Ultrasensitive circulating tumor DNA analysis enables precision medicine: experimental workflow considerations

Figures & data

Figure 1. Overview over the ctDNA analytical workflow. The schematic overview illustrates dependencies between study design and experimental choices

Figure 2. Overview of preanalytical steps and variable parameters in ctDNA analysis. The use of ctDNA as biomarker is affected by a wide range of preanalytical factors. Experimental steps and common parameters that can vary are shown

Table 1. Overview of comparative preanalytical studies

Reference	Collection tube	Extraction method	Other study variables	Quality controls and analysis
Alidousty et al., 2017 [62]	Test of study	Automated	Time before plasma isolation	F, C.E., qPCR
Barra et al., 2015 [91]	EDTA	Automated	Time before plasma isolation, temperature	qPCR
Barrett et al., 2014 [63]	EDTA	Test of study	Temperature, time between plasma isolation and DNA extraction	dPCR
Barták et al., 2019 [64]	Test of study	Test of study		F, methylation-specific qPCR
Board et al., 2008 [65]	EDTA	Test of study	Time before plasma isolation	qPCR
Breitbach et al., 2014 [66]	EDTA	Test of study		C.E., qPCR
Denis et al., 2015 [67]	Test of study	Magnetic bead-based	Time before plasma isolation, shipping	dPCR
Devonshire et al., 2014 [109]	EDTA added to sample	Test of study	Inhibition of qPCR	qPCR, dPCR
Diefenbach et al., 2018 [108]	EDTA	Test of study		C.E., dPCR
Fleischhacker et al., 2011 [68]	EDTA	Test of study		qPCR
Gerber et al., 2020 [90]	Test of study	Column based	Time before plasma isolation, temperature	F, C.E., qPCR, dPCR, WGS
Henao Diaz et al., 2016 [69]	Test of study	Column based	Time before plasma isolation, temperature	C.E., WGS
Hidestrand et al., 2012 [70]	Test of study	Column based	Time before plasma isolation, temperature, shipping	qPCR
Kim et al., 2014 [71]	n/a	Test of study		qPCR
Kloten et al., 2017 [72]	EDTA	Test of study		F, C.E., qPCR, dPCR
Markus et al., 2018 [110]	Test of study	Test of study	Temperature	dPCR, WGS
Mauger et al., 2015 [114]	EDTA	Test of study		F, C.E., qPCR
Mazurek et al., 2013 [73]	EDTA	Test of study		qPCR
Medina Diaz et al., 2016 [74]	Test of study	Column based	Time before plasma isolation, temperature, shipping	qPCR, BEAMing, SafeSeqS
Mehrotra et al., 2017 [75]	Test of study	Column based	Time before plasma isolation	F, qPCR, targeted sequencing
Nikolaev et al., 2018 [98]	Test of study	Column based	Time before plasma isolation, temperature	F, C.E., qPCR
Norton et al., 2013 [101]	Test of study	Column based	Time before plasma isolation, shipping	dPCR
Norton et al., 2013 [104]	Test of study	Column based	Time before plasma isolation, temperature, shipping	qPCR
Ordonez et al., 2013 [117]	EDTA	Test of study	Time before plasma isolation, temperature	qPCR
Page et al., 2013 [76]	EDTA	Test of study	Centrifugation, time before plasma isolation	F, C.E., targeted sequencing
Parpart-Li et al., 2017 [88]	Test of study	Column based	Time before plasma isolation, temperature	F, C.E., dPCR, targeted sequencing
Perez-Barrios et al., 2016 [77]	EDTA	Test of study		F, C.E., dPCR
Raymond et al., 2017 [106]	Test of study	Column based	Shipping, time between plasma isolation and DNA extraction	F, PCR and G.E., targeted sequencing
Risberg et al., 2018 [92]	Test of study	Column based and automated	Centrifugation, time before plasma isolation, temperature	dPCR, TAm-Seq
Rothwell et al., 2016 [78]	Test of study	Column based	Time before plasma isolation, shipping	qPCR, targeted sequencing, WGS
Sato et al., 2018 [100]	Test of study	Column based and automated	Time between plasma isolation and DNA extraction, temperature	F, C.E., MBP-QP
Schmidt et al., 2017 [79]	Test of study	Test of study	Time before plasma isolation	qPCR
Sherwood et al., 2016 [80]	Test of study	Test of study	Centrifugation, time before plasma isolation	qPCR
Sorber et al., 2017 [111]	n/a	Test of study		F, qPCR, dPCR
Sozzi et al., 2005 [99]	n/a	n/a	Time between plasma isolation and DNA extraction	DNA Dipsticks, qPCR
Swinkels et al., 2003 [81]	EDTA	Not performed	Centrifugation	qPCR
Toro et al., 2015 [82]	Test of study	Column based	Time before plasma isolation	dPCR
van Dessel et al., 2017 [83]	Test of study	Column based	Time before plasma isolation	F, dPCR
van Ginkel et al., 2017 [84]	Test of study	Test of study	Centrifugation, time before plasma isolation	Test of study, dPCR
Ward Gahlawat et al., 2019 [102]	Test of study	Column based	Time before plasma isolation, temperature	F, C.E., dPCR
Warton et al., 2017 [85]	Test of study	Column based	Time before plasma isolation	qPCR, G.E.
Warton et al., 2018 [112]	EDTA	Test of study		qPCR
Wolf et al., 2016 [86]	Test of study	Test of study		F, C.E., qPCR, targeted sequencing
Wong et al., 2013 [103]	Test of study	Column based	Time before plasma isolation, temperature, shipping	MALDI-TOF MS, MPSS
Zhao et al., 2019 [87]	Test of study	Column based	Time before plasma isolation	qPCR, S, sequencing

Abbreviations: n/a, not available or not stated; F, fluorometric measurement with Qubit (Invitrogen), Quant-iT (Invitrogen), and/or Quantus Fluorometer (Promega); C.E., capillary electrophoresis with Fragment Analyzer (Aligent Technologies), Experion Automated Electrophoresis system (Bio-Rad Laboratories), Bioanalyzer (Agilent Technologies) and/or TapeStation (Agilent Technologies); qPCR, quantitative PCR; dPCR, digital PCR; WGS, whole genome sequencing; S, spectrophotometric measurement with NanoDrop (Thermo Scientific); G.E., gel electrophoresis; MBP-QP, mutation-biased PCR quenching probe; MPSS, massively parallel shotgun sequencing.

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