2021-S-0006 Standard for the Use of GenBank 

for Taxonomic Assignment of Wildlife

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OSAC 2021-S-0006

Standard for the Use of 

GenBank for Taxonomic 

Assignment of Wildlife

Wildlife Forensic Biology Subcommittee

Biology Scientific Area Committee

Organization of Scientific Area Committees (OSAC) for Forensic Science
2021-S-0006 Standard for the Use of GenBank 

for Taxonomic Assignment of Wildlife

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OSAC Proposed Standard

OSAC 2021-S-0006

Standard for the Use of GenBank for 

Taxonomic Assignment of Wildlife

Prepared by 

Wildlife Forensic Biology Subcommittee

Version: 2.0

November 2021

Disclaimer:

This OSAC Proposed Standard was written by the Wildlife Forensic Biology Subcommittee of 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 forensicscience 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.

To be placed on the OSAC Registry, certain types of standards first must be reviewed by a Scientific and Technical 

Review Panel (STRP). The STRP process is vital to OSAC’s mission of generating and recognizing scientifically 

sound standards for producing and interpreting forensic science results. The STRP shall provide critical and 

knowledgeable reviews of draft standards or of proposed revisions of standards previously published by standards 

developing organizations (SDOs) to ensure that the published methods that practitioners employ are scientifically 

valid, and the resulting claims are trustworthy.

The STRP panel will consist of an independent and diverse panel, including subject matter experts, human factors 

scientists, quality assurance personnel, and legal experts, which will be tasked with evaluating the proposed standard 

based on a comprehensive list of science-based criteria. 

For more information about this important process, please visit our website at: 

https://www.nist.gov/topics/organization-scientific-area-committees-forensic-science/scientific-technical-reviewpanels
2021-S-0006 Standard for the Use of GenBank 

for Taxonomic Assignment of Wildlife

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Standard for the Use of GenBank for Taxonomic Assignment of Wildlife

Foreword 

This standard defines the requirements that shall be met when comparing evidentiary sequences to 

those in GenBank for taxonomic assignment of non-human samples. The aim is to provide a framework 

that will result in consistency in the wildlife forensic DNA community. Use of these standards is 

expected for forensic scientists with a working understanding of DNA sequencing.

This standard was developed by the Biology/ Wildlife Forensic Biology Subcommittee of the 

Organization of Scientific Area Committees. This standard is intended to assist those using GenBank for 

the taxonomic identification of wildlife in forensic casework. 

All hyperlinks and web addresses shown in this document are current as of the publication date of this 

standard. 

Keywords: GenBank, BLAST, DNA, Public sequence databases, Taxonomic identification, Wildlife
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for Taxonomic Assignment of Wildlife

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Table of Contents 

1. Scope 5

2. Normative References 5

3. Terms and Definitions 5

4. Requirements 7

Annex A (informative) Bibliography 11
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for Taxonomic Assignment of Wildlife

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Standard for the Use of GenBank for Taxonomic Assignment of Wildlife 

1. Scope 

This standard covers the requirements and recommendations for analysis and selection of DNA 

sequences retrieved from the National Center for Biotechnology Information’s GenBank and

their subsequent use as reference material for taxonomic identification of wildlife1. This standard does 

not cover the use of DNA sequences from other public sequence databases (e.g., BOLD, UNITE), the 

protocol for downloading sequences from GenBank for inclusion in in-house databases, or the use of 

custom BLAST searches against GenBank. However, the criteria can be conceptually applied to other 

sequence databases.

2. Normative References 

NCBI Field Guide Glossary available at 

https://www.ncbi.nlm.nih.gov/Class/FieldGuide/glossary.html#

Madden T. (2013). “The BLAST Sequence Analysis Tool.” In: The NCBI Handbook, 2nd ed. Bethesda, 

MD. Available from https://www.ncbi.nlm.nih.gov/books/NBK153387/

ANSI/ASB Standard 019, First Edition. Wildlife Forensics General Standards, 2019. 

ANSI/ASB Standard 029, First Edition. Report Writing in Wildlife Forensics: Morphology and 

Genetics, 2019 

3. Terms and Definitions 

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

3.1 

alignment 

An arrangement of two or more nucleotide or protein sequences that is used to illustrate similarity 

among those sequences. 

3.2 

Basic Local Alignment Search Tool 

BLAST 

The a) BLAST algorithm, and b) a suite of database search programs that implement variations of 

this algorithm to generate alignments between a nucleotide or protein sequence in a query, and 

nucleotide or protein sequences within a database. 

3.3 

expectation value 

e-value 

The number of distinct alignments expected by chance; the default sorting metric in BLAST search 

results. 

1 For the purposes of this document, “wildlife” species are defined as non-human multicellular animals and plants, 

whether wild, captive-bred, or domesticated. 
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3.4 

GenBank 

A public repository of DNA sequences maintained by the National Center for Biotechnology Information, 

part of the U.S. National Institutes of Health.

3.5 

hit(s) 

Sequence(s) returned from GenBank when performing a BLAST search. Also known as a “subject 

sequence.” 

3.6 

interspecific 

Between members of different species. 

3.7 

intraspecific 

Between members of the same species. 

3.8 

National Center for Biotechnology Information 

NCBI 

The U.S. National Center for Biotechnology Information (NCBI) is located in Bethesda, Maryland and is 

part of the United States National Library of Medicine (a branch of the National Institutes of Health). 

NCBI houses a series of databases relevant to biotechnology and biomedicine and provides several 

bioinformatics tools for searching and analyzing the housed data.

3.9 

phylogram 

A branching diagram that illustrates relationships amongst organisms. Phylograms are typically 

generated using genetic sequences and/or morphological characters. 

3.10 

query 

(n) The nucleotide or protein sequence that has an unknown source (i.e., evidence sequence), or (v) the 

action of searching an unknown sequence against a database. 

3.11 

query coverage 

The percent of the query sequence length that is included in the aligned segment with a hit. 

3.12 

sequence identity 

The percentage or number of nucleotides or amino acids that are identical between two sequences. 

3.13 

subject sequence(s) 

A nucleotide or protein sequence(s) returned from a GenBank BLAST search. Also known as a “hit”.

3.14 

taxonomic identification 

Analyses to establish the classification of biological evidence to family, genus, species, etc. These 

analyses are based on class characters (e.g., morphological, genetic) that are diagnostic for the 

taxonomic level in question. 
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3.15 

topology 

The branching structure of a phylogram. 

3.16 

voucher specimen 

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

authority and is therefore valid for comparative purposes. Voucher specimens are of known identity, 

and are curated with available associated geographic, field collection, and life history data. 

4. Requirements 

Details about the operation of BLAST can be found in Madden (2013), and detailed information on the 

terms in the BLAST output can be found in the NCBI Field Guide Glossary. 

The following requirements and recommendations address criteria for the preparation and submission 

of evidentiary query sequences (4.1) and evaluation and interpretation of BLAST results from GenBank 

(4.2, 4.3), which should take into account whether the returned hit(s) is attributed to the correct 

species and whether the hit(s) is a close enough match for the taxon in question, appropriate level 

assignment (4.4) and reporting results from GenBank (4.5). 

4.1 Prior to performing a BLAST search, evidentiary query sequences: 

4.1.1 Shall be prepared by removing non-template flanking regions (e.g., primer); 

4.1.2 Shall meet sequence quality criteria as defined by the laboratory. Thus, laboratories are 

responsible for having these criteria clearly defined and ensuring their analysts follow these 

recommendations. 

4.1.3 Shall be examined to ensure it does not contain premature stop codons (e.g., by 

translation). 

4.2 To ensure that a hit(s) on which conclusions are based are of high quality, an initial assessment of 

the BLAST results: 

4.2.1 Shall ensure the hit(s) belongs to the expected broader taxonomic group (e.g., macerated 

plant tissue returns matches to sequences from the plant kingdom, not the bacterial 

kingdom). 

NOTE: In situations involving a complete unknown, it may not be possible to complete this assessment. 

4.2.2 Shall ensure that any hit(s) that is an anomaly among the returned results is not used. 

This would be indicated by being the only representative of its species interleaved 

among many in a different taxonomic group. This could be an indication of human error 

in sequence labeling during sequence preparation prior to GenBank upload. 

4.2.3 Shall ensure the hit(s) does not originate from an environmental sample (e.g., bulk soil 

extraction, bacterial swab) or low copy sample. 
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NOTE: The original publication can often be consulted to determine the source of the sequence. In some 

instances, this determination may not be possible. 

4.2.4 Should include a review for descriptors or characteristics that indicate the sequence was 

not reviewed prior to uploading in GenBank. 

NOTE: Sequences that have not been reviewed for quality may include descriptors such as “NGS”, “MPS”, 

“EST”, “shotgun”, “library”, and “WGS”; these may have been batch uploaded directly from the sequencing 

platform. Unedited sequences may also have a higher number of “Ns” or degenerate bases at the ends, or 

contain non-template flanking (e.g., primer, adapter) sequences. 

4.2.5 Should include a review for ambiguous bases. 

NOTE: Ambiguous bases should be treated with caution, as they can indicate poor-quality sequence, but 

they can also indicate heteroplasmic sites within a high-quality sequence. 

4.2.6 Shall ensure the hit(s) from a protein coding region does not contain premature stop 

codons. 

4.3 Any hit(s) on which conclusions are based shall be evaluated to determine if the returned 

sequence is attributed to the correct species based on the criteria listed below. This section is to 

determine if returned sequences are appropriate for interpretations as outlined in Section 4.4. 

These criteria confer either strong or moderate support to the attribution. If the returned 

sequence(s) does not meet at least the moderate criteria, they shall not be used for taxonomic 

assignment to the species level. 

4.3.1 Strong criteria (not all of these criteria have to be met, see section 4.5 for more information 

about how to evaluate relevant criteria): 

a) Sequence(s) is derived from a voucher specimen that bears a unique identifier.

b) Sequence(s), when downloaded, aligned with sequences from closely-related species 

and used to construct a phylogram, results in a species-level topology concordant with 

expectations from the peer-reviewed literature. 

c) Sequence(s) is from a study published in a peer-reviewed journal; the study addresses 

the phylogeny or taxonomy of the taxon of interest and the publication or accompanying 

metadata makes it clear that the source specimen(s) was morphologically identified by a 

taxonomic expert. 

d) Sequence(s) is part of a population genetic study for the given species published in a 

peer-reviewed journal. 

NOTE: Typically a population genetic study characterizes numerous individuals from the studied 

species in order to explore intraspecific variation (sample sizes will vary based on genetic 

variability and rareness of the species in question; published studies will have sample sizes that 

are appropriate for the species in question). The individuals may either be from the same 

geographic region, or from distinct populations within the known distributional range. 

4.3.2 Moderate criteria (not all of these criteria have to be met, see section 4.5 for more 

information about how to evaluate relevant criteria): 
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a) Sequence(s) is from a study published in a peer-reviewed journal; the study includes 

additional data establishing species identity (e.g., morphological evidence, museum

specimen), but it is not clear that the source specimen was a voucher (4.3.1a) or was 

morphologically identified by a taxonomic expert (4.3.1c). 

b) Sequence(s) is from a phylogenetic study in a peer-reviewed journal; the study 

addresses phylogeny or taxonomy of the taxon of interest and: 

i. includes most or all members of the genus in question, and 

ii. the locus shows resolution at the species level (see 4.4.2). 

c) Sequence(s) is one of multiple identical or near-identical sequences for the same 

locus and species from different submitters or geographic locations. 

d) Sequence(s) is not from a peer-reviewed study on the taxon of interest, but is 

accompanied by additional metadata concerning the source individual (e.g., location 

life history stage, name of collector, name of taxonomic expert who rendered the 

source individual’s identification). 

4.4 The following should be evaluated to determine the appropriate level for taxonomic 

assignment: 

4.4.1 Whether all likely candidate species in the taxonomic group in question are 

represented amongst the returned hit(s). 

NOTE: Complete taxon sampling is ideal, but often not feasible. If relevant taxa are missing, 

other loci or additional reference material should be considered. Species that are distantly 

related based on published phylogenies or those that do not occur in the geographic area of 

interest may be exempted from the comparison if sequences are not available. See section 4.5.2 in 

ASB 019 and section 3.5 in ASB 029. 

NOTE: Peer-reviewed literature or internal validation for the species/marker of interest 

provides the foundation for evaluating whether hits are appropriate and comprehensive 

enough to provide accurate interpretation for reporting. 

4.4.2 Whether the interspecific distance for the taxonomic group of interest at the surveyed locus 

is greater than intraspecific distance. 

NOTE: If inter- and intraspecific distances are similar, one should consider using a different 

locus or limiting identification to a higher taxonomic level. 

4.5 Reporting from BLAST results 

4.5.1 It is appropriate to report to the species level when all of these criteria are met: 

a) The evidentiary sequence(s) has been prepared as outlined in 4.1, 

b) The hit(s) on which conclusions are to be based: 

i. meets the quality criteria as defined in 4.2; 
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ii. meets at least two strong support criteria (as defined in 4.3.1), or at least one 

strong and one moderate (as defined in 4.3.2) support criteria; 

iii. has been evaluated against the criteria defined in 4.4; 

iv. and when aligned to the evidentiary query sequence, shows 99–100% identity 

(inclusive). 

NOTE: 99% is a conservative threshold, to be applied in instances where no other 

information is available for the target taxon. For most species, intraspecific distance will be 

greater than 1%; in cases where additional information (e.g., other loci, taxonomies based on 

morphological features) indicates species are well-separated, identities lower than 99% may still 

warrant a species level identification. 

NOTE: By default, BLAST results are sorted by E-value, which preferentially weights matches 

with higher query coverage, and max-score, based on sequence similarities. This can result in 

shorter sequences with higher percent identity being displayed after longer sequences with 

lower percent identity. The list may be sorted by the identity value to reveal the highestsimilarity matches. It is critical to consider both the percent identity and the length of the match 

when evaluating BLAST results. 

4.5.2 It is appropriate to report to a higher taxonomic level when all of these criteria are met: 

a) The evidentiary sequence(s) has been prepared as outlined in 4.1, 

b) The hit(s) meets the quality criteria as defined in 4.2, 

c) The hit(s) has been evaluated against the criteria defined in 4.4, 

d) The hit(s) does not meet the support criteria given in 4.5.1(b)ii, but is from a 

peer-reviewed publication and: 

i. The most similar sequences returned by a query are <99% identical and 

there is little definitive information on interspecific distance. 

OR 

ii. All top hits represent a single taxonomic level (i.e., genus, family, order), 

but there is a discrepancy at a lower taxonomic level (e.g., hits represent 

different species, but they all belong to a single genus). 
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Annex A (informative) 

This is not meant to be an all-inclusive list as the group recognizes other publications on this subject 

may exist. At the time this standard was drafted, these were the publications available for reference. 

Additionally, any mention of a particular software tool or vendor as part of this bibliography is 

purely incidental, and any inclusion does not imply endorsement. 

Bibliography 

1] Altschul SF. (2014). “BLAST Algorithm.” In: eLS, John Wiley & Sons, Ltd (Ed.). doi:

10.1002/9780470015902.a0005253.pub2.

2] ANSI/ASB Standard 019, Wildlife Forensics General Standards, First Edition, 2019. 

3] ANSI/ASB Standard 029, Report Writing in Wildlife Forensics: Morphology and Genetics, 

First Edition, 2019. 

4] ANSI/ASB Standard 048, Wildlife Forensic DNA Standard Procedures, First Edition, 2019.

5] Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW. (2013). 

“GenBank.” Nucleic Acids Research 41(D1):D36-42. Available from: 

https://www.ncbi.nlm.nih.gov/genbank/.

6] BLAST® Command Line Applications User Manual [Internet]. Bethesda (MD): National

Center for Biotechnology Information (US); 2008-. Available from:

https://www.ncbi.nlm.nih.gov/books/NBK279690/.

7] Brown TA. Genomes. 2nd edition. Oxford: Wiley-Liss; 2002. Chapter 16, Molecular

Phylogenetics. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21122/.

8] International Organization for Standardization. (2017). “ISO/IEC 17025:2005 General 

Requirements for the Competence of Testing and Calibration Laboratories.” 28 pp. 

9] Lee TRC, Anderson SJ, Tran-Nguyen LTT, Sallam N, Le Ru BP, Conlong D, Powell K, Ward A, 

Mitchell A. 2019. Towards a global DNA barcode reference library for quarantine 

identifications of lepidopteran stemborers, with an emphasis on sugarcane pests. Scientific 

Reports 9: 7039. Doi: https://doi.org/10.1038/s41598-019-42995-0. 

10] Lorenz JG, Jackson WE, Beck JC, Hanner R. (2005). “The problems and promise of DNA 

barcodes for species diagnosis of primate biomaterials.” Philosophical Transactions of the 

Royal Society B 360, 1869–1877. 

11] Madden T. (2013). “The BLAST Sequence Analysis Tool.” In: The NCBI Handbook, 2nd ed. 

Bethesda, MD. Available from https://www.ncbi.nlm.nih.gov/books/NBK153387/.