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Efficiency evaluation of the LT-DNA traces analysis modifications

Published:October 19, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.10.042

      Abstract

      Analysis of genetic profiles obtained from low template DNA samples (LT DNA) can be challenging because of increased probability of stochastic amplification artifacts occurrence. According to the recommendations of international genetic societies the quality of the LT-DNA traces results can be improved by applying low copy number (LCN) methods. Another strategy which allows to obtain better results of the analysis of LT-DNA traces is replicate the amplification of the same DNA sample and create consensus, composite (virtual pool profile) or real pool profile. The aim of the research was to analyze and compare the efficiency of modifications used in the testing of LT-DNA samples. Obtained results indicate that implementation of these methods in laboratory practice may lead to improvement in the quality of reported data from LT-DNA traces in genetic analyzes conducted to assist the justice system.

      Keywords

      1. Introduction

      Currently used STR amplification kits designed for forensic DNA profiling are much more sensitive than older generation systems used for genetic identification. It has therefore become possible to detect DNA profiles of samples containing a low level of genetic material (DNA starting template below 100 pg). Creating STR profile from LT-DNA template DNA evidence can provide important information in solving forensic cases. However, an analysis of genetic profiles obtained from this type of samples may be problematic. STR profiles of LT-DNA traces are often incomplete and the possibility of an occurrence of stochastic amplification artifacts (allele and locus drop-out, elevated stutter, heterozygote peak imbalance, allele drop-in) increase is observed [
      • Benschop C.
      • Haned H.
      • Sijen T.
      Consensus and pool profiles to assist in the analysis and interpretation of complex low template DNA mixtures.
      ,

      SWGDAM, Guidelines for STR Enhanced Detection Methods, (2014). Available at: http://media.wix.com/ugd/4344b0_29feed748e3742a5a7112467cccec8dd.pdf.

      ,
      • Cowen S.
      • Debenham P.
      • Dixon A.
      • et al.
      An investigation of the robustness of the consensus method of interpreting low-template DNA profiles.
      ]. For this reason, results of genetic typing can be difficult to interpret and therefore a careful analysis by an expert is require to correctly label STR alleles. The International Society for Forensic Genetics (ISFG) recommends particular caution in the reporting of results obtained by analysing these kind of traces [
      • Gill P.
      • Gusmao L.
      • Haned H.
      • et al.
      DNA commission of the International Society of Forensic Genetics: recommendations on the evaluation of STR typing results that may include drop-out and/or drop-in using probabilistic methods.
      ,
      • Gill P P.
      • Haned H.
      • Bleka O.
      • et al.
      Genotyping and interpretation of STR-DNA: low-template, mixturesand database matches-twenty years of research and development.
      ]. In some cases it is possible to improve the quality of genetic results of LT DNA samples using LCN techniques e.g. by increasing the amplification cycle number, reducing PCR reaction volume, purification and concentration of amplification products, increasing injection time or voltage during capillary electrophoresis. Another recommended solution, which can improve the quality of results of LT-DNA traces is performing at least three independent amplifications of the same DNA extract and creating consensus, composite (virtual pool profile) or real pool profile [
      • Benschop C.
      • Haned H.
      • Sijen T.
      Consensus and pool profiles to assist in the analysis and interpretation of complex low template DNA mixtures.
      ,
      • Parys-Proszek A.
      • Marcińska M.
      • Branicki W.
      • et al.
      Examination of LT-DNA traces – literature overview and general recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics (ISFG-PL).
      ]. Though implementation these modifications in workflow of DNA analysis is time-consuming and more expensive, it offers an opportunity to determine LT DNA genetic profile samples with a greater precision.

      2. Materials and Methods

      For the sensitivity analysis of the tested methods Control DNA 007 (Thermo Fisher Scientific) was selected ith the triplicates of following inputs: 200 pg, 100 pg, 70 pg, 50 pg, 40 pg, 30 pg, 20 pg and 10 pg DNA per reaction. Additionally, 39 different forensic LT-DNA traces were selected (blood, saliva, semen, contact traces). All forensic samples were subjected to magnetic DNA extraction with the Qiasymphony SP/AS workstation and the QIAsymphony DNA Investigator Kit (Qiagen). Genetic material was quantified using the Quantifiler Trio DNA Quantification Kit and a 7500 Real Time PCR System (Life Technologies). Three independent amplifications of the same extract were carried out using the same conditions: STR kit (NGM or NGM Select - Life Technologies), amount of DNA and analysis parameters. Samples after three independent PCRs and the samples containing mixture of above mentioned products with equal proportions were subjected to capillary electrophoresis using an ABI 3500xL DNA analyzer (Life Technologies). On the basis of the obtained results, a virtual pool profile -type consensus (containing repeated alleles identified at least twice in successive PCR reactions), as well as a composite (obtained by combining all detected alleles in the three replicates of PCR) and a real pool profile (obtained during electrophoresis of a sample combining in equal proportions its products of three amplification reactions) were created. In the next step, the remaining PCR products were purified on Amplicon Rx columns (Independent Forensic) and then subjected to electrophoresis. The research scheme is described in Fig. 1.
      Fig. 1
      Fig. 1Subsequent steps analysis LT-DNA sample.

      3. Results and discussion

      For most of the LT-DNA samples an improvement of results was observed in number of typed STR markers when additional modifications of analysis were performed. Locus/allele drop-out was the most common stochastic effect occurring in tested samples. Standard analysis of control DNA (007) with LOQ (limit of quantification) parameters resulted in drop-outs starting from 70 pg DNA input (mean allelic drop-out – 5%) and lower. After applying tested methods first drop-outs occurred from 50 pg DNA input (Amplicon Rx purification – 1%, consensus profile – 25% and real pool profile – 37%), 40 pg DNA input (composite profile – 12%), 20 pg DNA input (Amplicon Rx purification and real pool profile – 6%). All of the dilutions of Control DNA 007 in each modification showed no drop-in phenomenon and no increase in the height of stutters. The improvement in data quality was even more evident when analyzing the results with LOD (limit of detection) parameters. Noticeable increase in the intensity of the drop-out effect occurred only in samples with concentrations of 20 pg and lower. In contrast to LOQ parameters during data analyzing by LOD parameters drop-ins and elevated stutter for Control 007 samples were observed, mainly those purified on Amplicon Rx columns. The most numerous group of forensic samples with the positive results of genetic profiling above 75%, were the samples purified on Amplicon Rx columns and then report the real pool profile. However, due to the time-consuming, manual procedure, such analysis seems to be suitable for use only in justified cases. Consensus and composite methods also in many cases improve the quality of the results of LT-DNA samples, but especially composite method generates more drop-in artifacts. Intensification of stochastic effects in mixture LT-DNA samples often made it impossible to distinguish valid results from artifacts and posed a risk of making errors during reporting data. That is why it seems to be more effective to use consensus method (based on data that was confirmed at least twice in independent PCRs) than other tested modifications. The authors assume it is more justified to use the composite approach, real pool profile or column purification when analyzing LT-DNA traces from a single source trace. It should be emphasized that the decision to apply any modification to improve the quality of the results of LT-DNA trace depends on the data obtained from the DNA quantification and on the quality of the electrophoregrams of standard PCR reaction. Based on this information, it is the expert who decides the path to follow when analyzing LT-DNA samples in order to obtain to provide the most reliable genetic identification results.

      4. Conclusion

      The tested methods increased the efficiency of positive determination of STR markers in samples with a low amount of genetic material. As demonstrated above, in some cases, it is advisable to use additional purification of amplification products on Amplicon Rx columns, creating consensus and composite virtual profiles or a common real profile in LT DNA samples tested for the purposes of the judiciary. Though the use of mentioned modifications extends the time of sample analysis and generates high costs, it may increase the possibility of obtaining more reliable results of genetic tests.

      Funding source

      Supported by the Institute of Forensic Research in Krakow, Section of Forensic Genetics. The consent for study biological samples and implementation of a research project number III G/22/23 were approved by the Research Commission of Institute of Forensic Research in Krakow.

      Conflict of interest statement

      The authors declare that they have no competing interests.

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