OSAC 2022-S-0037 Standard for DNA-Based Taxonomic

Identification for Forensic Entomology

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OSAC 2022-S-0037

Standard for DNA-Based 

Taxonomic Identification in 

Forensic Entomology

Crime Scene Investigation & Reconstruction Subcommittee

Scene Examination Scientific Area Committee

Organization of Scientific Area Committees (OSAC) for Forensic Science
OSAC 2022-S-0037 Standard for DNA-Based Taxonomic

Identification for Forensic Entomology

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Draft OSAC Proposed Standard

OSAC 2022-S-0037

Standard for DNA-Based Taxonomic

Identification in Forensic Entomology

Prepared by 

Crime Scene Investigation & Reconstruction Subcommittee

Version 1.0

February 2023

Disclaimer:

This OSAC Proposed Standard was written by the Organization of Scientific Area Committees 

(OSAC) for Forensic Science following a process that includes an open comment period. This 

Proposed Standard will be submitted to a standards developing organization and is subject to 

change.

There may be references in an OSAC Proposed Standard to other publications under development 

by OSAC. The information in the Proposed Standard, and underlying concepts and methodologies, 

may be used by the forensic-science community before the completion of such companion 

publications.

Any identification of commercial equipment, instruments, or materials in the Proposed Standard is 

not a recommendation or endorsement by the U.S. Government and does not imply that the 

equipment, instruments, or materials are necessarily the best available for the purpose.
OSAC 2022-S-0037 Standard for DNA-Based Taxonomic

Identification for Forensic Entomology

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Foreword

This standard was developed to provide guidance to scene investigators and scene reconstructionist 

as a standard for DNA-based taxonomic identification in forensic entomology.

This document has been prepared by the Organization of Scientific Area Committees (OSAC) for 

Forensic Science Crime Scene Investigation and Reconstruction Subcommittee.

Keywords: forensic entomology, death investigation, taxonomy, DNA-based identification, polymerase 

chain reaction, DNA sequencing
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Identification for Forensic Entomology

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Table of Contents

1. Scope 5

2. Normative Reference 5

3. Terms and Definitions 5

4. Requirements 9

4.1 Laboratory Procedures 9

4.2 Sample Processing 9

4.3 DNA Extraction 10

4.4 Amplification 10

4.5 DNA Sequencing 10

5. Analysis and Interpretation 11

5.1 General 11

5.2 Taxonomic Identification 11

Annex A (informative) Bibliography 13

Figures

1 Phylogenetic Relationships................................................................................................................5
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1 1. Scope

2 This document outlines the current best practices to be used when employing DNA-based techniques to 

3 identify species, or higher taxonomic categories, of entomological origin for forensic investigation 

4 purposes. Potential applications include the estimation of some portion of the postmortem interval (PMI); 

5 however, methods for estimating PMI after a specimen has been identified are beyond the scope of this 

6 document. 

7 2. Normative Reference

8 There are no normative references.

9 See Annex A, (informative) Bibliography, for other references 

10 3. Terms and Definitions 

11 For purposes of this document, the following definitions and acronyms apply.

12 3.1

13 Allele

14 Any of the forms of the same gene that occur at the same locus on a homologous chromosome but differ in 

15 base sequence (OSAC Lexicon).

16 3.2

17 Amino acid

18 Any class of 20 molecules combined to form proteins in living things; the sequence of amino acids in a 

19 protein is determined by the exons of a gene (OSAC Lexicon).

20 3.3

21 Amplification

22 An increase in the number of copies of a specific DNA fragment; can be in vivo or in vitro (OSAC 

23 Lexicon).

24 3.4

25 Artifact

26 A non-allelic product of the DNA amplification or sequencing process (e.g., amplification of a pseudogene, 

27 non-template nucleotide addition, primer-dimer, or other non-specific product), an anomaly of the detection 

28 process (e.g., single or multichannel voltage spikes, or instrument noise), or a by-product of primer 

29 synthesis (e.g., “dye blob”) that may be observed on an electropherogram. Some artifacts may complicate 

30 the interpretation of DNA profiles when they cannot be distinguished from the actual alleles from a 

31 particular sample (OSAC Lexicon).

32 3.5

33 BLAST search

34 The Basic Local Alignment Search Tool (BLAST) is an algorithm for comparing and finding similarities 

35 between a query biological sequence and sequences deposited into the database and calculates a statistical 

36 significance of similarity. BLAST was developed and is currently run by the National Center for 

37 Biotechnology Information (NCBI) (Altschul et al, 1990).

38
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39 3.6

40 Casework samples

41 Entomological material recovered from the scene or otherwise believed to be associated with a crime (Catts 

42 and Goff 1992).

43 3.7

44 Controls 

45 Samples of known types run in parallel with experimental, reference, or evidence samples that are used to 

46 demonstrate that a procedure is working correctly (OSAC Lexicon). A negative control has no added 

47 template DNA; therefore, a result consistent with DNA present suggests contamination. A positive control 

48 has standard DNA of known genotype added; therefore, a result consistent with the expected genotype 

49 indicates that the protocol is reliable.

50 3.8

51 Deoxyribonucleic acid

52 DNA

53 A genetic material of organisms, usually double-stranded, is a biopolymer composed of nucleic acids,

54 identified by the presence of deoxyribose, a sugar, and the four nucleobases. DNA is a stable molecule; 

55 variations in the DNA sequence between individuals and species permit DNA testing to distinguish 

56 individuals and species from each other (OSAC Lexicon).

57 3.9

58 Electropherogram

59 The graphic representation of the separation of molecules by electrophoresis in which the data appear as 

60 peaks along a line; the format in which DNA typing results are often presented, with the X-axis displaying 

61 the observed allele(s) and the Y-axis recording the relative amount of DNA detected based on the signal 

62 measured in relative fluorescent units (RFU) collected during analysis (OSAC Lexicon).

63 3.10

64 Haplotype

65 A set of linked DNA variations, or polymorphisms, tend to be inherited together (e.g., the insect 

66 mitochondrial DNA molecule) (OSAC Lexicon).

67 3.11

68 Locus (plural loci)

69 A unique physical location of a gene (or specific sequence of DNA) on a chromosome (OSAC Lexicon.)

70 3.12

71 Peak height

72 The maximum Y-axis value obtained for a data peak measured in relative fluorescence units (OSAC 

73 Lexicon).

74 3.13

75 Phred score

76 A Phred quality score is a numerical value associated with each sequenced nucleotide that corresponds to 

77 the quality of that nucleotide that is logarithmically linked to error probabilities. For example, a Phred score 

78 of 10 has a base call accuracy of 90%, and a Phred score of 20 has a base call accuracy of 99% (Ewing et 

79 al., 1998).

80
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81 3.14

82 Polymerase chain reaction

83 PCR

84 A method whereby a specific sequence of nucleotides within a double-stranded DNA is amplified (OSAC 

85 Lexicon).

86 3.15

87 Reciprocal monophyly

88 A monophyletic group consists of a hypothetical common ancestor and all its descendants. Members of the 

89 group can be organisms or genotypes. Depicted on a phylogenetic tree, a monophyletic group is defined by 

90 a single branch from which all members, and no non-member, arise. Reciprocal monophyly exists for two 

91 species when the branches defining each, and no other species, arise from a single node on the phylogenetic 

92 tree. Figure 1 illustrates the terms applied to a taxonomic group according to the phylogenetic relationships 

93 of its member species (Schuh and Brower 2009).

94

95

96

97 Figure 1: Phylogenetic Relationships

98 A hypothetical phylogeny illustrates the classification of a taxonomic group when each member organism 

99 (or genotype) is more related to any other member than it is to any outside species (monophyletic), one 

100 lineage of the group is more related to species outside the group (paraphyletic), or two or more member 

101 lineages are more related to species outside the group (polyphyletic) (Tarone et. al. 2015). 

102 3.16

103 Paraphyly

104 A paraphyletic group contains a hypothetical common ancestor and some, but not all, of its descendants 

105 (Schuh and Brower 2009). 

106 3.17

107 Primers

108 A short polynucleotide chain, usually 18-30 bases long, targets a specific region of the template DNA and 

109 allows a DNA polymerase to initiate the synthesis of a complementary strand; Two primers are required to 

110 amplify a particular section of double-stranded DNA. The primers are complementary to opposite strands 

111 and are designed to bracket the region of interest for amplification. Polymerization is initiated at the 3’ end 

112 of the primer and extends in a 5 ́ to 3 ́ manner (OSAC Lexicon).

113
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114 3.18

115 Protein-coding gene 

116 The sequence of DNA which encodes for a protein. This usually includes a start and stop codon and is 

117 transcribed and translated into a protein (Watson et al., 2014).

118 3.19

119 Pseudogene

120 A DNA sequence that resembles a gene but has been mutated into an inactive form over the course of 

121 evolution. It often lacks introns and other essential DNA sequences necessary for function. Though 

122 genetically similar to the original functional gene, pseudogenes do not result in functional proteins

123 (National Cancer Institute Dictionary).

124 3.20

125 Reference genotype

126 A genotype from a specimen is considered to have been accurately identified to a given taxonomic level 

127 (add to OSAC lexicon).

128 3.21

129 Sequencing

130 A method for determining the order of bases in a DNA molecule. In Sanger sequencing, this is based on the 

131 selective incorporation of chain-terminating dideoxynucleotides by a DNA polymerase during in vitro DNA 

132 replication (Hillis et al. 1996).

133 3.22

134 Sister group(s)

135 A pair of taxa is inferred to be more closely related to each other than to any other taxon (Schuh and 

136 Brower 2009).

137 3.23

138 Stop codon

139 A three-base DNA sequence that codes for or signals the end of the protein sequence. The exact sequence 

140 of nucleotides terminating the protein-coding region depends on the particular genetic locus (Watson et al., 

141 2014).

142 3.24

143 Species

144 The level of taxonomic classification is denoted by a binomial (two-word) name in Latinate form (Notton 

145 2001).

146 3.25

147 Taxonomic identification

148 Analyses to establish the classification of an organism to family, genus, species, etc. These analyses are 

149 based on class characters diagnostic for the taxonomic level in question (OSAC Lexicon).

150 3.26

151 Voucher

152 Biological specimen that is representative of its species in accordance with the relevant taxonomic authority 

153 and is therefore valid for comparative purposes. Voucher specimens are of known identity and are curated 

154 with available associated geographic, field collection, and life-history data (OSAC Lexicon).
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155 4. Requirements

156 4.1 Laboratory Procedures

157 4.1.1 The laboratory shall have and follow procedures for cleaning and decontaminating facilities and

158 equipment.

159 4.1.2 Pre-PCR and post-PCR activities of the laboratory shall be separated by space to avoid 

160 contamination of questioned samples.

161 4.1.3 Equipment and supplies shall not be transferred from post-PCR to pre-PCR areas unless 

162 decontaminated, e.g., using bleach, with associated documentation.

163 4.1.4 Casework and non-casework-related laboratory procedures shall be separated by space or time. 

164 Cleaning and decontamination of the area between procedures shall occur when using the same laboratory 

165 area.

166 4.2 Sample Processing 

167 4.2.1 A detailed description of the specimen, including close-up images with a size scale, shall be 

168 recorded prior to any sample processing for DNA extraction. 

169 4.2.2 When possible, insect morphological characteristics should be preserved and therefore not destroyed 

170 for DNA extraction purposes. For soft larvae, a flap may be cut in the cuticle to minimize the destruction of 

171 surface features when removing internal tissue. For adults with bilateral symmetry, dissection of body parts 

172 for DNA extraction should be attempted first on only one side of the body (for example, one to three legs). 

173 The remains of any specimen used for DNA extraction should be preserved for future examination.

174 4.2.3 Specimens potentially useful for future molecular genetic analysis should be preserved appropriately 

175 (e.g., by freezing or in >90% ethanol). 

176 4.3 DNA Extraction

177 4.3.1 Written protocols shall exist and be available for inspection for all extraction methods used in the 

178 laboratory.

179 4.3.2 Each DNA extraction set shall include at least one reagent blank as a negative control, which is 

180 analyzed concurrently with casework samples.

181 4.3.3 The extraction of DNA from reference material shall be separated by time or space from the 

182 extraction of DNA from casework.

183 4.3.4 When extracted in the same space, samples suspected to have low quantities of DNA should be 

184 extracted before samples suspected to have high quantities of DNA.

185 4.4 Amplification

186 4.4.1 Written protocols shall exist for all amplification methods routinely used in the laboratory.
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187 4.4.2 Primers used for PCR amplification shall be documented in the case file, and primer sequences shall 

188 be available in laboratory documentation.

189 4.4.3 Each PCR run shall include a positive control, a PCR reagent negative control, and the DNA 

190 extraction reagent blank. 

191 4.4.4 All controls shall be amplified concurrently in the same instrument with the casework samples at all 

192 loci and with the same primers as the casework samples.

193 4.4.5 If the analysis includes genotyping reference specimens, these shall not be processed concurrently 

194 with casework specimens.

195 4.4.6 Results from casework samples shall not be accepted unless the positive control produces an 

196 expected genotype and unless the negative control(s) produces no genotype. 

197 4.5 DNA Sequencing

198 4.5.1 Written protocols shall exist for all sequencing methods routinely used in the laboratory.

199 4.5.2 Redundant sequence data, for example, by sequencing both the forward and reverse strand of a PCR 

200 amplicon, should be obtained.

201 5 Analysis and Interpretation 

202 5.1 General

203 5.1.1 Written protocols shall exist for all analysis and interpretation methods routinely used in the 

204 laboratory. These protocols shall include defined data quality indicators (e.g., Phred quality scores, signal 

205 intensities, peak heights). 

206 5.1.2 If contamination in the negative control is present above laboratory-established acceptance 

207 parameters, then the results related to that negative control shall not be used for interpretation.

208 5.1.3 If the evidence genotype includes all or part of the DNA sequence of a protein-coding gene, that 

209 sequence shall be translated to the implied amino acid sequence. A misplaced stop codon or other result 

210 inconsistent with the biological characteristics of the target gene protein will suggest that the genotype is an 

211 artifact or an incorrect locus such as a pseudogene.

212 Protocols covering sequencing shall minimally include:

213 a) the process for nucleotide sequence editing and comparison to reference sequences

214 b) the process by which sequence contamination (e.g., detectable results in the negative controls) is

215 evaluated and documented

216 c) the determination of minimum sequence quality (e.g., the Phred score of a peak on the 

217 electropherogram)

218 5.2 Taxonomic Identification
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219 5.2.1 Identification of a given taxonomic level shall be made by comparison to reference genotypes 

220 considered to be reliable for this purpose. The reference genotype(s) used to identify each specimen shall be 

221 noted in the report for analysis.

222 5.2.2 To be reliable, the reference genotypes shall:

223 a) be associated with voucher specimens (Wheeler 2003)

224 b) have been reported in a scientific publication that describes the specimen characters considered to 

225 be diagnostic of that taxon, either through direct documentation or by reference to the published 

226 taxonomic key, including the species in question that was used to make the identification

227 5.2.3 A BLAST search of the entire genotype database of the National Center for Biotechnology 

228 Information (or comparable database) is acceptable for a preliminary identification, but it shall not be the 

229 sole basis for the final taxonomic determination. 

230 5.2.4 The reference genotypes used for comparison to the evidence genotype should represent a selection 

231 of taxa appropriate for the setting and any tentative morphological identification of the casework specimen. 

232 Knowledge of the local carrion insect fauna is required to do this properly. 

233 This knowledge can include being able to identify a higher taxonomic category (e.g., taxonomic family) of 

234 the evidence without yet knowing the genotype and the species in that higher taxonomic category that 

235 potentially occur in the circumstances of the scene (e.g., at that location and that time of year). Because 

236 intraspecific genotype variation is common, identification is not necessarily made by an exact match to a 

237 reference. 

238 Failure to use an appropriate taxon selection could result in the evidence being apparently identified as the 

239 most closely related species in the database (Wells and Stevens 2008) when the true species is not included 

240 in the database. Morphological identification of a problematic species pair may narrow the need for a 

241 comprehensive database but still requires the inclusion of the species in question and their nearest genetic 

242 relatives.

243 5.2.5. Multiple protocols exist for making the comparison between evidence and reference genotypes. 

244 For species identification, a common method is a phylogenetic analysis (Harvey et al. 2008), with the 

245 identification indicated by a sister-group relationship (Schuh and Brower 2009) between the evidence 

246 genotype and a reference species. When using this approach, the reference database should represent the 

247 genotype(s)/haplotype(s) of multiple unrelated individuals of each species. 

248 Reliable species identification by phylogenetic analysis depends on a pattern of reciprocal monophyly for 

249 the gene(s) being analyzed. Therefore, particular care should be made to search the published literature and 

250 databases for evidence of non-monophyly (see Figure 1). Paraphyly of mitochondrial DNA is not unusual 

251 for closely related animal species. 

252 The reporting of phylogenetic results shall include the phylogenetic data file and the software analysis log.

253
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254 Annex A

255 (informative)

256 Bibliography

257 1] American National Standards Institute/American Academy of Forensic Sciences Standards 

258 Board. ANSI/ASB Standard 048, First Edition. Wildlife Forensic DNA Standard Procedures. 2019. 

259 www.asbstandardsboard.org/wp-content/uploads/2019/05/048_Std_e1.pdf.

260 2] Altschul, S.F. et al. “Basic local alignment search tool.” J Mol Biol. Vol. 215, no. 3, 5 Oct 1990, 

261 pp. 403-10. DOI: 10.1016/S0022-2836(05)80360-2. PMID: 2231712.

262 3] Catts, E.P. and M.L. Goff. “Forensic entomology in criminal investigations.” Annual Review of 

263 Entomology, vol 37, 1992, pp. 253-272.

264 4] Ewing, B. et al.. “Base-calling of automated sequencer traces using Phred. I. Accuracy 

265 assessment.” Genome research, vol. 8, 1998, pp. 175-185.

266 5] Harvey, M.L. et al.. “A global study of forensically significant calliphorids: implications for 

267 identification.” Forensic Science International, vol. 177, 2008, pp. 66-76.

6] Hillis, D.M. et al. “Nucleic acids IV: sequencing and cloning.” Molecular Systematics. 2

nd 268 Ed, 

269 edited by Hillis, D.M., Moritz, C., Sinauer, M.B. 1996, pp. 321-381

270 7] Notton, D., et al.. “Best practice in the use of the scientific names of animals: Support for editors 

271 of technical journals.” Bulletin of Zoological Nomenclature, vol. 68, no. 4, 2011, pp. 313-322.

272 8] Schuh, R.T., and A.V.Z Brower. Biological Systematics. Cornell University Press, 2009

273 9] Tarone, A.M., et al.. “Molecular Biology In Forensic Entomology.” Forensic Entomology: 

274 International Dimensions and Frontiers. Edited by J.K. Tomberlin and M.E. Benbow. 2015. Figure 

275 reproduced by permission of Taylor and Francis Group, LLC, a division of Informa PLC. 

10] Watson, J.D. et al.. Molecular Biology of the Gene. 7

th 276 Ed. Cold Spring Harbor Laboratory 

277 Press, 2014.

278 11] Wheeler, T.A. The role of voucher specimens in validating faunistic and ecological research. 

279 Biological Survey of Canada (Terrestrial Arthropods) Document Series, vol. 9, 2003, pp. 1-21.

280 12] Wells, J.D., and J.R. Stevens. “Applications of DNA-based methods in forensic entomology.” 

281 Annual Review of Entomology, vol. 53, 2008, pp. 103-120.