Comparative study on the effects of reduced PCR reaction volumes and increased cycle number, on the sensitivity and the stochastic threshold of the AmpFlSTR Identifiler® Plus kit

Bessekri, M.W.; Aggoune, A.; Lazreg, Sihem; Bucht, Rebecca E.; Fuller, Varden

doi:10.1016/j.fsigss.2013.10.156

Cited by 17 publications

(17 citation statements)

References 12 publications

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“…Cycle increases, however, may introduce some confounding factors, e.g. heightened susceptibility to contamination and more drastic differences in inter-and intra-locus peak height balance [16,[21][22][23][24][25][26]. Concerns are justi ed when analyzing 6.6 pg of DNA, as the resulting data is less predictable and more di cult to interpret than standard single source, multi-cell, samples [27].…”

Section: Introductionmentioning

confidence: 99%

Revisiting Single Cell Analysis in Forensic Science

Watkins

Myers²,

Xavier

et al. 2020

Preprint

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Background Forensic science has yet to take full advantage of single cell analysis. Its greatest benefit is the ability to alleviate the challenges associated with DNA mixture analysis, which, despite the emergence of probabilistic genotyping, remains a significant hurdle in forensic science. Many of the factors that cause complexity in mixture interpretation are absent in single cell analyses — multiple contributors and varied levels of contribution and allele masking. Recent technological innovations have been developed within the scientific community that mitigate the historical risks of single cell analysis in forensic casework. This study revisits single cell analyses in the context of forensic identification, introducing previously unseen depth to the characterization of data generated from single cells using a novel pipeline that includes recovery of single cells using the DEPArray™ NxT and amplification using the PowerPlex Fusion 6c kit with varied PCR cycles (29, 30, and 31); the resulting allelic signal was assessed using analytical thresholds of 10, 100 and 150RFU. Results The mean peak heights across the sample sets generally increased as cycle number increased, 75.0 ± 85.3, 147.1 ± 172.6, and 226.1 ± 298.2 RFU, for 29, 30, and 31 cycles, respectively. The average proportion of allele/locus dropout was most significantly impacted by changes in the detection threshold, whereas increases f PCR cycle number had less impact. Overall data quality improved notably when increasing PCR from 29 to 30 cycles; however, less improvement and more volatility introduced at 31 cycles. The average random match probabilities (RMP) for the 29, 30, and 31 cycle sets at 150RFU are 1 in 2.4 × 1018 ± 1.46 × 1019, 1 in 1.49 × 1025 ± 5.8 × 1025, and 1 in 1.83 × 1024 ± 8.09 × 1024, respectively. Conclusions This study introduces and optimizes an analytical pipeline to generate ample high quality data from single cells that can be used in forensic analyses, thus removing the need for complex mixture interpretation. An analysis of the impact of PCR cycles on single cells revealed a significant overall improvement in the amount and quality of interpretable data when increasing from 29 to 30 cycles, while no such improvement was noted when increasing to 31 cycles.

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Section: Introductionmentioning

confidence: 99%

Revisiting Single Cell Analysis in Forensic Science

Watkins

Myers²,

Xavier

et al. 2020

Preprint

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“…In our comparison of LTDNA PCR strategies for DNA extracted from telogen hairs, an increased number [34] of PCR cycles (the LCN method) produced a higher number of alleles concordant with reference samples than standard Profiler Plus PCR, standard PCR with a MinElute post-PCR clean-up, and half-volume PCR with double the concentration of Taq polymerase. However, this came at the cost of a higher number of non-concordant alleles (and thus non-concordant profiles).…”

Section: Discussionmentioning

confidence: 97%

“…To our knowledge, this is the first study to compare three commonly applied LTDNA PCR strategies (LCN, post-PCR clean-up, and reduced volume reaction) with a standard PCR on the same samples with the exception of Bessekri et al [34] who only compared reduced volume PCR with an increase in PCR cycles from 28 to 29. While it is well known that each of these strategies can improve the sensitivity of LTDNA analysis in comparison to standard methods, their performance relative to each other is unknown.…”

Section: Discussionmentioning

confidence: 99%

Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs

McNevin

Edson

Robertson

et al. 2015

Forensic Sci Med Pathol

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“…For the full volume; 7,5 mL of PowerPlex ESI 16 amplification master mix is added to 17,5 mL of each DNA dilution [11]. The amplification volume is reduced using 3 mL of master mix and 7 mL of DNA template [2]. This experiment was designed to simultaneously assess and compare: sensitivity, stochastic occurrences and intralocus peak imbalance with decreasing DNA quantities.…”

Section: Methodsmentioning

confidence: 99%