Keywords

Introduction

The current oversight for forensic DNA analysis is provided through the National Institute of Standards and Technology (NIST) via review by OSAC, the Organization of Scientific Area Committees. This group facilitates implementation of forensically sound science and technical methods by drafting and evaluating standards that are to be used by forensic science laboratories (www.nist.gov). Previously, the Technical Working Groups sponsored by the Federal Bureau of Investigation (FBI) were performing this role by providing recommendations for implementation of new and current technology in the forensic science workplace. In 1989, the Guidelines for a Quality Assurance Program for DNA Analysis was prepared by TWGDAM and then revised three times after the TWGDAM working group of private and public sector forensic scientists was renamed to SWGDAM, the Scientific Working Group for DNA Analysis Methods in 1999. Prior to 1989, there were no national recommendations for forensic science laboratories to follow.

The DNA Advisory Board (DAB) began its 5-year project to create a set of federal quality standards for forensic laboratories in 1995. These standards include the quality manual areas of forensic science laboratory organization, personnel, facilities, evidence tracking and storage, validation, test procedures, equipment calibration and maintenance, technical review and reports, proficiency testing and corrective action, laboratory audits, safety guidelines, and use of subcontractors for testing of backlogged samples (https://strbase.nist.gov/validation/Intro_to_DAB_Standards.pdf). Two sets of standards were created by the DNA Advisory Board. In October 1998, The Quality Assurance Standards for Forensic DNA Testing Laboratories (Forensic Standards) became active to provide oversight to the activities of DNA laboratories that analyze criminal casework (https://strbase.nist.gov/QAS/Final-FBI-Director-Forensic-Standards.pdf). The Quality Assurance Standards for Convicted Offender DNA Databasing Laboratories was initiated in April 1999 for the analysis of DNA known reference samples collected from convicted offenders (https://strbase.nist.gov/QAS/Final-FBI-Director-Databasing-Standards.pdf).

DNA is a molecule that defines the function of genes but also contains noncoding regions that vary from individual to individual that are useful for human identification. Deoxyribonucleic acid (DNA) is composed of two polynucleotide chains carrying genetic instructions for all known organisms and their functions. DNA is a double helix comprised of two antiparallel strands that form the backbone of the helix by creating a sugar-phosphate structure that are bound together by complementary nucleotide base pairing, adenine (A) to thymine (T) and guanine (G) to cytosine (C). The regions of interest for short tandem repeat analysis have been prescreened and preselected for genetic variability in human populations. The estimated frequency of each allele at any given chromosome locus has been calculated in sample ethnic populations and have been shown to vary slightly in frequency based on original founder populations. The high discrimination power or ability to individualize comes from both the great number of allele possibilities at any given locus and the larger number of loci used in combination to calculate estimated relative frequency of any given DNA profile compared to the unrelated random individuals in a population. The level of STR analysis is now so sophisticated that a human DNA profile is unique to an individual unless they have a genetic identical twin.

The integrity of DNA data is essential for establishing a link to a correct DNA source. Deoxyribonucleic acid or DNA is the biological molecule used to identify potential donors to biological evidence. It is useful as a forensic tool to indicate associations of individuals to biological evidence such as blood, semen, saliva, urine, and epithelial cells. Although an association can be identified by DNA testing, the timing of deposit and the context to the case often is in question. After evidence collection, DNA is extracted, quantified, amplified, and detected by capillary electrophoresis in the forensic science laboratory. Analysis software is used for the viewing and interpretation of DNA fragments to make a conclusion about a potential inclusion or exclusion of a DNA source. Additional DNA mixture interpretation software may be used as an aid to interpret complex DNA samples. The basic steps used to generate a human DNA profile are carefully quality controlled to be sure that a scientifically accurate result can be achieved. This leaves the court debate to the meaning of the DNA in the case rather than with the scientific validity of the DNA result itself. The basic steps of this process can be broken down into the component parts: (a) DNA collection, (b) DNA extraction, (c) DNA quantitation, (d) DNA amplification, (e) capillary electrophoresis, (f) data interpretation, and (g) statistical assessment.

DNA Methods

DNA collection. DNA evidence collection refers to the recognition and collection of biological evidence that may be pertinent to the case. Forms of DNA evidence include blood, saliva, semen, urine, touch DNA, bone, tissue, hair, vomit, feces, perspiration, and tears. These forms of evidence may be found as part of property crimes, homicides, sexual assaults, child abuse cases, robbery, kidnapping, etc. DNA collection strategies include the use of personal protective equipment (PPE) to avoid contamination, and therefore error, in the final DNA results. Saliva can contain droplets with DNA molecules that can fall on evidence or a surface and be mixed inadvertently into the crime scene sample (Aparna and Shanti Iyer 2020; Pandeshwar and Das 2014). Therefore, masks and face shields are essential for prevention of DNA contamination by evidence collection personnel. Likewise, shed epithelial cells from the skin and hairs may be falsely included in DNA evidence should it be deposited on the evidence, and full disposable crime scene suits, gloves, and head coverings are required to prevent this phenomenon (Zajac et al. 2019). The key to high-quality DNA results in criminal casework relies on best practices for quality control at the crime scene for evidence recognition, collection, and preservation (Goray et al. 2012; Szkuta et al. 2017; Pilli et al. 2013).

DNA collection methods and techniques vary for DNA recovery rates as does the expected amount of DNA yield from different tissue and fluid sources (Tan and Yiap 2009). The estimated DNA yield from various tissue sources is the following: blood (200 uL, 4–12 ug), cultured cells (5 million, 15–20 ug), liver (25 mg, 10–30 ug), heart (25 mg, 5–10 ug), and spleen (10 mg, 5–30 ug) (www.qiagen.com). The quantity of DNA recovered from biological evidence is affected by the environment that it is exposed to, and therefore, a range of DNA yield is presented in most forensic studies. The estimated DNA content for forensic biological samples in liquid blood is 20–40 ug/mL, in liquid semen is 150–300 ug/mL, and in liquid saliva is 1–10 ug/mL (Lee and Ladd 2001). Collection method recovery rates vary but include the following estimated percent recovery for epithelial cells from polyester blend fabric: cotton swab (80%), blotting paper (55%), foam-tipped swab (50%), scotch tape (45%), gauze (20%), fingerprint lift tape (15%), Whatman filter paper (15%), Sirchie lift tape (10%), duct tape (5%), positive nylon membrane (1%), and Post-it Note (0%) (https://www.ncjrs.gov/pdffiles1/nij/grants/236826.pdf). Since the method of recovery of DNA molecules varies from forensic science laboratory to laboratory, there may be some level of error introduced by the sampling method or subsequent processing that could lead to suboptimal or no DNA results dependent on the facility or analyst preference (Garvin et al. 2013; Hebda et al. 2014; Singh et al. 2018).

Case documentation. Submission form(s) are included in the case folder to document the requesting agency evidence submissions for testing. On occasion, the incorrect test can be requested for the type of evidence, and one quality control step in both standard DNA and postconviction DNA testing is to evaluate the evidence, the test requests, and the unsubmitted items for potential error. If evidence was never submitted for testing but could have been probative and relevant, there may be the opportunity to reevaluate the case circumstances. Other quality control measures include ensuring pages are labeled with the case number, initials, and date; photographs and diagrams are included and labeled correctly; the proper worksheets are used and filled out completely; and communication and correspondence forms are included in the case folder. For each and every step of the DNA testing process, there should be a quality worksheet that is signed and dated and records the process for the case folder. Peer review internal to the forensic laboratory where analysts review each other’s case documentation and supervisors and quality managers also review documentation is designed as a quality control measure to reduce sloppy and incomplete documentation or typographical errors.

Reagent preparation. Quality and technical manuals are required in forensic science laboratories to show analysts how reagents are to be made and to reduce variability in preparation techniques. Receipt of reagents and chemicals by the forensic biology section of the laboratory is documented in a chemical log. The tracking of reagent quality includes catalogue numbers, manufacturer lot numbers, suppliers, and testing of new reagents with old reagent results to ensure that a comparable result is achieved. Once the reagent has been tested and is appropriate for laboratory use, it can be implemented in the general preparation of forensic test reagents thereafter. Expiration dates are checked regularly as well (e.g., https://www1.nyc.gov/assets/ocme/downloads/pdf/technical-manuals/qaqc-procedures-manual/reagents.pdf).

DNA extraction. The DNA extraction process requires either the manufacturer of the kit or the forensic laboratory (or both) quality controls the reagents used to purify DNA. All of the reagents and disposable plastics are required to be of molecular biology grade quality indicating they are DNase, RNase, and protease-free (e.g., https://www.mt.com/dam/RAININ/PDFs/TechPapers/test_protocol.pdf). These enzymes, if present, will damage the recovery of the DNA and, therefore, must be removed from all surfaces, disposable plastics, and reagents to ensure DNA of high quality and purity is recovered from the biological evidence. Even the water used in reagent preparation must be of molecular biology grade to be quality enough to use in the DNA extraction process. DNA is a large polymeric molecule that is too large to enter the cell membrane. In order to utilize external DNA, some bacteria secrete DNases (enzymes that biodegrade DNA by hydrolysis) outside of the cell to digest DNA into nucleotides. The nucleotides can then move into the bacterial cell membrane via transport proteins. The bacteria use nucleotides to make nucleic acids and as a source of nitrogen, phosphate, and carbon (https://microbiologyinfo.com/deoxyribonuclease-dnase-test/). In addition, a positive DNA sample of known quality and identity is used as a calibration control or reference standard for the DNA extraction process. A negative reagent control is processed simultaneously with the samples and the positive control to test the purity of the reagents and indicate no contaminant DNA is present in the reagents (Morton and Collins 1995). The purpose of DNA extraction is to chemically remove any inhibitory contaminants in the evidentiary sample and purify the DNA molecules away from other cellular components such as proteins, lipids, and carbohydrates so that the template is available for PCR replication.

DNA quantitation. This step is to determine the yield of the DNA from the evidentiary sample (Arya et al. 2005). qPCR assays vary, but in general they are a two-step PCR method that amplifies DNA target loci and includes a fluorescent dye accumulation step to visualize amplification in real time. This allows the analyst to determine both the quantity and quality of the DNA sample prior to generating a full human DNA profile using STR analysis kits and a three-step PCR cycling process called end point PCR. This step requires a manufacturer certification for qPCR kit reagents as well as internal control samples to the assay to check for proper functionality. Positive and negative PCR amplification controls for qPCR assays are commonly included in the kits or can be purchased separately. Human DNA of known concentration is purchasable from molecular biology suppliers and can be purified DNA from American Type Culture Collection (ATCC) certified human cell lines (www.atcc.org); negative controls can be purchased as purified DNA from other animal and plant species to test for PCR amplification specificity. A negative reagent control is performed for the assay by addition of kit reagents to a sample well minus the DNA template to test for purity and no contamination of reagents. In addition, synthetic DNA (internal positive control, IPC) is added to the reagents in the kit as a PCR amplification and pipetting control to indicate that specific DNA template is able to be replicated with the test reagents regardless of whether the evidentiary DNA is amplifiable. The calibration of reagents across the plate is simultaneously performed with the synthetic DNA as the results should be comparable from sample well to sample well (Ewing et al. 2016; Swango et al. 2007; Raymaekers et al. 2009).

DNA amplification. The PCR process is a molecular biology technique to replicate DNA synthetically in a sterile tube. The components of a PCR reaction include buffer, free nucleotides, short complementary PCR primers to the region of interest for amplification, purified genomic DNA template, and magnesium chloride as a cofactor for the Taq polymerase enzyme and Taq polymerase itself. In the PCR process, the DNA double helix is denatured by heat, the PCR primers bind to the complementary regions during the annealing step, and then the final new strand synthesis step occurs. This three step thermal cycling process continues for 25–32 cycles until millions of copies of the original target sequence are represented. The specificity of primer binding is due to DNA sequence homology on the template strand; mutations can result in primer mispairing or absence of replication (Zhu et al. 2020; Green and Sambrook 2019). STR analysis kit details and the history of their development are reviewed in a later section in this chapter.

PCR amplification of genetic loci is performed by using manufacturer kits. The evolution of these kits to those with expanded multiplexes has not changed the quality control measures. A positive human DNA sample is included in each kit to verify the kit functions properly, and the positive control genotype is consistent from run to run. A negative control is recommended as a “blank” sample well containing reagents but lacking DNA template and, therefore, should produce no DNA profile. PCR replication can be difficult to achieve with low quantity and low quality DNA templates due to variability in template sampling and preferential amplification of higher-quantity target sequences. This results in stochastic effects of the minor component in low level samples and minor components in a mixture (Miller Coyle 2015). Internal validation studies of new DNA typing kits are critical to show that the manufacturer claims are true regarding efficiency and accuracy. The internal validation studies are performed “in-house” in the current laboratory setting to determine how it performs in the hands of the analyst. Validation studies include performance checks on method reliability, reproducibility, sensitivity, and specificity. For each new kit that is developed, forensic laboratories need to validate and peer-review validation studies to show forensic community consensus as part of meeting court admissibility criteria (Ewing et al. 2016; Zhou et al. 2016; Gopinath et al. 2016).

Capillary electrophoresis. Capillary electrophoresis is a semi-automated method for selective size separation of DNA fragments based on size and charge. DNA is heat denatured and placed in purified deionized formamide to maintain a single-strand configuration to the DNA fragment. Only one strand of DNA has been fluorescently labeled in the PCR amplification process, so that is the strand that can be visualized in the capillary electrophoresis step. A liquid polymer is used as the sieving medium to separate the DNA fragments into a fluorescently labeled and visible “barcode” that fills the glass capillaries in the capillary electrophoresis system. Allelic ladders and internal lane size standards control for the accurate sizing of DNA fragments by providing systematic sized patterns to compare the unknown DNA fragments against. Capillaries are sensitive to heat and mobility of DNA fragments can vary per capillary; thus, an internal lane size standard (or comigration control) corrects for mobility shift. Beyond the use of these standards, data may not be reproducible from PCR reaction to PCR reaction due to slight differences in efficiency rates at each locus and due to template sampling differences in the electrokinetic injection step (Krivácsy et al. 1999; Opekar et al. 2016).

Data interpretation. A technical review is performed to check that the reports and conclusions are correct. The review includes that proper controls were used; the controls gave appropriate results, and the conclusions are in agreement with data. The allele calls and statistical calculations are then verified by a second, qualified individual. Supervisor oversight includes review of a percentage of the casework that is produced at the forensic science laboratory. The challenges to DNA reside in complex DNA mixture interpretation, and there is considerable variability in data interpretation from forensic laboratory to forensic laboratory (Butler et al. 2018; Buckleton et al. 2018). Each laboratory has set its own policy to follow for data interpretation above and below an analytical instrument threshold, for establishing the true number of contributors to a DNA mixture, and for assessment of contaminant alleles.

Analyst bias has been recognized as a subconscious or conscious attempt to include an individual in a DNA result simply because an individual has been identified as a candidate suspect. Bias is represented by selective inclusion and/or nondisclosure of other candidates observed in the data either due to error, intent, or laboratory policy. Most importantly for trial, scientific accuracy is needed to provide effective interpretation of the data and effective counsel. Improvements in data interpretation with mixtures have been made with probabilistic genotyping software analysis systems (e.g., TrueAllele, STRmix) (Greenspoon et al. 2015; Perlin et al. 2015; Bauer et al. 2020; Perlin et al. 2011; Bright et al. 2019; Buckleton et al. 2019; Moretti et al. 2017). While computer software can assist in making statistical inferences, they do also need to be fully understood to recognize the benefits, limitations, and inherent error rates associated with each program. Precision is the ability to obtain the same result every time a test is run; however, the test result may not be accurate. So the ability to obtain both a precise and scientifically accurate result is the desired objective, and finding the optimal method is key while realizing technology may go through several iterations before arriving to the point of optimum.

A case in point, here, is the use of probabilistic genotyping software that is designed to eliminate confirmation bias when analyzing data by eye; however, the analyst may have bias in establishing the number of contributors to specify for the software analysis: a situation of concern. Less has been written about confirmation bias in DNA analysis; however, some good forensic studies have been done (Skellern 2015; Mattijssen et al. 2016; Dror 2012, 2015; Dror et al. 2015; Nakhaeizadeh et al. 2014; Brauner 2012; Dror and Hampikian 2011). Certain aspects such as the effect of adjusting the analytical threshold (an invisible line above which alleles are reported and below which alleles are not reported) and determining the effect on reporting number of contributors appear absent in many forensic laboratory validation studies and would affect the interpretation of whether or not an individual may be included in a DNA mixture (Kirby et al. 2017). The setting of the analytical threshold is established in historical policy when forensic laboratories first established their DNA units; however, this will be worth revisiting as probabilistic genotyping software programs are installed and validated now, with new and better technology.

An article by Gill et al. (2006) clarifies for the DNA analyst factors for identifying the number of contributors in the mixture. “The number of alleles observed per locus, circumstances of the case, and the possibility of related contributors go into deciding how many contributors to condition on.” The allele counting method (assuming a heterozygote as donor) yields a minimum estimate of number of contributors per genetic locus. Scientifically speaking, the maximum alleles observed at any given locus should yield the minimum estimated of detected number of contributors to the DNA mixture. Given the allele counting method, this article goes further to explain the issue of number of contributors and how that assessment relates to determining a likelihood ratio (LR) for probabilistic genotyping. “It is not always easy to specify hypotheses in complex cases where multiple perpetrators or victims may be present. The DNA result itself may indicate that different explanations may be possible. Furthermore, it is possible that Hp (prosecutor hypothesis) and Hd (defense hypothesis) could be very different from each other. For example, under Hp we might consider (victim and suspect); whereas with Hd we might examine more complex scenarios with 3 contributors (3 unknowns, U0 + U1 + U2). There is a common misconception that the number of contributors under Hp and Hd should be the same. There is no requirement for this.” The article suggests “the smallest number of unknown contributors needed to explain the evidence are usually the ones to maximize the respective likelihoods.” In courtroom testimony, often a DNA analyst will state that a DNA result is conservative (“an assignment for the weight of the evidence that is believed to favor the defense”); however, the maximum number of contributors may not be assigned correctly to the DNA mixture due to the policy of consensus profiling.

Evidence of additional contributors in replicate PCR amplifications, alleles detected below the analytical threshold, spurious alleles, contamination, and detection of minor DNA elements by differential electrokinetic injection all amount to the same thing: additional alleles are present that cannot be accounted for by the standards submitted and should be disclosed in reports, testimony, and statistics. The effect of including the maximum possible contributors based on allele counts in any one of the PCR replicates (duplicates or triplicates) is to, first, acknowledge the scientific observation and, second, to generate the best possible probability estimate when using probabilistic genotyping methods. Historically, forensic laboratories were greatly concerned over reporting out trace levels of DNA contamination. The development of new DNA standards has been recently completed to address the validation of DNA probabilistic genotyping software systems and for DNA mixture interpretation to improve consistency in results determination from forensic science laboratory to forensic science laboratory. While PCR amplification and capillary electrophoresis artifacts and parameters still present a problem for data interpretation to DNA analysts, the new standards should be an aid to increase uniformity in DNA interpretation of mixtures from biological evidence.

History of STR analysis. Approximately three million nucleotide bases (noncoding regions) with multiple copies of short tandem repeat sequences construct the DNA backbone (e.g., CAGTCAGTCAGT; three repeats). These regions are called “variable number of short tandem repeats (VNTRs).” If a sufficient number of STR loci are tested (profiled), then the evidence of a person’s identity (and unique STR identifier) is enhanced because the likelihood of two unrelated people having the same number of repeated sequences in these regions becomes vanishingly small (https://nij.ojp.gov/topics/articles/what-str-analysis).

The original form of STR analysis used single PCR reactions to test per locus information, multiplexing to combine loci rapidly followed. The first multiplexes had three or four loci per PCR reaction. The primary commercial suppliers for the early systems were (a) Promega Corporation (Madison, WI) that used a silver staining method to visualize loci (CTT: CSF1PO, TPOX, TH01; CTTV: CSF1PO, TPOX, TH01, VWA; FFV: F13A1, FESFPS, VWA; FFFL: F13A1, FESFPS, F13B, LPL; GammaSTRTM: D16S539, D7S820, D13S317, D5S818) and (b) Applied Biosystems (Foster City, CA) that used a fluorescent dye technology AmpFlSTR Green I (Amelogenin, TH01, TPOX, CSF1PO and AmpFlSTR Blue: D3S1358, VWA, FGA (https://strbase.nist.gov/multiplx.htm)).

Expanded multiplexes quickly followed with the Promega Powerplex series: PowerPlex ES: D3S1358, TH01, D21S11, D18S51, SE33, Amelogenin, VWA, D8S1179, FGA; PowerPlex 16 HS: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, VWA, D8S1179, TPOX, FGA; PowerPlex 18D: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, Amelogenin, VWA, D8S1179, TPOX, FGA, D19S433, D2S1338; PowerPlex ESX 16: Amelogenin, D3S1358, TH01, D21S11, D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1045, VWA, D8S1179, FGA, D2S441, D12S391, D19S433; PowerPlex ESX 17: Amelogenin, D3S1358, TH01, D21S11, D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1045, VWA, D8S1179, FGA, D2S441, D12S391, D19S433, SE33; PowerPlex ESI 16: Amelogenin, D3S1358, D19S433, D2S1338, D22S1045, D16S539, D18S51, D1S1656, D10S1248, D2S441, TH01, VWA, D21S11, D12S391, D8S1179, FGA; PowerPlex ESI 17: Amelogenin, D3S1358, D19S433, D2S1338, D22S1045, D16S539, D18S51, D1S1656, D10S1248, D2S441, TH01, VWA, D21S11, D12S391, D8S1179, FGA, SE33; and PowerPlex 21: Amelogenin, D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, VWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA. Also, Applied Biosystems “filer” series: AmpFlSTR Profiler Plus: D3S1358, VWA, FGA, Amelogenin, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820; AmpFlSTR Profiler Plus ID: D3S1358, VWA, FGA, Amelogenin, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820; AmpFlSTR COfiler: D3S1358, D16S539, Amelogenin, TH01, TPOX, CSF1PO, D7S820; AmpFlSTR Sinofiler (available only in China): D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D5S818, D13S317, D16S539, D2S1338, D19S433, VWA, D12S391, D18S51, Amelogenin, D6S1043, FGA; AmpFlSTR Profiler: D3S1358, VWA, FGA, Amelogenin, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820; AmpFlSTR SEfiler: D3S1358, VWA, D16S539, D2S1338, Amelogenin, D8S1179, SE33, D19S433, TH01, FGA, D21S11, D18S51; and AmpFlSTR SEfiler Plus: D3S1358, VWA, D16S539, D2S1338, Amelogenin, D8S1179, SE33, D19S433, TH01, FGA, D21S11, D18S51. These kits were a series integrated for the Combined DNA Indexing System (CODIS) core 13 loci required for comparisons with the National DNA Index System (NDIS) DNA database of convicted offender samples.

The latest generation kits are large megaplexes and specialty application kits. Included in the Promega series are PowerPlex 16: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, VWA, D8S1179, TPOX, FGA; PowerPlex Fusion (includes 22 loci, amelogenin for gender identification, and a Y chromosome locus): Amelogenin, D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, VWA, D21S11, D7S820, D5S818, TPOX, DYS391, D8S1179, D12S391, D19S433, FGA, D22S1045; and the Y chromosome specific STR kits: PowerPlex Y: DYS391, DYS389I, DYS439, DYS389II, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390, DYS385a/b; and PowerPlex Y23: DYS576, DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS481, DYS549, DYS533, DYS438, DYS437, DYS570, DYS635, DYS390, DYS439, DYS392, DYS643, DYS393, DYS458, DYS385a/b, DYS456, Y_GATA_H4. Applied Biosystems megaplexes include AmpFlSTR Identifiler: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, Amelogenin, D5S818, FGA; AmpFlSTR Identifiler Direct: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, Amelogenin, D5S818, FGA; AmpFlSTR Identifiler Plus: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, Amelogenin, D5S818, FGA; AmpFlSTR NGM: D10S1248, VWA, D16S539, D2S1338, Amelogenin, D8S1179, D21S11, D18S51, D22S1045, D19S433, TH01, FGA, D2S441, D3S1358, D1S1656, D12S391; AmpFlSTR NGM SElect: D10S1248, VWA, D16S539, D2S1338, Amelogenin, D8S1179, D21S11, D18S51, D22S1045, D19S433, TH01, FGA, D2S441, D3S1358, D1S1656, D12S391, SE33; AmpFlSTR GlobalFiler: D3S1358, VWA, D16S539, CSF1PO, TPOX, Yindel, Amelogenin, D8S1179, D21S11, D18S51, DYS391, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, D2S1338; AmpFlSTR VeriFiler: D10S1248, D1S1656, Amelogenin, D2S1338, D22S1045, D19S433, TH01, D2S441, D6S1043, D12S391; AmpFlSTR MiniFiler: D13S317, D7S820, Amelogenin, D2S1338, D21S11, D16S539, D18S51, CSF1PO, FGA; AmpFlSTR Yfiler: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y_GATA_H4, DYS437, DYS438, DYS448. Promega Corporation and Applied Biosystems are no longer the only STR kit suppliers; manufacturer’s now include QIAGEN N.V. (Venlo, Netherlands) and Biotype (Dresden, Germany) (https://strbase.nist.gov/multiplx.htm).

The STRBase website compiles relevant information on current scientific literature for autosomal and Y chromosome STR marker systems, fact sheets from various kit manufacturers, statistics on tri-allele patterns, mutation rates observed for each chromosome locus, the sequence information for each short tandem repeat for motifs/nucleotide base combinations, genomic map positions, and allele size ranges (https://strbase.nist.gov). This website of compiled useful STR marker systems has been available since 1997. Each chromosome locus is fully described and links to the original publications, and authors are conveniently provided. PCR primer sequences for each kit are published, and concordant studies comparing various kit results have been compared for consistency between genotyping STR kit manufacturers. Where non-concordance was detected, NIST worked with the manufacturers to refine the science so that all data sets would be comparable for uploading into the NDIS DNA database system.

Specialty STR kits have been optimized for the detection of alleles from highly degraded DNA samples. The ability to type degraded DNA specimens was improved by redesigning the STR marker amplicons so that a smaller-sized polymerase chain reaction (PCR) product was created at each locus. This kit was called the AmpFlSTR MiniFiler PCR Amplification Kit. The kit contains reagents for the amplification of eight miniSTRs which are the largest-sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA) (Mulero et al. 2008; Bright et al. 2011). The MiniFiler kit was validated for casework use (Luce et al. 2009; Hill et al. 2007) and has been used on degraded skeletal remains and cigarette butts (Ip et al. 2014) and for war remains identifications successfully (Marjanović et al. 2009). A Chinese forensic STR kit called Sinofiler was specifically released for the Chinese forensic science laboratories (Shuqin Huang et al. 2010). The kit includes the STR loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D13S317, D16S539, D2S1338, D19S433, vWA, D18S51, D6S1043, D12S391, D5S818, and FGA. After evaluation for the Chinese Han population, the kit was deemed suitable. There was no statistical departure from expectation of Hardy–Weinberg Equilibrium (HWE) for all loci but D6S1043. There was no linkage disequilibrium in all pairs of loci examined. This kit was validated for this particular population group and country (Liu et al. 2014).

Future directions for STR technology. Two new trends are present in STR analysis. The first is to revive kinship analysis using DNA test methods and place it in a larger category called forensic genealogy. Interesting genealogy studies include a study of surnames and the founder male lineages (27 total) for the Old Order Amish males in Pennsylvania (Pollin et al. 2008). This study could account for 98% of the male lineages associated with the Lancaster area. Also, surname analysis and YSTR tests have been successfully applied for predictive value in Chinese Han populations to the level of 65% accuracy and increases to 80% accuracy for top four surnames (Shi et al. 2018). This approach can be useful as a “dragnet” concept where surnames can be predicted from Y chromosome haplotypes. Geography and restricted populations fare better with this approach as Y chromosome ancestral “footprints” are detectable in some populations to the level that they stretch back to 500 AD to an ancestral founder population; others are more difficult to use predictively but could still have usefulness for DNA investigative leads (Whiting and Miller Coyle 2019).

Familial DNA search methodology is different than ancestral surname searches since surname searches rely on the inheritance of paternal lineages by Y chromosome ancestry. Y chromosome ancestry is not one hundred percent accurate as there are some common Y chromosome haplotypes that are not apparently traceable back in historical records to the same family group. While seeking ancestors to your family genealogical tree can be useful with this method, it still requires some verification by investigation of property records, birth, marriage, and death certificates and other genealogical records to confirm a likely family relationship. The concept of a coincidental match in haplotype analysis using STR markers has to be considered as a fortuitous occurrence or the association was not documented back many generations ago in the family tree (https://www.ancestry.com). The coincidental match can be challenging to interpret using YSTR technology, but the DNA method is still highly valuable as an exclusionary tool.

Familial search, however, is a search of DNA databases by law enforcement for similar but not exact DNA profiles indicating a genetic relative that can provide a possible investigative lead in the case (https://criminal.findlaw.com/criminal-rights/familial-dna-searches.html). This DNA database search technique was able to identify through a DNA relative and investigation the Golden State Killer in 2018, approximately 30 years after his homicides began. This was the only effective manner in which he was identified and apprehended. Traditional DNA searches are performed to identify an exact high stringency DNA match, but with familial search techniques, partial matching at reduced stringency is permitted in the software-driven search to provide a candidate list of possible DNA matches. These individuals are then further evaluated and traditionally investigated to establish their possible role in the crime. Most state and federal authorities continuously collect known reference DNA samples from convicted offenders that are continuously uploaded into searchable DNA databases. If there is no familial search match, it may be due to the fact that there are no genetic relatives apprehended and convicted; therefore, there are no leads in that particular database. Law enforcement, however, does have the opportunity to access private ancestry-related DNA databases and uses those reference populations to also search for candidate leads. There are some justifiable concerns by civil liberties advocates that criticize familial DNA search technology as an invasion of personal privacy. The argument is that familial DNA searching affects the privacy of unconvicted genetic relatives, who are innocent of the crime, and this violates the Fourth Amendment in the Unites States Constitution which protects individuals against unreasonable searches. For this reason, some states such as Maryland have banned the use of familial search technology from being used; others like California have passed state legislation to allow familial searching in violent crime with policies in place for correct application and usage.

The second trend in human identification technology is to update and revise new DNA standards to fit with current technology and quality management strategies. The newest policies for year 2020 can be found at this website: https://www.nist.gov/news-events/news/2020/05/two-new-forensic-dna-standards-added-osac-registry. The Organization of Scientific Area Committees or OSAC is an organization with professional and scientific members that have expertise in 25 different forensic disciplines and have expertise in the areas of peer-reviewed scientific research, measurement science, statistics, and legal policy. The role of OSAC is to build the foundational science of DNA methods and promote the use of DNA standards for evaluating and implementing new technology.

In 2015, OSAC began by drafting new DNA standards that were then submitted to the Academy Standards Board (ASB) of the American Academy of Forensic Sciences (AAFS) and evaluated again by OSAC prior to posting on the registry. The new standards are (1) ANSI/ASB Standard 020, Standard for Validation Studies of DNA Mixtures, and Development and Verification of a Laboratory’s Mixture Interpretation Protocol and (2) ANSI/ASB Standard 040, Standard for Forensic DNA Interpretation and Comparison Protocols. The verification of a laboratory’s mixture interpretation protocol must demonstrate that a laboratory’s protocols produce consistent and reliable conclusions with DNA samples different from the ones used in the initial validation studies. This implies that a test period is required on adjudicated casework samples to validate that the research samples and the authentic casework samples are similar and function appropriately with the new protocol. The new standards also limit forensic science laboratories and prevent them from interpreting DNA mixtures that exceed validated methods especially regarding use on increasingly complex mixtures with additional contributors. The majority of current DNA units in forensic science laboratories still only interpret three person DNA mixtures from biological evidence. The new OSAC DNA standards are still complementary to the FBI’s DNA Quality Assurance Standards. They also build upon the Scientific Working Group on DNA Analysis Methods work, the SWGDAM guidelines. There still may be forensic science laboratory compliance issues as implementation of the OSAC DNA standards remains voluntary. Many forensic science laboratories are already working toward proper implementation, however, as it is best practice to follow the latest edition of DNA standards by OSAC to promote quality and scientific integrity in laboratory practices for forensic human DNA identification methods.

Conclusions

Quality control preserves the integrity of the DNA test result in STR analyses. Without reagent, equipment, and method checks and balances, there may be “protocol drift” that allows the analyst to deviate from the written document or method protocol thus creating variance from case to case. Part of public confidence building in forensic science laboratories is to convince the public that the scientific method is valid, accurate, and consistent from case to case regardless of who is on trial. Some of the most spectacular forensic fraudulent cases stem from this problem, where laboratory administration review and oversight is not sufficient to catch incorrect documentation or inconsistent DNA results. The Swecker and Wolf report (http://wwwcache.wral.com/asset/news/state/nccapitol/2016/09/07/15994768/254822-Swecker_Report.pdf) documents the type of quality assurance and quality control issues that can arise in presumptive and confirmatory blood identification tests and also created discrepancies in blood serological and DNA reports that became evident in the courtroom to defense counsel and science experts alike. The exhaustive fifteen thousand or more case reviews by outside experts and a three judge panel identified two hundred and thirty cases that resulted in retrials, acquittals, and exonerations simply based on poorly written and poorly enforced protocols. A key element to this issue was the fact that “inconclusive” was not written into the blood identification procedure as a test result option; when forced to choose, sometimes the analyst selected the incorrect result. The Inspector General of New York State issued an executive summary of a leading forensic science serology and DNA unit as well that found fraud in sexual assault kit evaluations for semen evidence. Along with poor quality screening of evidence by a fraudulent serologist, the DNA unit was inconsistent in the manner in which DNA mixtures were interpreted (https://ig.ny.gov/sites/g/files/oee571/files/2016-12/OCMEFinalReport.pdf). The manner in which the analyst decided the true number of contributors (NOC) to the DNA mixture was at issue; some were calculating NOC based on alleles called by the analysis software; others were including evidence of additional contributors below the analytical threshold.

All scientific methods have an error rate, and a process must be in place to guarantee that reagents, equipment, and methods are functioning properly to reduce the error rate. The four main areas highlighted in this chapter (DNA collection methods, polymerase chain reaction (PCR) factors, electrokinetic injection considerations, and DNA data interpretation) are detailed to indicate there are checks and balances to the procedures that are built into the forensic science laboratory procedures. Forensic science laboratories are also provided the guidance documents by the DNA Advisory Board and the Organization of Scientific Area Committees designed to enhance DNA test result consistency. Still, there are some criticisms to the quality control process in forensic DNA testing. “Negative controls also can’t rule out contamination of individual samples” (http://www.injusticeinperugia.org/viewfromwilmington.html). This statement is true. Most of the quality control in the DNA unit is designed to detect gross contamination events or reagent and equipment failures, but it is possible to have a single independent tube become contaminated, and it may go undetected in the surveillance system of the quality manager. However, there are internal forensic science laboratory DNA databases of the laboratory personnel that DNA results are screened against prior to providing a DNA report to a submitting agency to attempt to screen out accidental DNA contamination during the laboratory processing steps. Crime scene personnel and emergency services workers must be subpoenaed to provide an elimination known reference DNA sample for comparison to casework samples.

In addition to OSAC standards, enforcement by laboratory administration, and the personal ethics of the individual analyst, laboratory auditing is another method for reviewing the quality of a forensic laboratories work product. The American Society of Crime Laboratory Directors Laboratory Accreditation Board (ASCLAD-LAB) (www.ascld.org) is a not-for-profit professional society of crime laboratory directors and forensic science managers whose function is “to foster professional interests, assist the development of laboratory management principles and techniques; acquire, preserve, and disseminate forensic based information; maintain and improve communication among crime laboratory directors; and to promote, encourage, and maintain the highest standards of practice in the field.” ASCLD inspects forensic laboratories and accredits their practices and facilities every 5 years to review and maintain the integrity of the scientific processes used to evaluate forensic evidence in criminal casework. The ASCLD auditing procedure reviews the forensic laboratory process as a whole and makes recommendations for quality management improvement; however, the reviews are randomized and based on whatever becomes evident during the audit as the two week process can only provide a “spot check” on the complexities and total volume of casework that is processed in all of the units of the forensic science laboratory.

Many forensic science laboratories have also applied for and been granted ISO17025 accreditation status which is an external auditing process that is provided to all clinical and manufacturing laboratories. ISO/IEC 17025 standardization enables “laboratories to show that they operate competently and generate valid results, thereby promoting confidence in their work both nationally and around the world. It also helps facilitate cooperation between laboratories and other bodies by generating wider acceptance of results between countries. Test reports and certificates can be accepted from one country to another without the need for further testing, which, in turn, improves international trade” (https://www.iso.org/ISO-IEC-17025-testing-and-calibration-laboratories.html). These claims are true, and the need for global standardization is very helpful for sharing DNA data between country borders. If the DNA data is of high quality and can be shared, increased casework can be solved and more missing persons identified through quality controlled global DNA databases and STR analysis methods.