Identification of forensically important Diptera from...
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Identification of forensically important Diptera from
Belgium and France using the Barcode of Life Data
Systems (BOLD) and GenBank
Gontran Sonet1, Kurt Jordaens2,3, Yves Braet4, Luc Bourguignon4, Eréna Dupont4,
Thierry Backeljau1,3, Marc De Meyer2 & Stijn Desmyter4
4 National Institute of
Criminalistics and Criminology,
Belgium
1 & 2 JEMU – Joint Experimental Molecular Unit,
Royal Belgian Institute of Natural Sciences &
Royal Museum for Central Africa, Belgium
3 University of Antwerp,
Belgium
Lucilia caesar (Linné 1758)
© Krzysztof Szpila, Nicolaus Copernicus University, Torun
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INTRODUCTION Forensic entomology
© Crime Scene Unit of South-Western Finland, Turku Police Department
Pohjoismäki et al. Forensic Science International 2010
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INTRODUCTION Dipteran species of forensic interest in Western Europe
Calliphoridae
Calliphora vicina Robineau-desvoidy, 1830
© jorgemotalmeida Viseu Portugal Calliphora vomitoria (Linnaeus, 1758)
© Roger Thomason Mossbank UK
Lucilia sericata
(Meigen 1826)
© Alvesg Lisbon Portugal
Chrysomya albiceps
(Wiedemann, 1819)
© Nikita Vikhrev Moscow Russia
Protophormia terraenovae
(Robineau-desvoidy, 1830)
© jari.flinck Helsinki Finland
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Muscidae
Muscina levida (Harris 1780)
© Tim worfolk Devon UK
Musca autumnalis De Geer, 1776
© Nick Upton Wiltshire UK
INTRODUCTION Dipteran species of forensic interest in Western Europe
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Sarcophagidae
Sarcophaga spp.
INTRODUCTION Dipteran species of forensic interest in Western Europe
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Reference dataset
INTRODUCTION DNA-based specimen identification
sp1
sp2
sp3
Unknown species
Query
sequence
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INTRODUCTION Validation of a reference dataset
Lucilia sericata
Protophormia
terraenovae
Reference sequences
available in BOLD
New in
this
study
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Reference dataset
INTRODUCTION Validation of a reference dataset
sp1
sp2
sp3
sp4
New reference
sequence
Before applying the DNA barcoding identification method to
samples of a certain location, it is necessary to validate the method
with samples from that specific location.
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Material & Methods Specimens
85 specimens from 27 localities in Belgium and France
Adults identified using morphology 16 species of forensic interest
Calliphoridae 14 Calliphora vicina Robineau-desvoidy, 1830
16 Calliphora vomitoria (Linnaeus, 1758)
7 Chrysomya albiceps (Wiedemann, 1819)
1 Cynomya mortuorum (Linnaeus, 1761)
2 Lucilia ampullacea Villeneuve, 1922
16 Lucilia sericata (Meigen 1826)
12 Protophormia terraenovae (Robineau-desvoidy, 1830)
Muscidae 1 Eudasyphora cyanella (Meigen, 1826)
1 Musca autumnalis De Geer, 1776
5 Muscina levida (Harris 1780)
5 Muscina prolapsa (Harris, 1780)
1 Neomyia cornicina (Fabricius, 1781)
1 Polietes lardarius (Fabricius, 1781)
Fanniidae 1 Fannia sp1
1 Fannia sp2
1 Fannia sp3
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Material & Methods Searches in BOLD and GenBank
Barcode fragment (642-658 bp):
85 sequences,
42 haplotypes
GenBank
BOLD: Public Records
341 580 sequences;
45 368 nominal species
11 732 interim species 24 May 2013
BOLD: Species Level Records
(including early releases)
1 367 662 sequences
127 679 species
53 394 interim species 24 May 2013
+ keyword
“barcode”
1 2 3 4
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Material & Methods Searches in BOLD and GenBank
Best matches
>99% similarity
with query
(max. 100)
Correct match
Incorrect match
Query
Ambiguous
identification
Unambiguous
identification
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Results Local reference dataset
1st COI records
Muscina prolapsa (Harris, 1780)
© cgm crex Sweden
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Cynomya mortuorum (Linnaeus, 1761)
© brundlefly
Results Local reference dataset
2nd COI record
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Neomyia cornicina (Fabricius, 1781)
© Hakon Cyprinoid Norway
Polietes lardarius (Fabricius, 1781)
© Nick Upton Wiltshire UK
Eudasyphora cyanella (Meigen, 1826)
© Tim Worfolk Devon UK
Potential forensic interest
Results Local reference dataset
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Search procedure: 1 & 2 3 4
Matches > 99% similarity
(% of queries):
86%
95%
62%
(% of species): 69% 88% 31%
Results and Discussion Searches in BOLD and GenBank
References considered in the search procedures:
1&2 Public records (GenBank & BOLD)
3 Public + early releases (BOLD)
4 Records labelled as BARCODE (GenBank)
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Search procedure: 1 & 2 3 4
Presence of correct best
match(es) when > 99%
similarity :
100% 100% 100%
Ambiguous identifications
(% of queries):
50%
62%
27%
(% of species): 27% 43% 20%
Results and Discussion Searches in BOLD and GenBank
References considered in the search procedures:
1&2 Public records (GenBank & BOLD)
3 Public + early releases (BOLD)
4 Records labelled as BARCODE (GenBank)
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6
64
3 1 0
10
20
30
40
50
60
70
incorrect
correct 1&2 3 4
Results and Discussion Searches in BOLD and GenBank
Number of best matches > 99%
obtained for Calliphora vomitoria Search procedure:
1&2 Public records (GenBank & BOLD)
3 Public + early releases (BOLD)
4 Records labelled as BARCODE (GenBank)
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10
55
5 2
2
2
0
10
20
30
40
50
60
incorrect
correct 1&2 3 4
Results and Discussion Searches in BOLD and GenBank
Muscidae ??
=
Number of best matches > 99%
obtained for Calliphora vicina Search procedure:
1&2 Public records (GenBank & BOLD)
3 Public + early releases (BOLD)
4 Records labelled as BARCODE (GenBank)
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2
16
2 4
5
0
5
10
15
20
25
incorrect
correct
1&2 3 4
Results and Discussion Searches in BOLD and GenBank
Lucilia caesar
& L. illustris
=
Number of best matches > 99%
obtained for Lucilia ampullacea Search procedure:
1&2 Public records (GenBank & BOLD)
3 Public + early releases (BOLD)
4 Records labelled as BARCODE (GenBank)
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Results and Discussion Searches in BOLD and GenBank
15
4
3
0
20
40
60
80
100
120
incorrect
correct
1&2 3 4
Lucilia
cuprina
=
Number of best matches > 99%
obtained for Lucilia sericata Search procedure:
1&2 Public records (GenBank & BOLD)
3 Public + early releases (BOLD)
4 Records labelled as BARCODE (GenBank)
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Conclusion
Validating public reference libraries using reference sequences of the
local entomofauna allowed us to:
1. make sure that the local entomofauna is well represented in the
reference library
2. “red flag” cases of unreliable identifications and
3. determine ways to address each problematic identification:
• correction of mislabelled reference sequences (dynamic libraries)
• adaptation of the identification method (distance threshold)
• usage of alternative markers (hybridisation, introgression, etc.)
Value of the Species Level records of BOLD
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Acknowledgements
The authors would like to thank:
• you for your attention,
• the organisers of the conference and
• the Belgian Science Policy Office (Belspo)