Final Report on the Analysis of Samples
Submitted by Tyler Huggins
Wildlife Forensic DNA Laboratory Case File 12-019
Information
The samples were submitted
by Tyler Huggins for species identification.
Purpose
At
the request of Mr. Tyler Huggins, we tested the samples listed in the Items Receipt section
to
determine the species of origin, sex
and individual mitochondrial and
microsatellite profiles.
Items Receipt
The following items were received from Mr. Huggins via Canada Post Express (Waybill: LT 731
704 091 CA) on May 1st,
2012:
Item Label
Description
Laboratory ID
#1 Hide
with
hair Huggins_1
The
following items were received from Mr. Huggins via Canada Post Xpresspost (Tracking number (LT 683 902
055 CA) on August 2, 2012:
Item Label Description
Laboratory ID
S000072569
|
Hair
and hide in vial with clear liquid
|
Huggins_2
|
S000072565
|
Hair
and hide in vial with clear liquid
|
Huggins_3
|
The
following items
were received on
October 5, 2012:
Item Label Description
Laboratory ID
S000072570
|
Bristle
swab in lysis
buffer
|
570
|
S000072576
|
Bristle
swab in lysis
buffer
|
576
|
S000072578
|
Bristle
swab in lysis
buffer
|
578
|
The
items were placed in
a secure
evidence room of the
DNA
Building, Trent University on receipt.
Summary of Results
Laboratory
ID
|
Species Identified from
mitochondrial analysis
|
Gender
|
Individual DNA Profile
|
Human Control
Region Haplotype
|
Huggins_1
|
Ursus americanus &
Homo sapiens
|
Black bear
Female;
no human gender
|
Black bear microsatellite
profile at 14/15 loci No human microsatellite profile
|
Haplotype A
|
Huggins_2
|
Ursus americanus
|
n/a
|
n/a
|
n/a
|
570
|
Homo sapiens
|
n/a
|
n/a
|
Haplotype A
|
576
|
Homo sapiens
|
n/a
|
n/a
|
Haplotype A
|
578
|
Homo sapiens
|
n/a
|
n/a
|
Haplotype A
|
Evidence Disposition
The
samples were consumed
for
DNA extraction; the DNA has been retained for
further
analysis.
Examination
Huggins_1
On initial examination, the sample consisted of a piece of hide with hair attached.
The whole tissue was treated to extract DNA. The extracted DNA was processed
to determine the species of origin,
mitochondrial control region
haplotype, gender and individual microsatellite
ID.
Huggins_2
On initial examination, the
sample consisted of a piece of hide with hair attached. A single hair was treated to extract DNA. The extracted DNA was processed to
determine the species of origin.
570, 576 & 578
On initial examination, the samples consisted of bristly swabs in vials with lysis buffer. The samples were treated to extract DNA. The extracted DNA was processed to
determine the human mitochondrial control region.
RESULTS
DNA
Extraction
and
Quantification
DNA was extracted from the samples using
QIAamp (Qiagen) extraction protocol and extracted
DNA was quantified using
a fluorometer-based Picogreen assay. A total amount of 300 ng of DNA was
extracted
from sample
Huggins_1. A total amount
of
3
ng
of
DNA was
extracted
from
sample
Huggins_2. The total amount of DNA extracted from samples 570, 576, and 578 is approximately 62.5
ng
each.
Mitochondrial DNA Analysis
– Sample: Huggins_1
To determine the species of origin for sample Huggins_1, mitochondrial DNA was amplified using universal mammalian cytochrome b primers, black bear species specific control region primers
and human specific control region primers (Figure A3 and Figure A4). The cytochrome b region
sequence and bear specific
control region sequence generated from sample Huggin_1 demonstrated low
sequence divergence and statistically significant grouping with control Black Bear (Ursus americanus)
sequences from the
National Centre
of Biotechnology
Information (NCBI) database. The human
specific control region sequence generated from sample Huggin_1 demonstrated low sequence divergence and
statistically significant grouping with control human
(Homo sapiens) sequences from the
National Centre
of
Biotechnology Information
(NCBI) database.
Based on the amount of human and black
bear
mitochondrial DNA amplification,
it was
estimated that approximately
10%
of
the functional
mitochondrial
template DNA
from sample
Huggins_1 originated from Black Bear (Ursus americanus) (Figure A1) and approximately 15% of the
functional mitochondrial
template
DNA
from sample Huggins_1 originated
from human
(Homo
sapiens) (Figure A2).
Analysis of the human mitochondrial control region sequence obtained from Huggins_1 (based
on 402 bp consensus based on forward and reverse of
the
mtDNA HVS1 non-coding region) was 100% identical at sites 15999-16400
of the complete mtDNA genome of Accession
#JQ705199
(used in the
publication by Behar et al., 2012). Further analysis indicates that it is a European haplotype; it occurs
with
highest frequency in
East Europe (11%) and Caucasus (10%) (i.e. it initially originated from near
the Caucasus
mountains
regions between the
Black
and Caspian Seas). It
is
not known to
have originally occurred in East Asia, Southeast Asia, Australia, Oceania (i.e. New
Zealand, Papua New
Guinea, etc.), North America,
South
America or Central America.
Nuclear DNA Analysis – Gender
Determination
–
Sample: Huggins_1
To determine
the gender of the
originating
animal for sample Huggins_1, the X- and Y- specific
alleles of the amelogenin gene were amplified using both bear specific and human specific primers. There was amplification of Huggins_1 DNA using bear specific primers (Figure A5). However, there
was
no amplification
of Huggins_1 nuclear DNA using the human specific
primers. To
ensure that the
black bear specific amelogenin primers were not amplifying
human DNA in the sample, control human DNA was also amplified. As seen
in Figure A6,
there was no amplification of human nuclear DNA in sample Huggins_1. Therefore, it was determined that nuclear DNA from
sample Huggins_1 originated from a female
Black Bear (Ursus americanus).
Nuclear DNA Analysis – DNA Profile –
Sample: Huggins_1
Nuclear DNA from Huggins_1 was amplified at the following 15 microsatellite loci using black
bear specific primers: G10J, G10B, G1D, G10H, G10L, Amelogenin, MU05, G1A, G10C, G10U, G10X, MSUT6, G10M, MU59, and MU50. The sample produced a profile at 14 out of the
15 loci (Figure
A7.1 and Figure A7.2).
Nuclear DNA from Huggins_1 was amplified at the following 16 microsatellite loci using human specific primers: D8S1179,
D21S11, D7S820, CSF1PO, D3S1358,
TH01, D13S317, D16S539,
D2S1338, D19S433, vWA, TPOX, D18S51, Amelogenin, D5S818, and FGA. The sample did not
produce a microsatellite profile at any of the 16 loci. Based on the amount of human nuclear DNA
amplification,
it
was estimated that the total amount of functional nuclear template human DNA from
sample Huggins_1 is considerably less
that 15%, as derived from the estimation
of
mitochondrial DNA.
Mitochondrial DNA Analysis
– Sample: Huggins_2
To
determine the species of origin for sample Huggins_2 mitochondrial DNA was amplified
using black bear species specific
cytochrome b primers (Figure A8) and human species specific control
region primers
(Figure A9). The human DNA amplified to
trace
levels and did
not generate sufficient
DNA
to derive a DNA sequence. The cytochrome b sequence generated from
sample Huggins_2
demonstrated low sequence divergence and statistically significant grouping
with control Black Bear (Ursus americanus)
sequences
from
the National
Centre of Biotechnology Information
(NCBI) database.
Based on the amount of human and black
bear
mitochondrial DNA amplification,
it was
estimated that approximately 10% of the functional template DNA from sample Huggins_2 originated
from Black Bear (Ursus americanus) (Figure A8). Due to the presence of trace amounts of amplified
human
DNA, the amount of functional human DNA was estimated to
be less than 0.1% (Figure A9).
Mitochondrial DNA Analysis
– Samples: 570, 576,
578
To determine the control region sequence for samples 570, 576, and 578, mitochondrial DNA was amplified using human species specific control region primers at 30 cycles (Figure A10). DNA from the laboratory technician (labeled “Lab Tech”) who conducted the tests was also amplified. The
human
specific
control region
sequence
generated
from sample
570, 576, 578, and Lab Tech
demonstrated low sequence divergence and statistically significant grouping
with
control human (Homo
sapiens) sequences from the National Centre of Biotechnology
Information (NCBI) database.
Based on 423 bp consensus sequence based on forward and reverse of the mtDNA HVS1 non-
coding region, DNA obtained from samples 570, 576, and 578 cannot be excluded as originating from
the same source. Furthermore, human
mitochondrial
DNA obtained
from
Huggins_1 cannot be
excluded as originating from the same source as 570, 576 and 578. Based on 7 single nucleotide
differences,
DNA
from sample Lab Tech can be
excluded as the human
DNA
in Huggins_1.
It
is concluded that the species of origin that is the major contributor of nuclear and
mitochondrial DNA in sample Huggins_1 is a
female black bear, Ursus americanus. The sample gave a
microsatellite profile. The sample also yielded human mitochondrial DNA with a
control region sequence. Huggins_1 gave no
detectable nuclear DNA. Huggins_2, a single hair, gave primarily black bear mitochondrial DNA. There was only a trace of human
mitochondrial DNA
compared to Huggins_1. Human mitochondrial DNA obtained
from samples Huggins_1, 570, 576, and 578 cannot be excluded as originating
from the
same source; however, DNA from sample Lab
Tech can be excluded as being present in Huggins_1 .
Signed
Tasnova Khan, MSc Dr.
B. White, PhD Forensic Scientist Forensic Supervisor November 15,
2012 November 15,
2012
Appendix 1: Technical Section
DNA (deoxyribonucleic acid)
Living organisms are made of
cells
and within most
of their
cells
are chromosomes made up
of
deoxyribonucleic acid (DNA). DNA is the blueprint that encodes all the materials needed for the development and function of each unique organism.
Strands of DNA are made up of four different building blocks known as nucleotides, a DNA sequence
can
be determined by knowing the order in which these nucleotides are arranged. While the majority of the nucleotide
sequence does not vary
between organisms, small portions vary significantly enough
that identification
of
species and individuals within a species can be carried out.
DNA Extraction and Quantification
DNA was extracted from the case items using a QIAamp (Qiagen) extraction protocol. Extracted DNA
from the case items was quantified using a fluorometer-based picogreen assay on the BMG FluoStar Galaxy 96- well plate system.
Mitochondrial DNA
For species identification the control region and cytochrome b for Ursus americanus and the control
region for Homo sapiens within the mitochondrial DNA were amplified from the case sample. For
the cytochrome
b and control region for Ursus americanus, species specific primers which are designed to amplify the cytochrome
b and control region in Ursus americanus
were used. For the control region for Homo sapiens, species specific primers which are designed to amplify the control region in Homo sapiens were used. The amplification products
were separated on an agarose gel to visualize
results. If amplified DNA was produced,
it is
then sequenced on an
ABI 3730 DNA Analysis System.
The
cytochrome b and control region sequences were then compared to several cytochrome b and control region sequences, originating from control sequences of Ursus americanus and Homo sapiens. Phylogenetic analysis was subsequently carried out to show which of the sequences within the database was most closely related to those from the case sample, thus indicating
the
species of origin. Sequences were analysed using the software programs Sequence Analysis 5.4 and the phylogenetic software package MEGA 4
and
the NCBI BLAST database.
Gender Identification
DNA from the case item was amplified at the X- and Y- specific alleles of the amelogenin gene.
The
amplified products were separated on an agarose gel. The gender-specific amplified products were separated on
an ABI 3730 DNA Analysis System and sized using GeneMarker 1.9 software.
Individual Identification – DNA Profiling
Within DNA are stretches of short repeated sequences. The length of these stretches varies depending on
the number of copies of the repeat found at a specific region (locus) of the DNA. In DNA profiling relies on the fact that the number of repeats at a locus varies between individuals, the different variations (number of repeats)
are
known as alleles. For any given locus an individual will have a
certain number of repeats (a certain allele).
Since animals inherit one copy of each chromosome from each parent, it means that the individual has two alleles for each locus.
Scientists have designed a method that allows them to probe these specific loci on animal chromosomes, and determine which alleles an individual has at each locus. The result is a distinctive pattern of alleles for the loci that were used.
A locus by itself is usually not unique to an individual;
if,
however, more loci are included, the odds are greater that the samples
are
from the same animal. A Partial profile occurs when not all the loci tested produce a marker giving an incomplete profile for the sample.
With fewer loci
available for comparison it has a lower capability to distinguish between individuals. For a sample to be excluded as originating from the same individual as another sample the profiles must differ at more than one locus.
The DNA from the case items was amplified at
16 human loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, Amelogenin, D5S818, and FGA. Amplified products
were separated on an ABI 3730
DNA
Analysis System and sized
using GeneMarker 1.9 software.
Appendix 2: Data
Figure A2: Amplified product of control human DNA and DNA from sample Huggins_12-019_1 using Homo sapiens specific control region PCR primers at 30 cycles
Figure A3: Screenshot of a portion of the sequence obtained from Huggins_1 using Ursus americanus specific control region PCR primers
Figure A4: Screenshot of a portion of the sequence obtained from Huggins_1 using Homo sapiens specific control region PCR primers
Figure A6: Allele report for gender test using black bear specific amelogenin primers. Sample 1: Female human sample. Sample 2: Male human sample. Sample 3: Huggins_1 sample.
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