe-
cies (Clostridium tetani E88, A. massiliensis, L. reuteri, E.
faecalis, F. bacterium M10-2) that may facilitate the
establishment of PMI prediction models. Finally, we
developed an ANN model that predicted PMI with an
MAE of 14.5
4.4 h during 15-day decomposition and
1.5
0.8 h within 24-h decomposition. This study pro-
vides directions for further research on leveraging
machine learning models with microbial-based data sets
for estimating PMI.
Experimental procedures
Sample collection and experimental set-up
In this experiment, 240 mice (strain C57BL/6, 18–25 g,
males, 6–10 weeks) were acquired from the Experimental
Animal Centre of Xi’an Jiaotong University and cohoused
and given a 2-week normal diet. After sacrifice by cervi-
cal dislocation, the mice were placed on sterile plates
with ambient temperature (Ta, 25
1.5 C) and moderate
relative humidity (RH, 50
7%). This work used only
dead animals that had been sacrificed as a by-product of
other research and was deemed not to be animal
research by the Institutional Animal Use and Care Com-
mittee of Xi’an Jiaotong University. Mice were euthanized
humanely under approved approval No. 2017-288. We
recorded a visual body score estimate for the head and
torso according to Megyesi and colleagues (2005))
(Table 1). Ten time points (0 h, 8 h, 12 h, 1 day, 2 days,
4 days, 7 days, 10 days, 13 days and 15 days) were set
spanning 15 days, and at each time point, brains, hearts
and ceca were removed from 24 mice under strict aseptic


operation and stored at −80 C until further processing.
The organs could not be removed from the remains
because of advanced corruption occurring from 15 days
after death. In total, 144 samples (D2Brain, D4Brain,
D7Brain, D10Brain, D13Brain, D15Brain, D2Heart,
D4Heart, D7Heart, D10Heart, D13Heart, D15Heart,
D2Cecum, D4Cecum, D7Cecum, D10Cecum,
D13Cecum and D15Cecum) were used to compare the
differences in microbial composition between different
organs. A total of 240 cecum samples (H0Cecum,
H8Cecum, H12Cecum, D1Cecum, D2Cecum, D4Cecum,
D7Cecum, D10Cecum, D13Cecum and D15Cecum)
were used in deep learning algorithms to estimate PMI.
DNA extraction, PCR amplification and high-throughput
sequencing
Total genomic DNA was extracted from 25 mg of organ
samples (brains, hearts and ceca) using QIAamp® DNA
Mini Kit (Qiagen, Germany) following the protocols
recorded in the handbook. Final purified DNA was eluted
in a final volume of 100 μl. The extracted bacterial geno-
mic DNA was estimated by measuring the absorbance at
260 nm (A260) using a NanoDrop spectrophotometer
(Thermo Scientific, Inc., Waltham, MA, USA) before
downstream processing. Only samples harbouring at
least 1 ng μl−1 DNA and yielding a clean spectrogram
with a peak at 260 nm were included in subsequent
amplification steps.
For IonS5™XL sequencing, total genomic DNA was
subjected to PCR amplification targeting an informative
portion of the 16S rRNA variable region 3–4 (V3, V4)
using the bacterial primers 341F (50 -
0
CCTAYGGGRBGCASCAG-3 ) and 806R (50 -
0
GGACTACNNGGGTATCTAAT-3 ). Negative controls
included no template controls for DNA extraction and
PCR amplification. PCR was performed using a T100™
Thermal Cycler (Bio-Rad, Hercules, USA) with the follow-
ing cycling parameters: 95 C for 5 min, followed by
34 cycles of 94 C for 1 min, 57 C for 45 s, 72 C for
1 min, and a final elongation step of 72 C for 10 min and,
finally, 16 C for 5 min. PCR products were detected by
agarose gel electrophoresis. The target bands were
removed under a UV lamp and recovered by a GeneJET
Gel Extraction Kit (Thermo Scientific). Using the Ion Plus
Fragment Library Kit 48 rxns (Thermo Scientific), we fin-
ished library construction. After Qubit quantification and
library testing, each amplicon pool was sequenced using
one lane of the Ion S5™XL plate.
High-throughput sequencing reads were merged and
quality filtered using Cutadapt (Langille et al., 2013a)
(V1.9.1, Langille, 2013b). After removing low-quality
sequences, reads were divided into groups according to
the barcode. When the barcode and primer sequences
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
Microbiome and AI in death time estimation 13
were removed, only the raw data remained. To obtain
clean data, chimaeras were filtered and trimmed
according to the species annotation database in the
VSEARCH pipeline. High-throughput sequencing reads
were classified into OTUs in the UPARSE pipeline
(v7.0.1001) within a 0.03 difference (equivalent to 97%
similarity) (Haas et al., 2011).
The Mothur method and SSUrRNA database in
SILVA132 were utilized for taxonomy assignment, which
was performed at various levels according to representa-
tive sequences of each OTU. The representative
sequences of all OTUs were then aligned to the
Greengenes reference alignment using MUSCLE (Quast
et al., 2013) (Version 3.8.31, http://www.drive5.com/
muscle/), and this alignment was used to construct a phy-
logenetic tree. Data from each sample were homoge-
nized, with the standard of the least amount of data in
the samples. Alpha diversity was measured by the Shan-
non, Chao1, Simpson and ACE indexes in the bacterial
communities in Mothur software (Tai et al., 2016).
All statistical analyses were conducted in the R envi-
ronment (v2.15.3; http://www.r-project.org/). To assess
the microbial diversity and abundance, the alpha (α)
diversities of OTU richness and various indexes
(Shannon index, Chao 1, ACE, Simpson) were calcu-
lated, while the microbial beta diversity was estimated
according to the UniFrac distance between the samples.
To measure the sequencing depth, the coverage index
was used. The PD_whole_tree index described the phy-
logenetic diversity of the microbiome in the samples. Beta
diversity was calculated to compare differences between
microbial community profiles, and the data are shown as
the results of PCoA. Principal component analysis, PCoA
and NMDS coordination analysis were performed to visu-
alize the similarities or dissimilarities of variables that
best represented the pairwise distances between sample
groups, which were displayed by the WGCNA package,
stat packages and ggplot2 package in R software
(v2.15.3).
Statistical analyses were performed to find significant
differences in microbial community structure between
groups. To identify microbial markers in specific organs
or at specific time points, Student T-test, MetaStat, Sim-
per analyses and LEfSe analysis were implemented.
UPGMA (Li and Xu, 2007) is a hierarchical clustering
method that follows a three-step procedure. First,
UPGMA begins with the two closest OTUs and combines
them to form a new OTU. Second, the arithmetic mean
distances between the new OTU and all other OTUs
were recalculated. Finally, the process was repeated,
and a complete phylogenetic tree was obtained. Analysis
of molecular variance was utilized to test discrepancy
among groups based on weighted or non-weighted
UniFrac distance matrix in Mothur software. The
14 R. Liu et al.
PICRUSt (Langille et al., 2013a) together with the KEGG
database as a reference was utilized to predict meta-
genomic functional content based on the 16S rRNA
sequences (Kanehisa et al., 2014).
Computational methods
Data materials. The data for this project were produced by
collecting postmortem organic samples of mouse cadavers
during decomposition in controlled environmental condi-
tions. DNA collected from each sample was analysed using
16S rDNA high-throughput sequencing, which allowed for
species detection and relative abundance calculation in all
samples. Therefore, the raw data included the relative
abundance of bacterial sequences in postmortem organs
(brains, hearts and ceca) and PMI labels. The sample sizes
of the brain, heart and cecum data sets were 48, 48 and
240 respectively. Brain and heart data included six PMI
points (including 48, 96, 168, 240, 312 and 360 h after
death). Four more time points (including 0, 8, 12 and 24 h)
were obtained in cecum data set.
Data preprocessing. Each sample in the brain data set
and heart data set contained the relative abundance values
of 3414 different taxa. Moreover, the individual sample in
the cecum data set contained 4099 different taxa. However,
the relative abundance values of many taxa were mostly
zero in these three data sets, and data preprocessing was
required. This preprocessing process consisted of three
steps. First, the taxon whose relative abundance was zero
in most samples in each data set was removed. Specifi-
cally, samples from each data set were grouped according
to the time point of death. Hence, there are 10 groups, six
groups and six groups of samples in the cecum data set,
brain data set and heart data set respectively. For each
data set, if the relative abundance of a taxon was zero in
more than 60% samples in all groups, then this taxon was
deleted in this data set. Second, taxa with low relative
abundances were removed. Briefly, if the mean relative
abundance of a taxon was lower than 3 in all groups of a
data set, then it was removed from this data set. Third, the
taxon with low relative abundance variance was removed.
For each taxon, we calculated the variance of the mean rel-
ative abundance in each group. Then, the taxa
corresponding to the first 80% of the variance in each data
set were regarded as informative features and reserved. All
removed features are considered as noise and low contri-
bution features in this study. Finally, samples in the cecum
data set, brain data set and heart data set contained
891, 160 and 116 OTUs respectively.
Data set selection. RF, SVM and ANN models were
applied to these three data sets to predict the PMI of the
mice postmortem model. Each data set was divided into
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
two parts: 70% was the training data and the remaining
30% was the testing data. The accuracy of the models
was measured by the MAE, which calculated the devia-
tion of the predicted from observed values and represen-
ted the average prediction error in the same unit of the
original data. The efficiency of the models was evaluated
by the goodness-of-fit test (R-squared, R2) (Rights and
Sterba, 2019). In particular, each model was run and
evaluated 15 times in each data set, and the mean value
of the MAE and R2 was shown in Fig. 1.
Significant microbial markers selection. In this study, the
identification of strain biomarkers was built on an SVM-
RFE model and RF regression model. Again, each model
was run 15 times, and their feature importance gave the
ranking of features. Finally, we summed up all rankings
of corresponding features in each experiment of the two
models. Then, features were reordered to generate the
final feature ranking list.
Deep learning algorithms applied in PMI estimation. To
determine the most robust model, we applied SVM-RFE
model, RF regression model and ANN model to the brain,
heart, cecum postmortem microbial data sets and evalu-
ated the model performance with the MAE and R2 of PMI
prediction.
RF (Breiman, 2001; Mamoshina et al., 2018) is a clas-
sification and regression model. This model assembles
many tree-structured predictors and aggregates their
results (majority vote for classification and averaging for
regression). Tree-structured predictions are generated
based on the bootstrap samples of the training data, and
the candidate feature subset of this tree is randomly
selected (Svetnik et al., 2003). Each tree grows to its
maximum depth without pruning. In addition, the RF
model also assesses the importance of different features
(Breiman, 2001; Kamin
ska, 2019). In this study, a RF
model was established with regression trees. For every
regression tree, training samples are chosen with
replacement from the original training data set. A candi-
date feature subset is randomly selected from the feature
set, and each node of this tree will choose one feature
from this subset to split. The splitting criterion of a regres-
sion problem minimizes the mean squared error at the
node. During the process of generating a forest, the size
of the candidate feature subset is fixed. For the regres-
sion problem, the output of this forest is formed by aver-
aging all regression trees. The importance of features
(Archer and Kimes, 2008; Genuer et al., 2010) was deter-
mined using out-of-bag observations. Specifically, the
importance of a variable was measured by calculating
the increase in the prediction error (mean square error)
on the condition that only that variable is permuted.

A SVM based on recursive feature elimination (Guyon
et al., 2002; Sahran et al., 2018) is a popular feature
selection method. In this study, we used the linear SVM-
RFE to perform feature selection. The steps involved in
the SVM-RFE algorithm can be briefly described as fol-
lows: (i) train a linear SVM based on the training data set;
(ii) compute the ranking criteria of features according to
their weights in the SVM model. Specifically, ranking
criteria ci of the feature is ci = (wi)2, and wi is the
corresponding weight of this feature; (iii) rank the features
by their importance and eliminate the feature with
smallest ranking criterion; (iv) update the feature rank list;
and (v) repeat these processes. Moreover, a cross-
validation algorithm with 10-fold is combined with SVM-
RFE to determine the number of features adaptively.
ANN model (LeCun et al., 2015; Sahran et al., 2018) was
used in this study with four neuron layers (including two hid-
den layers). The ANN model tends to make every neuron
have a similar importance, so it is not suitable for feature
selection. The input layer has 45 neurons that correspond to
the biomarkers in cecum as described previously. Two hid-
den layers have 23 and 12 neurons respectively. The output
layer has only one neuron to yield the predicted value of
PMI. In the last three layers, the rectified linear unit activa-
tion function was used to enhance model non-linearity. The
error function in this model is the mean square error. More-
over, to minimize this error function, the backpropagation
algorithm is used to adjust the weights of connections in the
network. The performance of the model is evaluated based
on the MAE and goodness-of-fit of the testing data.
Evaluation of the regression model. We calculate the
MAE and R2 on the testing data to assess the regression
model performance. To simplify, we assume that the
observed value and prediction value of N samples are yi
and y^i ði = 1,2, …, NÞ. The MAE of all these samples is cal-
PN
culated as MAE = N1 jy^i −yi j . This evaluation criterion
i=1
can intuitively measure the error of time prediction. R2
describes how well the model fits the observations
PN
ðy^i − yÞ
2
(Lewis-Beck and Skalaban, 1990). In detail, R2 = P
i =1
N Benbow, M.E., Pechal, J.L., Lang, J.M., Erb, R., and
ðyi − yÞ2
i =1
, where y^i , yi and y are the prediction value, observed
value and the mean value of observations respectively.
Abbreviations
AI artificial intelligence
ANN artificial neural network
MAE mean absolute error
PMI postmortem interval
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
Microbiome and AI in death time estimation 15
RF random forest
SVM support vector machine.
ACKNOWLEDGEMENTS
The authors thank Prof. Jiru Xu for scientific design and meth-
odology direct. The authors also thank Shanghai Majorbio
Bio-pharm Technology and Novogene for insightful assistance
with the DNA sequencing and metagenomics analysis. This
study was funded by the Council of the National Natural Sci-
ence Foundation of China (Nos. 81730056, 81722027,
81273339, 11801435 (MS)). The funders had no role in the
study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
AUTHOR CONTRIBUTIONS
Ruina Liu, Qi Wang and Zhenyuan Wang contributed to the
conception and design of the work. Ruina Liu, Haohui Zhang,
Di Wu, Kai Yu, Wumin Cai and Gongji Wang contributed to the
acquisition of data. Ruina Liu, Yuexi Gu and Mingwang Shen
contributed to the analysis and interpretation of data. All co-
authors contributed to the critical revision of the manuscript.
All co-authors contributed to the final approval of the manu-
script. Ruina Liu and Yuexi Gu contributed to the drafting the
article. All the authors fulfil the ICMJE criteria for authorship.
DATA AVAILABILITY STATEMENT
The data sets used and analysed during the current study
are available from the corresponding author on reasonable
request.
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APPENDIX
Mathematics
The deviation of support vector regression mode
1.Prediction function the more accurate this
prediction is
f ðxi Þ = wT xi + b:
2.Original problem
This is an SVM regression problem with the pipeline, and
the objective function is
1 Xn
min kwk2 + C lε ð f ðxi , yi ÞÞ
w,b 2
i=1
The loss function of this problem is defined as
18 R. Liu et al.
  
0, ifj f ðxi Þ −yi j ≤ ε _ ^ _ ^ _ ^
lε ð f ðxi , yi ÞÞ = L w, b, ξ i , ξi , αi , αi , βi , βi
j f ðxi Þ−yi j −ε, otherwise
X
n 
^ _
 X
n 
^ _

The quadratic programming problem is obtained as = αi −αi yi −ε αi −αi
i=1 i=1

n 
X _  1X n X n 
^ _
 ^ _ 
1 ^ − αi − αi α j − α j xTi x j
min_ kwk2 + C ξi + ξ i 2 i=1 j=1
^
2 i=1
w, b,ξ, ξ
_
Then, the dual problem of the original problem is
s:t: f ðxi Þ− yi ≤ ε + ξi
^
yi −f ðxi Þ ≤ ε + ξi X
n   X
n  
^ _ ^ _
_ max
_ ^
αi −αi yi −ε αi −αi
αi , αi i = 1
ξi ≥ 0 i=1
^
1 X n X n   ^ _ 
ξi ≥ 0 −
^ _
αi − αi α j − α j xTi x j s:t:
2 i=1 j=1
By introducing Lagrange multipliers, the Lagrangian X
n 
^ _
 _ ^
αi −αi = 0 0 ≤ αi ≤ C 0 ≤ αi ≤ C
function is constructed as
i=1

 _ _ _ 
^ ^
^
L w, b, ξi , ξi , αi , αi , βi , βi The KKT condition needed to satisfy is

n  _  X   8
1 X ^
n _ _
>
_

= kwk2 + C ξi + ξ i + αi wT xi + b −yi −ε −ξi >

> f ð x Þ − y ≤ ε + ξ
>
> i i i
2 >
> ^
i=1 i=1
>
> yi − f ðxi Þ ≤ ε + ξi
Xn   X n _ _
X
n >
>
^ ^ ^ ^ >
> _
+ αi yi −wT xi − b− ε− ξi − βi ξ − βi ξ i >
> ^
>
i=1 i=1 i=1 > , ξi ≥ 0
>
>
ξ i
>
> _
>
> ^
> αi , αi ≥ 0
>
Then, the dual problem of the original problem is >
> _
>
> ^
max_α ,α^ ,β_ ,β^ minw,b,_ξ ,^ξ . Deriving the partial derivation of >
> βi ,βi ≥ 0
>
>  
i i i i i i
>
> _
_ >
>
_
^
> αi wT xi + b −yi − ε−ξi = 0
>
L with respect to variables w,b, ξi , ξi , we can obtain that >
>
>
>
<^  ^

Xn Xn αi yi −wT xi −b −ε− ξ i
∂L _
>  
=w+ αi x i −
^
αi x i = 0 >
>
∂w >
> Pn _

i=1 i=1 >

> w =
^
α −α i xi
>
>
i
∂L X n _ X n >
> 
i=1

αi −
^
αi = 0 >
>P
= >
>
n _
∂b i = 1 >
>
^
αi − αi = 0
i=1 >
>i =1
_ >
>
∂L _
>
> _
_= C −αi −β i = 0
>
>
_
>
> C = α + β
∂ξi >
>
i i
>
> ^
>
> C =
^
α + β
∂L ^ >
>
i i
= C −αi − βi = 0
^
>
> _
^ > ^
> αi αi = 0
∂ξi >
>
>
> _
: ^
βi βi = 0
Thus, it is deduced that

n 
X _
 3. Prediction function
^
w= αi −αi xi
Finally, the form of the prediction function is
i=1
n  _
X 
^
αi −αi = 0 f ðxÞ = wT x + b
n  
i=1
_ _ X ^
_

C = αi + β i = αi −αi xi T x + b
^
i=1
^
C = αi + β n 
X _

^
= αi −αi κðxi , xÞ + b
Substituting it into L i=1

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology
 Microbiome and AI in death time estimation 19
In this study, the kernel function is taken as a linear Supporting Information
kernel function κ(xi, x) = xiTx. Hence, the prediction func-
  Additional Supporting Information may be found in the online
P
n
^
_

tion is f ðxÞ = αi − αi xi T x + b , and the vector xi satis- version of this article at the publisher’s web-site:
i=1
_ Appendix S1: Supporting Information
^
fied to αi −αi 6¼ 0 is the support vector.

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology