A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology

Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single-cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30–150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well-performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%–75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 μL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single-cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.     

Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA

DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. 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). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.     

The development of reduced size STR amplicons as tools for analysis of degraded DNA

New multiplex PCR sets of commonly used short tandem repeat (STR) markers have been developed to produce PCR products that are reduced in size when compared to standard commercial STR kits. The reduction in size of these amplicons can facilitate the examination and analysis of degraded DNA evidence by improving amplification efficiency. This "miniSTR" approach will permit current forensic practitioners to use STR markers and instrumentation already present in their laboratories and to generate genotyping data that is directly comparable to reference samples and searchable through the FBI's Combined DNA Index System (CODIS) databases. This paper discusses the development of these new primer sets and presents some initial results in the analysis of degraded and aged DNA samples. A method for removal of problematic fluorescent dye artifacts is also described. Comparison studies in over 100 samples have verified that these miniSTR primers can provide fully concordant results to commercial STR kits and can provide improved signal from degraded DNA specimens. These miniplex sets should prove valuable in the analysis of samples where allele dropout and reduced sensitivity of larger STR alleles occurs.     

Rapid amplification of commercial STR typing kits

Forensic DNA typing is currently conducted in approximately 8–10 h. The process includes DNA extraction, quantitation, multiplex PCR amplification, and fragment length detection. Today's commercial multiplex short tandem repeat (STR) typing kits are not optimized for rapid PCR thermal cycling. Current protocols require approximately 3 h for amplifying a multiplex containing 15 STR loci plus amelogenin. With the continuing development of miniaturization technologies such as microfluidic and micro-capillary devices, there is a desire to reduce the overall time required to type DNA samples. Such miniature devices could be used for initial screening at a crime scene, at a border, and at airports. There is also the benefit of reducing the required PCR amplification time for labs typing single-source reference samples. Surveys of fast processing polymerases working in combination with rapid cycling protocols have resulted in the development of a ‘rapid’ PCR amplification protocol. Results are obtained in less than 36 min run on a standard peltier-based thermal cycler employing a heating rate of 4 °C/s. Capillary electrophoresis characterization of the PCR products indicates good peak balance between loci, strong signal intensity and minor adenylation artifacts. Genotyping results are concordant with standard amplification conditions utilizing a standard 3 h (non-rapid) thermal cycling procedure. The rapid assay conditions are robust enough to routinely amplify 0.5 ng of template DNA (with 28 cycles).     

Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples

The amplification and typing conditions for the 13 core CODIS loci and their forensic applicability were evaluated. These loci are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. Results were obtained using the multiplex STR systems AmpFlSTR Profiler Plus and AmpFlSTR COfiler (Applied Biosystems, Foster City, CA), GenePrint PowerPlex (Promega Corporation, Madison, WI), and subsets of these kits. For detection of fluorescently labeled amplified products, the ABI Prism 310 Genetic Analyzer, the ABI Prism 377 DNA Sequencer, the FMBIO II Fluorescent Imaging Device, and the Fluorlmager were utilized. The following studies were conducted: (a) evaluation of PCR parameter ranges required for adequate performance in multiplex amplification of STR loci, (b) determination of the sensitivity of detection of the systems, (c) characterization of non-allelic PCR products, (d) evaluation of heterozygous peak intensities, (e) determination of the relative level of stutter per locus, (f) determination of stochastic PCR thresholds, (g) analysis of previously typed case samples, environmentally insulted samples, and body fluid samples deposited on various substrates, and (h) detection of components of mixed DNA samples. The data demonstrate that the commercially available multiplex kits can be used to amplify and type STR loci successfully from DNA derived from human biological specimens. There was no evidence of false positive or false negative results and no substantial evidence of preferential amplification within a locus. Although at times general balance among loci labeled with the same fluorophore was not observed, the results obtained were still valid and robust. Suggested criteria are provided for determining whether a sample is derived from a single source or from more than one contributor. These criteria entail the following: (a) the number of peaks at a locus, (b) the relative height of stutter products, and (c) peak height ratios. Stochastic threshold levels and the efficiency of non-templated nucleotide addition should be considered when evaluating the presence of mixtures or low quantity DNA samples. Guidelines, not standards, for interpretation should be developed to interpret STR profiles in cases, because there will be instances in which the standards may not apply. These instances include (a) a primer binding site variant for one allele at a given locus, (b) unusually high stutter product, (c) gene duplication, and (d) translocation.     

TWGDAM validation of AmpFlSTR PCR amplification kits for forensic DNA casework

Laboratory procedures used in short tandem repeat (STR) analysis were subjected to various scenarios that assessed reliability and identified potential limitations. These validation studies were designed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board (DAB) (17,18). Various DNA samples were amplified by the polymerase chain reaction (PCR) using AmpFlSTR PCR Amplification Kits (i.e., AmpFlSTR Green I, Profiler, Profiler Plus, and COfiler kits), detected with ABI Prism instrumentation, and analyzed using GeneScan and Genotyper software. Data acquired in these studies reinforced an existing body of knowledge and expertise regarding application and interpretation of STR typing in the forensic science community. Consistent STR genotypes were detected in various body tissues and fluids. Inter-laboratory comparisons produced concordant genotype results. Quantitative interpretational aids for DNA mixtures were characterized. Ability of the typing systems to type potentially compromised samples reliably was evaluated. Nonprobative case evidentiary DNA was successfully amplified, genotyped, and interpreted. Potential limitations or cautionary factors in the interpretation of minimal fluorescence intensity were demonstrated. Differential amplification between loci was observed when PCR was inhibited; preferential amplification typically was not. Single AmpFlSTR locus amplification did not offer consistent benefit over AmpFlSTR multiplexing, even in cases of DNA degradation or PCR inhibition. During rigorous evaluation, AmpFlSTR PCR Amplification Kits reproducibly yielded sensitive and locus-specific results, as required in routine forensic analyses.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

PCR扩增3因素对低拷贝模板STR分型的影响研究

目的探讨低拷贝模板(low copy number,LCN)STR扩增方法,提高LCN检材的检验成功率。方法采用Profiler P lusTM试剂盒与9947A对照DNA,改变Taq酶量、体系、循环次数3个因素进行扩增检验,了解各变量对扩增检测的影响。结果对低拷贝模板DNA,单纯增加Taq酶量或反应体系,扩增效率改善不明显;增加循环数,显著提高检验灵敏度;低于0.01ng的模板DNA,同时增加扩增体系、Taq酶量、循环数在一定程度上提高扩增效率。结论对于影响扩增的Taq酶量、体系、循环次数3个因素中,循环数影响最大,但应慎用34次及以上循环数;三者同时增加,对于低于0.01ng模板DNA的扩增可有效改善。     

Full STR profile of a 67-year-old bone found in a fresh water lake

DNA extraction from and DNA typing of fresh water-exposed aged bone specimens poses a challenging task and is not very well examined. This study presents a new method to extract typable DNA from such problematic bone specimens. The procedure comprises low-heat drilling and cryogrinding, mild lysis conditions, and silica-column-based DNA cleaning. DNA quantity is assessed by quantitative PCR prior to short tandem repeat (STR) amplification. The procedure was employed with a 67-year-old tibia bone fragment recovered from a fresh water lake and succeeded to produce a full STR profile using the MPX-SP1 and MPX-SP2 mini-STR kits and a partial profile with 12 successfully amplified STRs using the Identifiler STR kit. The new method for the extraction of DNA from aged fresh water-exposed bone specimens presented herein was successfully applied to prepare DNA of sufficient quality and quantity to generate a full STR profile.     

Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes

Abstract:  With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus? STR reactions alone and in combination with AmpliTaq^® Gold. All reactions included the additional step of a post‐PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.     

DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones.     

Analysis of human fecal material for autosomal and Y chromosome STRs

Human stool samples from eight volunteers were stored under various conditions and extracted by three different procedures. Fecal material and tissue paper soiled with fecal material obtained from a crime scene were also extracted. Extracted DNA was amplified using the AmpFlSTR Profiler Plus, AmpFlSTR COfiler, and the AmpFlSTR Identifiler PCR amplification kits for the detection of the autosomal STR allelic patterns. DNA extracted from the male volunteers and from the soiled tissue paper evidence sample was also amplified using the Y-PLEX 6 and Y-PLEX 5 amplification kits. Analysis of the amplified products was carried out by capillary electrophoresis on the ABI PRISM 310 Genetic Analyzer. Autosomal and Y-STR profiles obtained from the fecal material were concordant with the results from the donors' buccal swabs.     

A study on the effects of degradation and template concentration on the amplification efficiency of the STR Miniplex primer sets

In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called "Miniplexes" to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to a commercial kit. We also conducted sensitivity and peak balance studies of three Miniplex sets. Lastly, we report a case study on two human skeletal remain samples collected from different environmental conditions. In both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles when compared to the larger sized amplicons from the commercial kit. Correct genotypes were obtained at template concentrations as low as 31 pg/25 microL. Overall, our data confirm that our redesigned primers can increase the probability of obtaining a usable profile in situations where standard kits fail.     

Validation and casework testing of the BioPlex-11 for STR typing of telogen hair roots

A new STR typing strategy has been developed allowing the simultaneous amplification and subsequent analysis of 11 polymorphic systems with amplicon sizes smaller than 270bp. The multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THO1, FGA and VWA) as well as four additional STR systems selected for their robustness (D2S1338, D12S391, TPOX and D5S818) together with the sex-specific locus amelogenin. After PCR amplification, the multiplex reaction is splitted into two sets of STR multiplexes by using biotin labelled primers only for one set. Using streptavidin-coated Sepharose beads five STR systems are separated from the other six systems prior to being analysed in two different runs on a capillary gel electrophoresis instrument. The multiplex system was developed and tested especially for the use in forensic casework if only limited amounts or highly degraded DNA is available, for instance, when isolated from telogen hair roots.     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.     

Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples

The analysis of LCN or highly degraded DNA samples presents a challenge for forensic science. Improving the quantity and/or quality of samples would greatly increase the profiling success rate from LCN and degraded samples. Whole genome amplification (WGA) is one method that has such potential. Two commercially available WGA kits, GenomePlex and GenomiPhi, were investigated for use on LCN and degraded DNA samples. Both kits amplified genomic DNA, producing microgram quantities from sub-nanogram templates. Profiling success of LCN DNA samples was increased, with improvements of over 700% from 10pg template DNA compared to non-WGA-amplified control samples. The amplification success with degraded DNA was also improved by WGA. Degraded DNA was simulated using restriction enzymes to demonstrate that the application of WGA can result in the typing of STR loci that could not previously be amplified. An increase in artefacts, such as stutter alleles and amplification biases, were observed in many samples. Results show that WGA is capable of increasing both the quality and quantity of DNA, and has the potential to improve profiling success from difficult samples in forensic casework.     

法医DNA标准物质备选细胞STR等位基因片段长度的定值

目的 研究建立法医DNA标准物质备选细胞基因组STR基因座等位基因片段长度标准定值的方法.方法 利用有机法提取HPF和HSSM细胞基因组DNA并进行STR复合扩增,将产物进行电泳检测.利用Gene Mapper软件分析电泳结果,记录STR基因座等位基因的数值,并对目前我国公安系统应用较为广泛的DNATyperTM15、IdentifilerTM两种试剂盒扩增产物进行相应的DNA片段长度(bp)统计以及定值.结果 HPF为男性个体细胞,HSSM为女性个体细胞.HPF和HSSM细胞DNATyperTM15系统等位基因片段长度范围分别为126.26±0.05～367.53±0.20bp和125.33±0.07～370.08±0.17bp,IdentifilerTM系统等位基因片段长度范围分别为117.22±0.04～340.02±0.08bp和117.21±0.03～323.86±0.09bp.结论 对STR基因座等位基因片段长度进行标准定值,可为法医DNA标准物质提供有效的溯源途径.     

Swiss Caucasian population data for the STR loci D2S1338 and D19S433 using the AmpFISTR SGM plus PCR amplification kit

Allele and genotype frequencies for the ten STR loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, FGA were determined in a Swiss Caucasian population sample (n=206) using the AmpFISTR SGM Plus Amplification kit. Electrophoresis was carried out on an ABI PRISM CE 310 Genetic Analyzer instrument. Previously, allele frequencies were published for the 13 STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO and D16S539 for the same samples (n=206) amplified with the AmpFISTR Profiler Plus and Cofiler PCR Amplification kits. Since the results for the eight loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, THO1, D16S539 shared between the AmpFISTR SGM Plus, Profiler Plus and Cofiler PCR Amplification kits already are published, only the allele frequencies for the two STR loci D2S1338 and D19S433 are reported in this paper. The two loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 15 loci (amplified with the Profiler, Cofiler, and SGM Plus amplification kits). The allelic frequency data can be used in forensic analyses to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.     

Validation of the Short Amplicon Multiplex Q8 Including the German DNA Database Systems*

Abstract: We have developed a concept to enable the analyzing of degraded stains with limited DNA template quantity. Therefore we have constructed a short tandem repeat (STR) multiplex including the German DNA database systems (Q8). The amplicon lengths are smaller than 280 bp. For the validation of Q8 over 50 degraded samples were investigated. Amplifications were performed with “low copy number” PCR, the number of PCR cycles was increased to 33 and the reaction volume was decreased to 12.5 μL. Compared with the MPX2 and Nonaplex kit, the average success rate was increased using the Q8 kit by approximately 20% and 30%, respectively. The efficiency of a sensitive STR multiplex with reduced amplicon lengths was confirmed in comparing the success rates of Q8 for typing degraded samples and samples with limited amount of DNA template while partial profiles were observed with the majority of the samples using commercially available kits.     

Next generation sequencing technology in Second World War victim identification

The killings during the Second World War (WWII), with nearly 100,000 victims, is one of the greatest losses of life in Slovenia’s modern history and most of the victims are still buried in hidden mass graves and remain unidentified. Identity, ancestry, and phenotypic SNPs, as well as STR markers are already used for solving various cases with Next Generation Sequencing (NGS) technology. In this study, the Precision ID GlobalFiler NGS STR panel was used to identify the WWII victim that could not be identified with capillary electrophoresis (CE) analyses because limited statistical support was obtained after amplification of autosomal STRs using CE STR kits. Bones and teeth were analysed and compared to family references (nephew and niece on paternal line). Prior to DNA isolation 0.5 g of powder was decalcified. The DNA was purified in a Biorobot EZ1 device. The nuclear DNA of the samples was quantified with the PowerQuant kit. Because the recommended posterior probability (PP) of 99.9% was followed with the goal of high confidence of correct identification, the NGS STR Panel was used, and after the analysis of additional STR loci the statistical calculation showed a PP of 99.99986%, showing that a large enough number of genetic markers were analysed when identifying the skeletal remains of the aunt. PP value endorsed the hypothesis that the tooth and bone samples were from individual related to the family references rather than from unrelated individual. In presented case, NGS technology proved to be a powerful tool for increasing the number of autosomal STRs needed for identification of WWII victims when linear markers cannot be used for comparison and only distant relatives are available for analyses.