Standard for Internal Validation of Human Short Tandem 

Repeat Profiling on Capillary Electrophoresis Platforms

Standard for Internal 

Validation of Human Short 

Tandem Repeat Profiling on 

Capillary Electrophoresis 

Platforms

Biological Methods Subcommittee

Biology/DNA Scientific Area Committee

Organization of Scientific Area Committees (OSAC) for Forensic Science
Standard for Internal Validation of Human Short Tandem 

Repeat Profiling on Capillary Electrophoresis Platforms

OSAC Proposed Standard

Standard for Internal Validation of 

Human Short Tandem Repeat Profiling 

on Capillary Electrophoresis Platforms

Prepared by 

Biological Methods Subcommittee

Version: 1.0

Disclaimer:

This document has been developed by the Biological Methods Subcommittee of the Organization of 

Scientific Area Committees (OSAC) for Forensic Science through a consensus process and is 

proposed for further development through a Standard Developing Organization (SDO). This document 

is being made available so that the forensic science community and interested parties can consider the 

recommendations of the OSAC pertaining to applicable forensic science practices. The document was 

developed with input from experts in a broad array of forensic science disciplines as well as scientific 

research, measurement science, statistics, law, and policy. 

This document has not been published by an SDO. Its contents are subject to change during the 

standards development process. All interested groups or individuals are strongly encouraged to submit 

comments on this proposed document during the open comment period administered by the American 

Academy of Forensic Science Standards Board (www.asbstandardsboard.org). 
Standard for Internal Validation of Human Short Tandem 

Repeat Profiling on Capillary Electrophoresis Platforms

TABLE OF CONTENTS

1. SCOPE..................................................................................................................................................5

2. NORMATIVE REFERENCES...........................................................................................................3

3. TERMS and DEFINITIONS ............................................................................................................3

4. REQUIREMENTS..............................................................................................................................5

5. CONFORMANCE ...............................................................................................................................7

6. ANNEX.................................................................................................................................................7
Standard for Internal Validation of Human Short Tandem 

Repeat Profiling on Capillary Electrophoresis Platforms

1. SCOPE

This document details requirements for performing an internal validation of a human short 

tandem repeat (STR) multiplex kit using capillary electrophoresis (CE).

2. NORMATIVE REFERENCES

The following referenced documents are the foundation of these requirements. For dated 

references, only the edition cited applies. For undated references, the latest edition of the 

referenced document applies.

FBI, Quality Assurance Standards for DNA Databasing Laboratories.

https://www.swgdam.org/#!publications/c1mix

FBI, Quality Assurance Standards for Forensic DNA Testing Laboratories.

https://www.swgdam.org/#!publications/c1mix

SWGDAM. SWGDAM Validation Guidelines for Forensic DNA Analysis Methods.

https://www.swgdam.org/#!publications/c1mix

3. TERMS and DEFINITIONS

3.1 Analytical threshold is the minimum height requirement at and abovewhich 

detected peaks on a STR DNA profile electropherogram can be reliably 

distinguished from instrument background noise; peaks above this threshold 

are generally not considered noise and are either artifacts or true alleles.

3.2 Artifact is a non-allelic product of the amplification process (e.g., stutter, nontemplated nucleotide addition, or other non-specific product), an anomaly of 

the detection process (e.g., single or multichannel voltage spikes or “pull-up”), 

or a by-product of primer synthesis (e.g., “dye blob”) that may be observed on 

an electropherogram.

3.3 Capillary electrophoresis (CE)is an electrophoretic technique for separating 

DNA molecules by their relative size based on their differential migration 

through a capillary tube filled with a liquid polymer.

3.4 Controls are samples of known types, run in parallel with experimental, 

reference, or evidence samples that are used to demonstrate that a procedure is 

working correctly.

3.5 Deoxyribonucleic acid (DNA) is a genetic material of organisms, usually 

double-stranded, a biopolymer composed of nucleic acids, identified by the
Standard for Internal Validation of Human Short Tandem 

Repeat Profiling on Capillary Electrophoresis Platforms

presence of deoxyribose, a sugar, and the four nucleobases. DNA is a stable 

molecule.

3.6 Drop-in is allelic peak(s) in an electropherogram that are notreproducible 

across multiple independent amplification events.

3.7 Drop-out is failure of an otherwise amplifiable allele to produce a signal above 

analytical threshold because the allele was not present or was not present in 

sufficient quantity in the aliquot that underwent PCR amplification.

3.8 Internal Validation is the accumulation of test data within the laboratory for 

developing the laboratory standard operating procedures and demonstrating 

that the established protocols for the technical steps of the test and for data 

interpretation perform as expected in the laboratory.

3.9 Locus (loci) is a unique physical location of a gene (or specific sequence of 

DNA) on a chromosome.

3.10 Peak height ratio is the relative ratio of two peaks at a given locus.

3.11 Polymerase chain reaction (PCR) is an enzymatic process by which a specific 

region of DNA is replicated during repetitive cycles that consist of the following: 

denaturation of the template; annealing of primers to complementary 

sequences at an empirically determined temperature; and extension of the 

bound primers by a DNA polymerase.

3.12 Precision studies are studies performed to evaluate the degree of mutual 

agreement among a series of individual measurements, values and/orresults.

3.13 Probabilistic genotyping is the use of biological modeling (i.e., statistical 

modeling informed by biological data), statistical theory, computer algorithms, 

and/or probability distributions to infer genotypes and/or calculate likelihood 

ratios.

3.14 Sensitivity studies are a set of critical studies performed during 

developmental and/or internal validation of DNA or other test methods 

designed to define the lower and upper limits/bounds of an assay to accurately 

detect an analyte.

3.15 Short tandem repeats (STR) are multiple copies of an identical (or similar) 

DNA sequence arranged in direct succession where the repeat sequence unit is 

2 base pairs (bp) to 6 bp in length.

3.16 Stochastic threshold is the peak height value in a DNA electrophoretic profile 

above which it is reasonable to assume that, at a given locus, allelic drop-out of 

a sister allele in a heterozygous pair has not occurred in a single source DNA 

sample.
Standard for Internal Validation of Human Short Tandem 

Repeat Profiling on Capillary Electrophoresis Platforms

3.17 Stutter is an artifact of PCR amplification typically observed one or more repeat 

units smaller or larger than an STR allele in a DNA electrophoretic profile, may 

result from strand slippage during PCR amplification. A stutter peak is generally 

of lower RFU than the allele peak.

3.18 Validation is the process of performing and evaluating a set of experiments 

that establish the efficacy, reliability, and limitations of a method, procedure or 

modification thereof; establishing recorded documentation that provides a high 

degree of assurance that a specific process will consistently produce an 

outcome meeting its predetermined specifications and quality attributes. May 

include developmental and/or internal validation.

4. REQUIREMENTS

4.1 The laboratory shall conduct an STR genotyping concordance study.

4.1.1 STR genotypes obtained from the new STR kit shall be compared to 

genotypes obtained from the previous kit for all overlapping loci,if 

applicable.

4.1.2 This study shall include the evaluation of concordance of the newSTR 

test kit utilizing the appropriate NIST Standard Reference Material 

for STR genotyping.

4.1.3 In the event that a discordant genotype is observed when comparing 

identical genetic markers, this discordant data shall be noted in the 

final validation document, and if possible a potential explanation forthe 

discordant data supported by data or other research studies shall be 

included in the final validation document.

4.2 The laboratory shall determine the sensitivity level and optimal DNA input range 

for each set of assay conditions/parameters (i.e. PCR cycle number, injection 

time/voltage, PCR reaction volume, etc.) with the new STR test kit.

4.2.1 A range of DNA input spanning lower and higher quantitiesthan 

typically analyzed by the testing laboratory shall be utilized.

4.3 The laboratory shall determine the analytical threshold for each dye channel of 

the new STR test kit using a range of sample types and DNA input quantities 

across multiple runs.

4.4 The laboratory shall characterize expected peak height ratio variation observed 

for each locus of the new STR test kit utilizing single source samples amplified 

over a range of DNA input amounts.

4.5 The laboratory shall characterize expected PCR stutter artifacts observed for each 

STR locus of the new STR test kit.
Standard for Internal Validation of Human Short Tandem 

Repeat Profiling on Capillary Electrophoresis Platforms

4.6 The laboratory shall determine a stochastic threshold for any manual binary 

interpretation method used to interpret the new STR test kit data or employ a 

probabilistic genotyping method.

4.7 The laboratory shall conduct a study utilizing mixed DNA samples that are 

representative of the number of contributors, DNA ratios, and DNA template 

input quantities expected to be interpreted by the testing laboratory.

4.7.1 The results of this study shall be used to establish mixture 

interpretation protocols for determining number ofcontributors, 

mixture ratios, and distinguishing major and minorcontributors.

4.8 The laboratory shall demonstrate allelic sizing precision and calling accuracy of 

the new STR test kit and capillary electrophoresis instrument through 

repeatability and reproducibility studies.

4.9 The laboratory shall determine the susceptibility of the genotyping process to the 

introduction and detection of exogenous DNA through the evaluation of controls 

(i.e. reagent blanks, negative and positive controls) and samples with known 

genotypes.

4.9.1 The laboratory shall document observed allelic drop-in.

4.10 The laboratory shall evaluate the new STR test kit using the laboratory’s testing 

procedures, and samples representative of those regularly analyzed by the testing 

laboratory.

4.10.1 The laboratory shall compare the results to previous resultsof 

known and case-type samples, where applicable.

4.10.2 This study shall include samples using all DNAextraction 

chemistries utilized by the laboratory.

5. CONFORMANCE

In order to demonstrate conformance with this standard, the laboratory shall have 

completed all applicable validation studies as described above:

5.1 Concordance

5.2 Sensitivity

5.3 Analytical threshold(s)

5.4 Peak height ratio variation

5.5 PCR Stutter artifacts
Standard for Internal Validation of Human Short Tandem 

Repeat Profiling on Capillary Electrophoresis Platforms

5.6 Stochastic threshold(s)

5.7 Mixed DNA samples

5.8 Precision and accuracy

5.9 Exogenous DNA and drop-in

5.10 Known and non-probative samples

6. ANNEX

Annex A – Bibliography

Bregu, J., Conklin, D., Coronado, E., Terrill, M., Cotton, R. W. and Grgicak, C. M. (2013), 

Analytical Thresholds and Sensitivity: Establishing RFU Thresholds for Forensic DNA 

Analysis,. J Forensic Sci, 58: 120–129. doi:10.1111/1556-4029.12008

Butler, J.M. Quality Assurance and Validation. In: Advanced Topics in Forensic DNA Typing: 

Methodology. Elsevier, 2011.

Gilder, J. R., Doom, T. E., Inman, K. and Krane, D. E. (2007), Run-Specific Limits of Detection 

and Quantitation for STR-based DNA Testing. Journal of Forensic Sciences, 52: 97–101. 

doi:10.1111/j.1556-4029.2006.00318.x

Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling 

Process. ENSFI 2010.

Scientific Working Group on DNA Analysis Methods. Interpretation Guidelines for Autosomal 

STR Typing by Forensic DNA Testing Laboratories. 

https://www.swgdam.org/#!publications/c1mix

Informational Web Sites: Additional information may be obtained from the following 

websites:

www.cstl.nist.gov/strbase

http://www.cstl.nist.gov/strbase/validation.htm

http://www.cstl.nist.gov/strbase/training.htm