Abstract
DNA profiles from degraded samples often suffer from information loss at the longer short tandem repeat (STR) loci. Sensitising the reactions, either by performing additional PCR cycles or increasing the capillary electrophoresis injection settings, carries the risk of over-amplifying or overloading the shorter fragments. We explored whether profiling of degraded DNA can be improved by preferential capturing of the longer amplified fragments. To this aim, a post-PCR purification protocol was developed that is based on AMPure XP beads that have size-selective properties. A comparison was made with an unselective post-PCR purification system (DTR gel filtration) and no purification of the PCR products. Besides a set of differently and serially degraded single source samples, unequal mixtures of degraded DNAs were analysed, in order to extract more genotyping information for the minor contributor without overloading the major component at the shorter amplicons. Purification by the AMPure protocol resulted in higher peak heights especially for the longer amplicons, while DTR gel filtration gave higher peaks for all amplicon sizes. Both purification methods presented more detected alleles, with the AMPure protocol performing slightly better, on average. In conclusion, the in-house developed AMPure protocol can be employed to improve STR profile analysis of degraded single source and (unequally) mixed DNA samples.
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Acknowledgments
This study was supported by a grant from the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO) within the framework of the Forensic Genomics Consortium Netherlands. The authors would like to thank Arnoud Kal for critically reading the manuscript.
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Supplementary Fig. 1
DNA integrity of Covaris-degraded samples. Three DNA samples were subjected to seven Covaris settings (Supplementary Table 1) to fragment the DNA. DNA quality is visualised on a QIAxcel system (Qiagen, Venlo, The Netherlands) using DNA size markers of 250 bp–8 kb (four fragments can be seen: 250 bp, 500 bp, 750 bp and 1 kb) and 25 bp–450 bp (fragments at 25 bp intervals) (JPEG 29 kb)
Supplementary Fig. 2
Post-PCR purification by AMPure XP magnetic beads (according to the manufacturer’s protocol) in various volume ratios of PCR product and beads (1:1.8 down to 1:0.4). These experiments are all based on 500 pg pristine DNA007 NGM PCR amplifications of which 24 μL was used during the AMPure purification and 1 μL purified PCR product was analysed by CE. A non-purified control sample is shown in the bottom row. On each row, the first (D10S1248; 70–125 bp) and the last locus (D2S1338; 280–360 bp) of the blue (6-FAM) channel are shown. The peaks of the control sample are labelled both with the allele call and the peak height; the other samples are labelled with the peak height only. The Y-axis is scaled to 6,000 rfu in all panels (JPEG 150 kb)
Supplementary Fig. 3
Average ratio of the fold increase in peak heights of the AMPure protocol and DTR gel filtration for a no or little degraded samples and b severely degraded samples. The loci are ordered by fragment length (from shorter ones on the left-hand to longer fragments on the right-hand side). The error bars represent the standard deviation. The horizontal line at ratio 1 represents an equal fold increase in peak height after DTR gel filtration and AMPure purification. a For the no or little degraded samples 224 ratios could be calculated, varying between 9 (for D8S1179 and vWA) and 18 (for D22S1045 and D12S391) ratios per locus. b For the severely degraded samples 157 ratios could be calculated, varying between 3 (for D12S391) and 18 (for D2S441 and D3S1358) ratios per locus. No ratios could be determined for the longer loci, due to the lack of genotyping information in the profiles of non-purified PCR products (JPEG 65 kb)
Supplementary Fig. 4
The effect of post-PCR purification by DTR gel filtration or AMPure purification on the number of donor alleles detected for non-purified PCR products. Gained alleles are shown as a positive number, saturated alleles as a negative value. Sample degradation methods are abbreviated as: C = Covaris, UV = UV-light, B = bone sample, FT = freeze/thaw cycles and S = swab overgrown with microbes. Alleles on short loci are shown in red, on mid-range loci in grey and on long loci in green (JPEG 84 kb)
Table S1
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ESM 1
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ESM 2
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Westen, A.A., van der Gaag, K.J., de Knijff, P. et al. Improved analysis of long STR amplicons from degraded single source and mixed DNA. Int J Legal Med 127, 741–747 (2013). https://doi.org/10.1007/s00414-012-0816-1
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DOI: https://doi.org/10.1007/s00414-012-0816-1