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Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization
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A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology.

Janine Schulte1, Michael A Marciano2, Eva Scheurer1

  • 1Institute of Forensic Medicine, University of Basel, Basel, Switzerland.

Journal of Forensic Sciences
|July 27, 2023
PubMed
Summary
This summary is machine-generated.

This study optimized short tandem repeat (STR) analysis for low template DNA, finding that reducing PCR volume improved peak height but increased artifacts. Specific kits like ESIFast and NGMDetect showed promise for single-cell genotyping.

Keywords:
DEPArray™ PLUSLT-DNAPCRSTRamplification volume reductionforensic single-cell analysislow template DNApolymerase chain reactionshort tandem repeat

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Area of Science:

  • Forensic Science
  • Molecular Biology
  • Genetics

Background:

  • Commercial STR amplification kits lack validation for low template DNA analysis.
  • Current methods for single-cell profiling rely on adjusting PCR cycles and analytical thresholds.

Purpose of the Study:

  • To determine optimal conditions for informative STR profiles from diluted DNA.
  • To evaluate these conditions on single cells using DEPArray™ technology.

Main Methods:

  • Compared four forensic STR kits with varying amplification volumes and DNA dilutions (down to 3.0 pg).
  • Tested two selected kits for single/pooled leukocyte and sperm cell genotyping.
  • Analyzed peak heights, artifact generation, and locus failure.

Main Results:

  • Reducing PCR volume (50%-75%) improved peak height but increased artifacts with diluted DNA.
  • Fusion 6C showed benefits from volume reduction for profile completeness.
  • ESIFast and NGMDetect offered a basis for consensus profiling in single/pooled cells, with locus dropouts as stochastic events.
  • 12.5 μL amplification volume yielded appropriate peak heights and stutter frequencies, with stutter peaks being the primary artifact in single-cell profiles.

Conclusions:

  • Optimized PCR conditions, including reduced amplification volume, can enhance STR profiling for low template DNA.
  • Specific STR kits demonstrate utility for single-cell genotyping, though stochastic events and artifacts require careful consideration.
  • Further research is warranted to refine low template DNA analysis techniques.