The author(s) shown below used Federal funding provided by the U.S. 

Department of Justice to prepare the following resource: 

Document Title: Evaluating the Skin Microbiome as Trace 

Evidence 

Author(s): Rob Knight, Jessica L. Metcalf, Jack Gilbert, 

David O. Carter 

Document Number: 251647 

Date Received: June 2018 

Award Number: 2014-R2-CX-K411 

This resource has not been published by the U.S. Department of 

Justice. This resource is being made publically available through the 

Office of Justice Programs’ National Criminal Justice Reference 

Service. 

Opinions or points of view expressed are those of the author(s) and 

do not necessarily reflect the official position or policies of the U.S. 

Department of Justice. 
 

 

 

 

 

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Final Summary Overview

October 1, 2015** – September 30, 2017

Award number 2014-R2-CX-K411

Evaluating the skin microbiome as trace evidence

Rob Knight1, Jessica L. Metcalf1,2+, Jack Gilbert3,4, David O. Carter5

1 – University of California San Diego, San Diego, CA

2 – University of Colorado, Boulder, CO

3 – University of Chicago, Chicago, IL

4 – Argonne National Laboratories, Chicago, IL

5 - Chaminade University, Honolulu, HI

**Due to a move from CU Boulder to UC San Diego, this grant started October 1, 2015.**

+Co-PI Metcalf joined the faculty at Colorado State University in Fall of 2016.

PURPOSE

The purpose of this project was to characterize basic transfer properties of an individual’s unique

skin microbial community to common surface materials, and to assess whether it is possible to

characterize an individual’s skin microbial handprint after a person is deceased. We had the

following specific objectives in mind.

Objective 1: Determine whether the sequence in which surfaces are touched by the same person

influences the detection of an identifiable skin microbial signature.

Objective 2: Determine whether the number of times a surface is touched by the same person

influences the detection of an identifiable skin microbial signature.

Objective 3: Determine whether individual skin microbial signatures are recoverable after

multiple people have touched an object.

Objective 4: Determine the stability of microbial signatures on surfaces over time.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
 

 

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Objective 5: Determine the magnitude of change in microbial skin communities during morgue

cooler storage (relative to initial samples at death scene).

PROJECT SUBJECTS

Subjects consisted of adult volunteers who were recruited through email listservs and

announcements at the University of California San Diego through IRB protocol #150846.

PROJECT DESIGN AND METHODS

The experimental design for Objectives 1-4 is outlined in Figure 1.

Prior to all experiments, we sprayed desk surfaces in the room with a 10% bleach solution and

allowed them to soak for 15 minutes before wiping off. Next we placed tiles on the bleached

desk surfaces and sprayed them with 10% bleach, and allowed them to soak for 20 minutes

before wiping off and rinsing with HPLC purified water.

Objective 1: The experiment was conducted with 13 participants touching 5 material types

(plastic, glass, ceramic, metal, and wood; Figure 1A). Each material type was touched in sets of

5 replicates in consecutive order, each touched 10 times. Tiles were swabbed immediately

thereafter.

Objective 2: On 3 separate days, plastic tiles were touched either 10 times, 20 times, or 30 times

by 20 participants (Figure 1B). Tiles were swabbed immediately thereafter.

Objective 3: The experiment was conducted with 6 pairs of participants on both plastic and

ceramic tiles since this material type has shown to be the most robust for recovering personalized

skin microbes. The first person in the pair touched a tile 30 times followed by the second person

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
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touching it 20 times an hour later (Figure 1D). The following day, the order of participants was

reversed. Tiles were swabbed immediately thereafter.

Objective 4: 12 participants each touched a plastic tile 20 times, rotating the tile between

touches such that each quadrant of the rectangular tile were touched equally by each part of the

hand (Figure 1C). One quadrant of the tile was sampled immediately following the 20 touches.

A second quadrant was sampled after five minutes, while the third and fourth quadrants were

sampled after 1 and 18 hours, respectively. A second set of experiments using just plastic tiles

was conducted to increase the sample size and extend the persistence time to several days.

Objective 5: In collaboration with the Honolulu Medical Examiners office, samples were

collected from deceased individuals and surrounding surfaces at five death scenes. Samples were

then collected again after arrival at the morgue, and continuously at 6 hour intervals until an

autopsy was performed.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
 

 

 

 

 

 

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Figure 1. Schematic describing the four skin trace evidence experiments described in Phases 1-4. (A) Five replicates

each of common surface materials, including ceramic, glass, metal, plastic and wood, were contacted with each

participant’s hand ten times. (B) The effect of the number of times each surface replicate was touched on the

accuracy of linking a surface to a participant was tested. (C) The length of time personalized skin microbes persisted

on a surface material was tested. (D) The ability to link personalized skin microbes to participants when a surface is

touched by two people (e.g. victim and perpetrator) was assessed.

Sample collection and data generation: To characterize each participant’s unique skin

microbial signature, skin from each participant’s hand was swabbed for five days leading up to,

during, and/or following the experiment. BD BBLTM CultureSwabTM (Becton Dickinson, USA)

sterile swabs were used to collect microbial DNA from participants’ hands and surfaces. Swabs

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
5

were immediately frozen at -20C until DNA extraction. Bacterial communities were

characterized by extracting DNA, amplifying the 16S rRNA gene, and sequencing on an

Illumina platform (MiSeq and HiSeq) following standard protocols

(http://www.earthmicrobiome.org/protocols-and-standards/).

DATA ANALYSIS

● Sequence data were processed using tools in the QIIME (v.1.9.1.) software package.

Processing included steps to assign sequences to samples, select out putative error-free

sequences, and rarefy sequences to an even depth such that each sample is represented by

the same number of sequences.

● We applied machine learning methods using a Random Forests classifier to match

participants to the tiles they touched.

● To detect the pairs of participants in the multiple-person experiment (Objective 3), we

used a Bayesian approach called SourceTracker (Knights et al. 2011) to estimate the

relative contribution of the top two individuals identified as sources to the microbial

profile of the sample. 

FINDINGS

● Skin microbes detected on ceramic and plastic surfaces were most robustly and

accurately linked to individuals (Figure 2).

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
 

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Figure 2. Prediction accuracies for material types that were touched by participants in sets of 5, each touched 10

times. The first replicate for each material type was the most accurate (blue) for predicting the correct participant 

with plastic and ceramic demonstrating the highest overall accuracy.

● Unlike in our first experiment, our ability to recover enough sequence data from surfaces

touched 10 times was poor in a second experiment in which we tested touch frequency;

only surfaces for 3 out of the 20 participants yielded enough sequence data for analysis.

However, 12 and 13 of the 20 surfaces touched 20 or 30 times respectively, successfully

sequenced. We have determined that our ability to recover enough signal to correctly

match the person with the tile they touched increases with 20 and 30 touches when

compared to 10 touches (Figure 3).

Figure 3. A heatmap showing the probability that a given individual contacted each plastic surface (dark = high

probability). An outline in green indicates a correct match; if the prediction misclassifies, an outline in blue indicates

the true donor and red indicates the misclassified prediction. The top 3 rows shows accuracies for the surfaces

touched 10 times, the following 13 rows shows accuracies for the surfaces touched 20 times, and the last 12 shows

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
 

 

 

 

 

 

 

 

 

 

 

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accuracies for the surfaces touched 30 times. The prediction accuracy increases from 0.67 for 10x (2/3) and 0.69 for

20x (9/13), to 0.83 for 30x (10/12).

● We found that we could recover the skin microbial signatures of one or both people in

many cases, and that some individuals leave a highly traceable signature against a

background of someone else’s signature while others do not (Figure 4).

Figure 4. Sourcetracker analysis shows the relative contribution of the top two individuals identified as sources to

the microbial profile of the sample. P1 and P2 refer to the first and second participant respectively. ‘Other’ indicates

that a participant in the experiment other than the two people who contacted the tile was identified as contributing to

the microbial profile of the sample. The four bars show alternating results for ceramic and plastic, respectively, with

the first set of bars showing the instance when P1 touched the surface first and the second set of bars showing the

instance when P2 touched the surface first. In most cases, the microbial signature of the person to last touch the

surface (P2: blue bar)) was most robustly detected while in a few cases signatures from from both P1 and P2 were

detected. However, one person’s skin microbiome signature was sometimes “dominant”, and would more accurately

match the surface microbes regardless of the order in which the surface was touched.

● We found that a person’s microbial signature can persist on a surface for at least one day.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
 

 

 

 

 

8

Figure 5. A heatmap showing the probability that a given individual contacted each plastic surface (dark = high

probability), arranged by time elapsed. An outline in green indicates a correct match; if the prediction misclassifies,

an outline in blue indicates the true donor and red indicates the misclassified prediction. The rows are ordered by

individual and time (e.g. the first 4 rows are all surfaces touched by the same individual, sampled after 1 second, 5

minutes, 1 hour, and 18 hours).

● We found that the skin microbial communities on a deceased individual, whether

sampled at earlier or later hours postmortem, still share unique/individualized microbial

communities found on that individual’s personal objects (Figure 6).

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
 

 

 

 

9

Figure 6. Samples from deceased individuals postmortem and their associated objects. Samples are colored by

individual. The largest spheres are of the personal objects, medium-sized spheres are of the skin swabs across all

time points (at scene of death, upon arrival at the morgue, and then at 6 hour intervals until an autopsy was

performed), and the smallest spheres are of the plastic sheets covering the deceased body.

IMPLICATIONS FOR CRIMINAL JUSTICE POLICY AND PRACTICE IN THE

UNITED STATES

By revealing new information about the transferability, stability, and individuality of human skin

microbiomes, this forensic science research has increased our knowledge of physical evidence,

providing basic information about the conditions under which criminal investigators can match

an individual’s skin microbial signature to objects or surfaces at crime scenes. This information

is a crucial first step to further developing these technologies such that they can be used by

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
10

general crime laboratories in the future. The current results are particularly exciting for criminal

investigation because they demonstrate that skin microbes can also serve as individual evidence

in a variety of scenarios. Most trace evidence, such as hairs and fibers, can only be used as class

evidence as opposed to individual evidence. The recovery of a blonde hair shaft, for example,

from a crime scene could be associated with anyone with blonde hair. Hair shafts very rarely

possess characteristics that are unique to an individual. Fibers provide similar information. A

blue cotton fiber recovered from a death scene could not be definitively linked to a blue cotton

sweatshirt owned by a suspect, for example. Therefore, using skin microbes to identify

individuals significantly expands the scope and power of trace evidence analysis. Our results also

reveal two candidate surface types, plastic and ceramic, to focus technology development for

recovering personalized skin microbial signatures as trace evidence at crime scenes.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.