Developmental validation of the PowerPlex(®) ESI 16/17 Fast and PowerPlex(®) ESX 16/17 Fast Systems.

The PowerPlex(®) ESI 16 Fast, ESI 17 Fast, ESX 16 Fast, and ESX 17 Fast Systems represent faster cycling versions (50min or less) of the PowerPlex(®) ESI and ESX Systems released by Promega in 2009 to accommodate the ENFSI and EDNAP groups' call for new STR multiplexes for Europe. In addition to amplification of purified DNA samples, these new faster cycling systems allow for direct amplification from single-source blood and buccal samples deposited on FTA(®) and nonFTA paper as well as from SwabSolution™ extracts of buccal swabs without the need for purification and quantitation. There are no changes to the autosomal primer pair sequences in the PowerPlex(®) ESI Fast and ESX Fast Systems compared to the original multiplexes, and full concordance at all autosomal loci and amelogenin was observed with data generated previously with the original PowerPlex(®) ESI and ESX Systems. This paper describes the developmental validation study performed on these new fast systems following guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and those of the DNA Advisory Board (DAB). Validation data demonstrate that these systems are sensitive for detecting low levels of DNA while also being capable of generating robust profiles from the high amount of input DNA present in direct-amplification samples. These systems are also tolerant to both high concentrations of PCR inhibitors as well as to slight variations in the final concentration of master mix and primer pair present in the amplification reaction that might be encountered due to pipetting error. The results of this validation study demonstrate that these systems may be used on multiple thermal cyclers and capillary electrophoresis platforms.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.

Developmental validation of the PowerPlex(®) 21 System.

The PowerPlex(®) 21 System is a STR multiplex that has been optimized for casework samples while still being capable of database workflows including direct amplification. The loci included in the multiplex offer increasing overlap with core loci used in different countries and regions throughout the world. The PowerPlex(®) 21 System contains D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, Amelogenin, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA. These loci represent all 13 core CODIS loci in addition to loci commonly used in Asia and Europe. A developmental validation study was completed to document performance capabilities and limitations of the PowerPlex(®) 21 System. Data from this validation work served as the basis for the following conclusions: genotyping of single-source samples was reliable across a range of template DNA concentrations with >95% alleles called at 50 pg. Direct amplification of samples from FTA(®) storage cards was successfully performed using the reagents provided with the system and modified cycling protocols provided in the technical manual. Mixture analysis showed that over 95% of minor alleles were detected at 1:9 ratios. Reaction conditions including volume and annealing temperature as well as the concentrations of primers, DNA polymerase, magnesium, and Master Mix were shown to be optimal and able to withstand moderate variations without affecting system performance. Reproducible results were generated by different users at different sites. Finally, concordance studies showed consistent results when comparing the PowerPlex(®) 21 System with other commercially available STR-genotyping systems.

Characterising the STR locus D6S1043 and examination of its effect on stutter rates.

The forensic analysis of DNA is most often undertaken by the amplification of short tandem repeats (STR) using the polymerase chain reaction (PCR). DNA amplification can result in production of the target allele amplicon and a by-product called stutter. Stutter is the result of the miscopy of the target allele and is typically one repeat smaller. Stutter is traditionally described as a ratio of stutter and allele height; stutter ratio (SR). The challenge to DNA profile interpretation is most serious whenever stutter products are of a similar height to the minor allelic peaks in a mixed DNA profile. An accurate assignment of peaks and the prediction of their height is important when objectively interpreting forensic DNA profiles. The longest uninterrupted stretch (LUS) of tandem repeats within the allele has previously been shown to be a good predictor of stutter ratio. LUS is determined by sequencing a range of observed alleles at a locus. The locus D6S1043 is a relatively new locus to appear in commercial forensic DNA testing kits. To date however, there has been no comprehensive report of sequencing of this locus. In this work, we sequence a sample of D6S1043 alleles to determine LUS values and investigate allele repeat number and LUS as explanatory variables for SR.

Mouse cell line authentication.

The scientific community has responded to the misidentification of human cell lines with validated methods to authenticate these cells; however, few assays are available for nonhuman cell line identification. We have developed a multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.

Haplotype data for 23 Y-chromosome markers in four U.S. population groups.

The PowerPlex Y23 kit contains 23 Y-chromosomal loci including all 17 of the markers in the Yfiler Y-STR kit plus six additional markers: DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643. We have typed 1032 unrelated population samples from four self-declared US groups: African Americans, Asians, Hispanics, and Western European Caucasians. An analysis of the population genetic parameters and the improvement of adding additional Y-STR markers to the dataset are described.

Developmental validation of the PowerPlex® 18D System, a rapid STR multiplex for analysis of reference samples.

As short tandem repeat markers remain the foundation of human identification throughout the world, new STR multiplexes require rigorous testing to ensure the assays are sufficiently robust and reliable for genotyping purposes. The PowerPlex(®) 18D System was created for the direct amplification of buccal and blood samples from FTA(®) storage cards and reliably accommodates other sample materials. The PowerPlex(®) 18D System allows simultaneous amplification of the 13 CODIS loci and amelogenin along with four additional loci: Penta E, Penta D, D2S1338, and D19S433. To demonstrate suitability for human identification testing, the PowerPlex(®) 18D System was tested for sensitivity, concordance, inhibitor tolerance, and performance with thermal cycling and reaction condition variation following SWGDAM developmental validation guidelines. Given these results, PowerPlex(®) 18D System can confidently be used for forensic and human identification testing.

Capillary electrophoresis and 5-channel LIF detection of a 26plex autosomal STR assay for human identification.

Multiplex polymerase chain reaction (PCR) is a common method used for DNA typing in forensic and paternity cases. There are numerous commercial short tandem repeat (STR) multiplex assays currently available to the forensic community. These assays amplify the core Combined DNA Index System (CODIS) STR loci for entry into the US. DNA database. Additional non-CODIS loci, which are considered genetically unlinked to the CODIS loci, can be useful in resolving challenging cases such as missing persons and mass disaster victim identification, paternity testing, and immigration testing. An STR multiplex has been successfully developed with 25 non-CODIS autosomal loci plus the sex-typing locus amelogenin for a total of 26 loci in a single 26plex amplification reaction. This chapter will focus on the preparation and the use of the 26plex assay with DNA samples for the purpose of human identification.

Authentication of African green monkey cell lines using human short tandem repeat markers.

BACKGROUND: Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification. RESULTS: The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line. CONCLUSIONS: A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.

STR sequence analysis for characterizing normal, variant, and null alleles.

DNA sequence variation is known to exist in and around the repeat region of short tandem repeat (STR) loci used in human identity testing. While the vast majority of STR alleles measured in forensic DNA laboratories worldwide type as "normal" alleles compared with STR kit allelic ladders, a number of variant alleles have been reported. In addition, a sequence difference at a polymerase chain reaction (PCR) primer binding site in the DNA template can cause allele drop-out (i.e., a "null" or "silent" allele) with one set of primers and not with another. Our group at the National Institute of Standards and Technology (NIST) has been sequencing variant and null alleles supplied by forensic labs and cataloging this information on the NIST STRBase website for the past decade. The PCR primer sequences and strategy used for our STR allele sequencing work involving 23 autosomal STRs and 17 Y-chromosome STRs are described along with the results from 111 variant and 17 null alleles.

Concordance and population studies along with stutter and peak height ratio analysis for the PowerPlex ® ESX 17 and ESI 17 Systems.

The PowerPlex(®) ESX 17 and ESI 17 Systems for short tandem repeat (STR) amplification were developed by the Promega Corporation to meet the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling (EDNAP) Group recommendations for increasing the number of STR loci included in the European Standard Set (ESS). The PowerPlex ESX 17 and ESI 17 Systems utilize different PCR primer combinations to co-amplify the following 17 loci: D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, vWA, SE33, and the sex-typing locus amelogenin. A total of 1443 U.S. population samples were evaluated with pre-commercialization versions of both kits. Stutter and heterozygote peak height ratios have been used to characterize kit performance. Typing results have been used to estimate the match probabilities provided by the chosen loci as well as in concordance studies. Full concordance between the typing results for the two kits was observed in 99.994% (49,055 out of 49,062) STR allele calls compared. All genotyping discrepancies were confirmed by DNA sequence analysis. As a result of these comparisons, a second forward primer for the D22S1045 locus has been added to the PowerPlex ESX 17 System to address a primer binding site mutation and the D1S1656 locus reverse primer in the PowerPlex ESI 17 System was modified to eliminate an amplification-efficiency reducing primer dimer.

Linkage disequilibrium analysis of D12S391 and vWA in U.S. population and paternity samples.

Recently, the European Network of Forensic Science Institutes voted to adopt five additional STR loci (D12S391, D1S1656, D2S441, D10S1248, and D22S1045) to their existing European Standard Set of seven STRs (TH01, vWA, FGA, D8S1179, D18S51, D21S11, and D3S1358). The D12S391 and vWA loci are located 6.3megabases (Mb) apart on chromosome 12. Ideally for use in forensic analyses, genetic markers on the same chromosome should be more than 50Mb in physical distance in order to ensure full recombination and thus independent inheritance. The purpose of this study was to evaluate if the closely located D12S391 and vWA loci are independent and, consequently, if these loci can be included in the product rule calculation for forensic and kinship analyses. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium between the D12S391 and vWA loci were tested using n=654 unrelated U.S. African American, Caucasian, and Hispanic samples, and n=764 father/son paternity samples. In the unrelated U.S. population samples, no significant departures from HWE were detected for D12S391 or vWA. No significant evidence of linkage disequilibrium was observed between the loci in the population samples. However, significant linkage disequilibrium was detected in U.S. African American, Caucasian, and Asian father/son samples with phased genotypes. No significant linkage disequilibrium was detected for U.S. Hispanic paternity samples. The use of phased father/son pairs allowed for robust detection of linkage disequilibrium between D12S391 and vWA. In unrelated population samples, linkage disequilibrium is present but more difficult to detect due to the large number of possible haplotype combinations and unknown allelic phase. For casework analyses that involve unrelated or related individuals, the single-locus genotype probabilities for D12S391 and vWA should not be multiplied to determine the match probability of an autosomal STR profile. Since the D12S391 and vWA loci are not independent, it is recommended that the observed combination of alleles at D12S391 and vWA should be treated as a non-independent diplotype for profile probability calculations. The observed haplotype frequencies for U.S. African American, Caucasian, Hispanic, and Asian populations are provided for match probability calculations.

A 26plex autosomal STR assay to aid human identity testing*.

A short tandem repeat multiplex assay has been successfully developed with 25 autosomal loci plus the sex-typing locus amelogenin for a total of 26 amplified products in a single reaction. Primers for the loci were designed so that all of the amplicons present were distributed from 65 base pairs (bp) to less than 400 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. A multiplex design strategy was developed to overcome challenges encountered in creating this assay. The limits of the multiplex were tested, resulting in the successful amplification of a wide range of genomic DNA sample concentrations from 2 ng to as low as 100 pg with 30 cycles of PCR. The 26plex has the potential to benefit the forensic community for reference sample testing and complex relationship evaluation.

Demonstration of rapid multiplex PCR amplification involving 16 genetic loci.

Current forensic DNA typing is conducted in approximately 8-10h. Steps include DNA extraction, quantification, polymerase chain reaction (PCR) amplification of multiple short tandem repeat (STR) loci, capillary electrophoresis separation with fluorescence detection, data analysis and DNA profile interpretation. The PCR amplification portion of the workflow typically takes approximately 3h with standard thermal cycling protocols. Here we demonstrate a rapid cycling protocol that amplifies 15 STR loci and the sex-typing marker amelogenin from the Identifiler STR typing kit in less than 36 min. This rapid protocol employs commercially available polymerases and the widely used GeneAmp 9700 thermal cycler. Complete concordance of STR allele calls (for 60 samples) between the rapid and standard thermal cycling protocols were observed although there was incomplete adenylation at several of the loci examined and some PCR artifacts were detected. Using less than 750 pg of template DNA and 28 cycles, STR peaks for all loci were above a 150 relative fluorescent unit (RFU) detection threshold with fully adequate inter-locus balance and heterozygote peak height ratios of greater than 0.84.

Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples.

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible-often directly next to the identified repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.

Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits.

The AmpFlSTR MiniFiler polymerase chain reaction amplification kit developed by Applied Biosystems enables size reduction on eight of the larger STR loci amplified in the Identifiler kit, which will aid recovery of information from highly degraded DNA samples. The MiniFiler Kit amplifies CSF1PO, FGA, D2S1338, D7S820, D13S317, D16S539, D18S51, and D21S11 as well as the sex-typing locus amelogenin. A total of 1308 samples were evaluated with both the MiniFiler and Identifiler STR kits: 449 African American, 445 Caucasian, 207 Hispanic, and 207 Asian individuals. Full concordance between Identifiler and MiniFiler Kits was observed in 99.7% (10,437 out of 10,464) STR allele calls compared. The 27 differences seen are listed in Table 1 and encompass the loci D13S317 (n = 14) and D16S539 (n = 10) as well as D18S51 (n = 1), D7S820 (n = 1), and CSF1PO (n = 1). Genotyping discrepancies between the Identifiler and MiniFiler kits were confirmed by reamplification of the samples and further testing using the PowerPlex 16 kit in many cases. DNA sequence analysis was also performed in order to understand the nature of the genetic variations causing the allele dropout or apparent repeat unit shift.