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BIABooster: On-line DNA concentration and size profiling with a limit of detection of 10 fg/µL. Application to highsensitivity characterization of circulating cell-free DNA Comtet-Louis Andriamanampisoa, Aurélien Bancaud, Audrey Boutonnet-Rodat, Audrey Didelot, Jacques Fabre, Frederic Fina, Fanny Garlan, Sonia Garrigou, Caroline Gaudy, Frederic Ginot, Daniel Henaff, Pierre Laurent-Puig, Arnaud Morin, Vincent Picot, Laure Saias, Valerie Taly, Pascale Tomasini, and Aziz Zaanan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04034 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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Analytical Chemistry

BIABooster: On-line DNA concentration and size profiling with a limit of detection of 10 fg/µL. Application to high-sensitivity characterization of circulating cell-free DNA 1

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Comtet-Louis Andriamanampisoa , Aurélien Bancaud , Audrey Boutonnet-Rodat , Audrey Didelot , Jacques 1

Fabre , Frédéric Fina

4,5,6

3,8

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1,*

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, Fanny Garlan , Sonia Garrigou , Caroline Gaudy , Frédéric Ginot , Daniel Henaff , 1

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Pierre Laurent-Puig , Arnaud Morin , Vincent Picot , Laure Saias , Valérie Taly , Pascale Tomasini , Aziz 3,8

Zaanan .

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Picometrics Technologies, 478 rue de la Découverte, 31 670 Labège, France LAAS-CNRS, Université de Toulouse, CNRS, Toulouse, France 3 INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, Paris, France. Equipe labellisée Ligue Nationale contre le cancer. 4 Laboratoire de Biologie Médicale, Unité de développement technologique; Assistance Publique Hôpitaux de Marseille, France. 5 ID-Solutions, 310 rue Louis Pasteur, 34790 Grabels, France 6 Service d’Anatomie Pathologique et Neuropathologie ; Timone II, Assistance Publique Hôpitaux de Marseille 7 Service de Dermatologie, Vénéréologie et Cancérologie cutanée; Assistance Publique Hôpitaux de Marseille, France. 8 Department of Digestive Oncology, European Georges Pompidou Hospital, AP-HP, Paris, France. 9 Aix Marseille University; Assistance Publique Hôpitaux de Marseille. Multidisciplinary Oncology & Therapeutic Innovations department, Marseille, France. 2

* To whom correspondence should be addressed. Tel: 33 531 61 50 90; Email: [email protected]

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ABSTRACT We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/µL in an operating time of 20 minutes. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves on-line concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces. Using a set of DNA ladders in the range 100-1500 bp, we report a sizing accuracy and precision better than 3%, and a concentration quantification precision of ~20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical researches involving cfDNA.

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The detection of alterations in cfDNA is likely to guide the administration of cancer drugs as first-line treatments

1

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or to fight emerging resistance or relapse in the near future. Additionally, the analysis of cfDNA concentration has often shown an increase in patients’ samples correlated with the disease stage or tumour load, and with overall survival at least in some cases

3–5

. Yet it has been reported that cfDNA concentration alone is not a

consistent biomarker due to its intrinsic variability in physiological conditions

6–8

. Associating cfDNA concentration

9

with an integrity index increases the predictive value of cfDNA analysis , suggesting that cfDNA size profiles could serve as a biomarker for patients’ monitoring. However, the concentration of cfDNA is often too low to be measured directly by gel electrophoresis, and indirect methods based on PCR amplification of a subset of genes 10

of variable length are used . Gel electrophoresis is the most popular technique to assess DNA size. Capillary gel electrophoresis reaches a 11

limit of detection (LOD) of ~10 pg/µL for a single fragment . High sensitivity kits of commercial electrophoresis 12

systems reach similar LODs, but they include a dilution step in a buffer enabling electrokinetic stacking . This feature has a practical interest, because the user only consumes 1-2 µL of sample. Nevertheless, this LOD of 10 pg/µL remains too high for cfDNA analysis. We have recently reported the principle of the µLAS microfluidic technology, standing for “µLAboratory for DNA 13

Separation”, to perform the operations of DNA concentration and separation simultaneously . It relies on the manipulation of DNA molecules in a pressure-driven viscoelastic flow in combination with a counterelectrophoresis. DNA undergoes a viscoelastic force oriented towards the channel walls, the amplitude of which depends on its molecular weight (MW). This technology can be operated in a linear channel to perform size 14

separation, an implementation similar to field flow fractionation . DNA concentration with µLAS is achieved by flowing the solution through a constriction. The funnel shape induces a spatial variation of the electric and hydrodynamic fields that modulates transverse viscoelastic forces, with a direct impact on DNA mobility. By setting the pressure drop and voltage appropriately, it is possible to switch from a low force migration mode with molecules located in the bulk of the channel, thus preferentially conveyed by hydrodynamics ahead of the constriction, to a high force regime dominated by counter-electrophoresis past the constriction because molecules are confined at the wall where the flow velocity is null. Therefore, DNA molecules can be concentrated 13

at the constriction (Fig. 1A) . We reasoned that the same physical phenomenon could be exploited in a capillary format by joining two capillaries of different diameters to produce a constriction (Fig. 1B). DNA size analysis is performed downstream in the narrow capillary with a fluorescence detector (Fig. 1C). This allows one to operate the technology with an existing instrument initially designed for capillary electrophoresis (CE), and to gain in automation and robustness. This enables an efficient stacking of DNA without prior sample dilution. We demonstrate the relevance of this technology, that we coin “BIABooster”, for DNA analysis with a LOD of 0.01 pg/µL for 1 kb fragments. We also show that BIABooster cfDNA profiling can be carried out for cancer patients and healthy individuals. The results, which are consistent with fluorimetry and droplet-based digital PCR assays, indicate that the concentration and size of cfDNA represent physiological information related to the clinical status of individuals. 3



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Figure 1: BIABooster system for DNA concentration, separation, and detection. (A) DNA concentration is achieved using a constriction and electro-hydrodynamic actuation. DNA transport is dominated by hydrodynamics (blue arrows) ahead of the constriction and by electrophoresis (red arrows) downstream, so DNA molecules accumulate at the constriction. (B) Experimental demonstration of the concentration was performed by welding two capillaries of different inner diameters (i.d.) of 100 and 20 µm (upper image). The fluorescence micrograph in the lower image shows the concentration of DNA molecules of ~5 kb at the junction (See Supplementary Video). (C) The final BIABooster device consists of three capillary segments with two junctions. Concentration and detection areas are marked with blue and red arrowheads, respectively. (D) This device is loaded in a capillary electrophoresis instrument equipped with a laser induced fluorescence detector (side module in the picture).

EXPERIMENTAL SECTION Reagents Sodium hydroxide (NaOH) and hydrochloric acid (HCl) are from Sigma-Aldrich (St. Louis, MO), YOYO-1 iodide (ref Y3601) from Life Technologies (Carlsbad, CA), DNA ladders from New England Biolabs (NEB) (Ipswich, MA. 100 bp ladder, ref N3231S), Interchim (Montluçon, France, 50 bp-GeneDireX, ref DM012-R500), or Biotools (Madrid, Spain, 100 bp-Biotools, ref 31006-4430). BIABooster running buffer is TBE 1X (Tris-HCl 90 mM, boric acid 90 mM, EDTA 2 mM, pH 8.5), with 13

polyvinylpyrrolidone 5% (PVP) to confer viscoelasticity . 30 µL of intercalating dye (Picometrics Technologies, France, ref 16-BB-DNA1K/01) were added to 3 mL of running buffer to make the analysis buffer. The mix solution was filtered at 0.45 µm while dispensing in Agilent CE vials. Single DNA fragments of 110, 300 and 500 bp were provided by ID-Solutions (Grabels, France). The fragments were calibrated by digital PCR by the supplier, respectively at 28, 43, and 15 pg/µL. 4


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Capillary assembly and instrumentation The BIABooster device is composed of two main units (upper panel in Fig. 1C), the injection chamber of internal diameter (i.d.) 320 µm, 2 cm long (~1.6 µL), and the downstream capillary of i.d. 50 µm for DNA separation and detection. Capillary assembly can be made by welding with a Fujikura LAZERMaster LZM-100 Laser Splicing System machine. First devices, as those used in Figure 1B, were assembled by Ceramoptec (Germany). BIABooster devices, as designed in Figure 1C and used for the rest of the study, were assembled at Picometrics. Silica capillaries were purchased from Molex (Polymicro Technologies™, TSP Capillary Tubing). Capillaries were cut at length using a CO2 laser at Molex. 5 mm of polyimide coating was also removed at the edges to be spliced. Alignment and splicing of the capillaries were performed using the automatic modes of the LZM-100 splicing system. After assembly, a PTFE tube i.d. 750µm and 8mm long was glued around junctions to make them robust, with an acrylate UV based glue. All analyses were performed using a G7100A capillary electrophoresis (CE) system (Agilent Technologies, Waldbronn, Germany) equipped with a Zetalif laser 488nm detector (Picometrics Technologies, France). The CE cassette was modified to install the BIABooster device with minimum bending at junctions by machining the cassette so as to place the optical head of the detector 9 cm downstream of the concentration junction (Fig. 1C). For imaging the concentration phenomenon at the junction (Fig. 1B), a blue LED and a camera were added inside an HP 3D G1600 CE instrument.

BIABooster DNA analysis Before use, devices were washed at 10 bar with HCl 0.1 M (4 min in each direction), NaOH 1 M (4 min in each direction), and distilled water (4 min in each direction). Between two sample analyses, capillaries were reconditioned at 10 bar using 0.1 M HCl for 2 min, water for 1 min and analysis buffer for 6 min. The consecutive steps of the method consist of injecting 0.85 µL of sample at 5 bar, followed by concentrating the DNA at 10 bar and 25 kV for 6.5 minutes, and then separating DNA fragments at 10 bar with a voltage decreasing from 25 kV to 0.1 kV in 15 minutes (see Fig. 2). During concentration and separation, inlet and outlet of the capillary device were dived into vials filled with analysis buffer. In these experimental conditions, all DNA molecules greater than 2 kbp migrate as a single peak. This result, which is consonant with the constant mobility of high MW DNA in gel electrophoresis, is accounted for by the even distribution near the wall of molecules longer than 2 kb.

Fragment Analyzer™ DNA analysis Fragment Analyzer™ analyses (Advanced Analytical Technologies Inc., USA) were performed with the high sensitivity kit (ref DNF474), with a 12 capillaries array (33cm effective length, ref A2300-1250-3355), according to manufacturer instructions. 5



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SNR and LOD calculation Serial dilutions of the NEB 100bp ladder in the range 50 to 0.5 pg/µL, or 1000 to 12.3 pg/µL, were prepared and separated by the BIABooster or the Fragment Analyzer™, respectively. Noise was estimated with OpenLAB CDS (Agilent Technologies) or PROSize 2.0 (Advanced Analytical) software. Peak-to-peak noise was determined over a lag of 1 minute before and after the peak signal in the OpenLAB CDS software. This procedure is not automated with PROSize 2.0, so peak-to-peak noise was estimated graphically by zooming on the screen. LOD was calculated by extrapolating the DNA concentration for a signal to noise ratio (SNR) of 3 from the less concentrated sample giving a SNR greater than 3. See supplementary information for graphical noise illustration and mathematical calculation of the LOD.

Quantification of DNA size and concentration Sizing calibration was set up with an automatic detection of the peaks in the standard DNA ladder, followed by linear interpolation between the consecutive bands of the ladder for size assignation. An example is given in 17

supplementary Fig. S1. DNA concentration was then determined using the method of Chamieh et al. , modified to take into account the fact that DNA velocity during the separation phase is not constant: briefly, for each DNA band of the ladder, we considered the correction factor given by the ratio between the fluorescence area and the known DNA concentration. The correction factor for any given size was then computed using a linear interpolation of this correction factor based on the standard ladder. An example is given in Fig. S1. We finally converted the fluorescence vs. time signal into a graph showing DNA concentration vs. fragment size. The full data treatment is automated in Scilab scripts that are available upon request. Fluorescence signal above 1.6 kb is a quantitative indicator of the presence of high molecular weight DNA which cannot be converted in concentration with this procedure. In cfDNA samples of this study, DNA greater than 1.6 kb is negligible.

Clinical samples and preparation Sample set 1 (Paris hospitals): This set contained 14 cfDNA samples from metastatic colorectal cancer (mCRC) patients and 9 from healthy individuals. mCRC samples came from a study performed at the European Georges Pompidou hospital (Paris, France) that enrolled patients between October 2012 and July 2015 (Placol Cohort). The Placol study received ethical approval from the “Ile-de-France ethics committee” (ID CRB: 2013-A00680-45) and all patients provided 18

written informed consent . Blood samples were collected before chemotherapy (N=5) or during treatment, before a new cycle of chemotherapy (N=9). Samples were chosen with either the G12V or the G13D mutation in the KRAS gene. Plasma samples from healthy individuals were purchased from Biopredict (N=4) or Wilmington and Biological Specialty Corporation, Colmar, US (N=5). DNA extraction and analyses were performed in the same conditions for cancer patients and healthy donors. 6


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Whole blood (4 EDTA tubes of 5 mL) was centrifuged at 3,000 g at room temperature for 10 minutes. Plasma was stored at -20°C and centrifuged a second time at 2,000 g at room temperature for 15 minutes in an Eppendorf 5430R centrifuge before DNA extraction. Cell-free DNA was extracted from 1-2 mL plasma using QIAmp® Circulating Nucleic Acid Kit (QIAGEN) according to the manufacturer’s instructions and resuspended into 50 µL of elution buffer. No carrier was added to the samples. ®

Purified DNA was quantified using Qubit dsDNA HS assay kit (ref Q32854; Life Technologies).

Sample set 2 (Marseille hospitals): Patients were enrolled in the study from Multidisciplinary and Therapeutic innovations department, and Service de dermatologie, vénéréologie et cancérologie cutanée, Assistance Publique Hôpitaux de Marseille. Patients with Non Small Cells Lung Cancer (NSCLC) (33 samples) were all metastatic. Melanoma patients (32 samples) were at various stages of the disease and treatment. There are also 3 samples from prostate cancer patients in the data set. All samples were collected for routine patients’ care and all patients previously signed a consent form allowing the use of the remaining samples for research purposes. Blood from 9 healthy volunteers come from Etablissement Français du Sang (convention EFS-PM n 21PLER2016-0088). Blood samples were collected in EDTA tubes. First centrifugation was performed less than 4 hours after blood sampling, at 2000 g for 10 minutes at room temperature. Plasma was then aspirated avoiding the intermediate phase, transferred into a conical tube, and centrifuged again at 4500 g for 5 minutes at room temperature. Plasmas were stored at -80°C before DNA extraction. Extraction was carried out with the IDXTRACT kit (id-Solutions, Grabels France) following the recommendations of the supplier. The extracted plasma volumes were 3 to 4 mL, and the final elution volume was 100 µL. The manufacturer has shown that this kit is equivalent to the QIAmp® Circulating Nucleic Acid Kit (Fig. S2). DNA samples are stored at -20°C in the elution buffer. Droplet-based Digital PCR Sample set 1: cfDNA was quantified using a KRAS assay as described elsewhere

18,19

. The KRAS amplicons are 60 bp long. A

multiplex assay is used, using TaqMan® probes, which allows to quantify both the mutated and the wild type alleles of the gene. A detailed protocol is given in Supporting Information. Briefly, reactions were performed in 25 µL, containing 5-9 µL of extracted DNA. Emulsions were generated using the RainDrop Digital PCR Source (RainDance Technologies, Billerica, US), and amplifications were performed with a BioRad® thermal cycler (MJ-Mini, S1000, or C1000 touch) for 45 cycles. After completion, emulsions were processed to measure the end-point fluorescence signal from each droplet using the RainDrop Digital PCR Sense. Data were analysed using the Raindrop Analyst II software as described by the manufacturer. cfDNA concentration was assessed successfully for 21 samples out of 22. 7



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Sample set 2 cfDNA was quantified using IDQUANT kit (id-Solutions, Grabels, France). This kit comprises a duplex ddPCR™ with TaqMan® probes which allows quantification of a single copy human gene (67 bp amplicon) as well as an exogenous DNA fragment of 110 bp used for inter-assay normalization of cfDNA extraction. Briefly, reactions were performed in duplicates in 21 µL containing 5 µL of cfDNA. Emulsions were generated using the droplet generator of the QX200 system (Bio-Rad), and amplifications were performed with a C1000 Touch™ Thermal Cycler (BIO-Rad) for 40 cycles. After completion, emulsions were processed to measure the end-point fluorescence signal from each droplet using the QX200 reader. Data were analysed using software 1.7 as described by the manufacturer. See more details in Supporting Information.

For both samples sets, a value of 3.3 pg/copy was used to convert concentration from copies/µL to pg/µL. The two dPCR assays used in this study give equivalent quantification results, as determined by a direct comparison on 20 melanoma samples from sample set 2 (supplementary Table S4).

Statistics Mann-Whitney tests and ANOVA analyses were performed using Analyse-it software (Analyse-it company, UK).

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RESULTS AND DISCUSSION BIABooster design and operating mode The BIABooster is operated in three consecutive phases with no time lag during the process. First, the sample is injected hydrodynamically with a pressure set to 5 bar for 80 s. Second, the sample is concentrated by setting the voltage to 25 kV and the pressure to 10 bar for 6.5 min. Third, DNA separation is achieved by progressively decreasing the voltage from 25 to 0.1 kV in 15 minutes (blue curve in Fig. 2). The entire protocol, which is automatically operated by the CE instrument, is carried out in less than 22 minutes. Note that the injection volume is typically 100 to 1000 fold greater than that in capillary or microchip electrophoresis

15,16

.

As an example, we show the separation of the NEB 100 bp DNA ladder diluted at 50 ng/mL (black curve in Fig. 2). For the fragment of 600 bp, which has the lowest concentration (right panel of Fig. 2), 1.5 pg corresponds to a peak of large signal-to-noise ratio (SNR). Notably the intensity of the low molecular weight bands of 100 and 200 bp was much lower than that of 600 bp. This effect stems from the enhanced diffusion of small molecules as well as the difference in velocity between fragments when passing in front of the detector, which leads to corrections for the determination of the concentration when quantifying unknown samples (see Experimental section).

Figure 2: BIABooster analysis of a 100 bp DNA ladder. The black dataset corresponds to the fluorescence signal collected for the NEB ladder at a concentration of 50 pg/µL. The blue curve corresponds to the applied voltage during analysis. The concentration phase occurs for 6.5 minutes (delimited by the red dashed line), and separation proceeds for 15 minutes. The same ladder is shown after agarose gel electrophoresis in the right panel. Mass values are for 0.5 µg/lane.

Sizing accuracy is better than 3% Sizing precision has been estimated using two methods. First, the separation of the NEB 100 bp ladder has been conducted four times on different days, each time in triplicate (Supplementary Table S1). The size of each of the thirteen peaks has then been determined as described in Experimental section. One-way ANOVA shows that there was no significant difference between experiments (Table S1). Coefficients of variation (CVs) were then calculated for each size from all the 12 individual values. All the CVs were smaller than 2%. Second, the sizes of 9



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the fragments of two other commercial ladders have been estimated and compared to their nominal values, both to confirm sizing precision and to assess sizing accuracy with DNA fragments different from those present in the NEB ladder. Sizing precision between duplicates of the same ladder was less than 1% for all the bands, confirming the results obtained with the NEB ladder (Supplementary Table S2). Accuracy has then been measured by comparing the average size value for each fragment to that expected from the specifications of the manufacturers (Table 1). Size error was comprised between 0 and 2.6%, with an average of 1.3%. Moreover, the separation performance of the technology appeared sufficient to detect two bands of 1150 and 1212 bp at “position 1200” in the 50 bpGeneDireX ladder. These two bands were not detected by agarose gel electrophoresis, but their presence has been confirmed by the manufacturer, though without stipulating their exact size (personal communication).

Table 1: Sizing accuracy. The different fragments of the 50 bp ladder from Gene DireX and the 100 bp ladder from Biotools have been characterized with the BIABooster system, using the NEB 100 bp ladder as standard. Each ladder has been run in duplicate. * The manufacturer confirmed that the 1200 band contains two fragments. Linearity and precision of DNA concentration measurements We then focused on the precision of BIABooster DNA concentration measurements. First, we used the BIABooster fluorescence traces recorded with the GeneDireX and Biotools ladders analyzed at nominal concentrations of 100 pg/µL and 50 pg/µL, respectively. By summing up the mass of each band determined against the NEB 100 bp ladder as a standard, we inferred the respective concentrations of these two samples to be 137 +/- 8 pg/µL and 73 +/- 11 pg/µL, giving a first estimate of precision better than ±20% (Table S2). In the same way, the 100 bp NEB ladder tested in duplicate showed a precision of 6%. Quantification accuracy was further explored by comparing the results of dPCR and BIABooster for three DNA fragments of 110, 300, and 500 bp. The respective concentrations were 28, 43, and 15 pg/µL according to dPCR, and 19, 39, and 9 pg/µL according to the BIABooster. BIABooster over dPCR concentration ratios are 0.7, 0.9, and 1.7 respectively. 10


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Interestingly we also measured the concentration of serial dilutions for these three fragments (Fig. 3) and 2

demonstrated that readout was linear in the range 0.3-40 pg/µL, as shown by the R coefficients average equals to 0.98.

Figure 3: Linearity of the concentration determined by the BIABooster. The graph shows the concentration of serial dilutions of three DNA fragments. The inset displays the same data in log-log scale.

Determination of the limit of detection The LOD, as defined by the DNA concentration for a signal to noise ratio (SNR) equals to 3, has been estimated by using serial dilutions of the 100 bp NEB ladder (fluorescence traces in supplementary Fig. S3). The total DNA concentration spanned 0.5 to 50 pg/µL (Table 2), and we focused on the 5 bands of 100, 200, 300, 400, and 1000 bp. Their respective LODs were 0.3, 0.07, 0.04, 0.02 and 0.01 pg/µL. The 30-fold difference in LOD between the 100 and 1000 bp fragments is readily accounted for by their difference in fluorescence intensity, as shown in Fig. 2. These values of the LOD were confirmed by performing the same analysis on the 110, 300, and 500 bp fragments characterized in Fig. 3. Their respective LODs of 0.08, 0.05, and 0.03 pg/µL were in good agreement with the estimates obtained with the 100 bp NEB ladder.

Table 2: SNR and LOD of the BIABooster system for five bands of the NEB 100 bp DNA ladder. nd: not detected. To compare this performance to a state-of-the-art electrophoresis system, we evaluated the LOD of the Fragment Analyzer™ system using the DNF-474 high sensitivity kit. The resulting LODs were 50 to 500 times higher than 11



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that of the BIABooster, namely 15.9, 3.6, 3.35, 3.3, and 5.4 pg/µL, for the 100, 200, 300, 400, and 1000 bp fragments, respectively (Supplementary Table S3). Application to purified cfDNA The BIABooster was then applied to the analysis of cfDNA extracted from blood plasma. We assayed samples from two research teams, each cohort including healthy individuals and patients with CRC, NLCSC, or melanoma. The BIABooster profiles typically contained a predominant thin peak around 150 bp, a second peak around 300 bp, which is usually smaller and wider, followed or not by a third even smaller and larger peak around 450-500 bp, as well as by various amounts of high MW DNA (greater than 1 kb, upper panel in Fig. 4A). This high MW fraction may come from genomic DNA of leucocytes during preanalytical stages or may reflect a physiological phenomenon. This profile was detected for cfDNA samples over a wide range of total DNA concentration spanning 1-500 pg/µL. It is consistent with previous reports based on highly concentrated cfDNA 20

samples analyzed with the bioAnalyzer™ (Agilent) . As a benchmark, when using such state-of-the-art electrophoresis systems, cfDNA profiles could only be assayed for highly concentrated samples with a total DNA concentration larger than ~500 pg/µL (Supplementary Fig. S4). To validate BIABooster cfDNA quantification, we compared the BIABooster to fluorimetry (Qubit™) and dPCR (see Experimental section). We first analyzed a set of samples composed of 8 healthy individuals and 14 metastatic colorectal cancers (sample set 1). The correlation between the Qubit™ and BIABooster total cfDNA was associated to a Pearson correlation coefficient of 0.5 (left panel in Fig. 4B). The median [25%-75% quantile] ratio between BIABooster and Qubit™ concentrations was 0.8 [0.5 - 1.6].

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Figure 4: Profiling cfDNA with BIABooster. A) typical cfDNA profiles for concentration from 1 to 500 pg/µL (751650 bp range). These cfDNA samples are from melanoma patients. Red lines represent base lines used to measure fluorescence intensities B) left plot shows correlation between cfDNA concentrations of sample set 1 determined by the BIABooster system and fluorimetry (Qubit™). In the middle and right panels, the same comparison is carried out with dPCR for sample set 1 and 2, respectively. Dotted lines represent the median ratio between the two plotted concentrations. Next, we investigated the correlation between dPCR and BIABooster concentrations for the same samples (central panel in Fig. 4B). The Pearson correlation coefficient was 0.65, with a median [25%-75% quantile] ratio between BIABooster and dPCR concentrations equals to 1.3 [0.9 – 2.3]. We finally focused on 32 melanoma samples of another sample set (sample set 2), the concentration of which had been characterized by another dPCR assay. The correlation plot was quite clear again with Pearson correlation coefficient of 0.5. The median ratio [25%-75% quantile] between BIABooster and dPCR concentrations was 0.6 [0.3 – 1.4]. These results support the relevance of our technology to measure total cfDNA concentration. To investigate further the origin of this difference in the concentration ratios between the two sample sets, 20 of the 32 melanoma samples were also quantified using the dPCR assay used for sample set 1. The median [25%75% quantile] concentration ratio between the two dPCR assays for these samples was 0.93 [0.7 – 1.3], showing that the two dPCR assays are equivalent (see Table S4). Therefore, the observed difference between the two ratios is rather due to noise in the data, as suggested by the overlapping [25%-75% quantile] intervals, than to a bias linked to dPCR assays or to samples.

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Quantitative cfDNA profiling for biomarker identification Total cfDNA concentration has often been described as a potential biomarker for solid cancer

3,5,20

. Indeed, in

sample set 1, cfDNA concentration appears as an effective biomarker to distinguish metastatic colorectal cancer before a new cycle of chemotherapy (n=14) from healthy individuals (n=8, p-value=0.006; Fig.5, left, upper row). In sample set 2, the same conclusion could be drawn for NLCSC patients (n=33), and for the 3 prostate samples present in this sample set (Fig.5, left, lower row). In contrast, discrimination between melanoma samples (n=32) and healthy individuals (n=9) is not as significant, although present. Note that the concentration distribution of healthy individuals was different between the two sample sets. This difference appeared clearly from a Mann-Whitney test, with a p-value

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