VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations

Abstract

Background:

In the context of ex vivo gene therapy or chimeric antigen receptor T cell (CAR-T) cell therapy, vector copy number (VCN) analysis in transduced cells by lentiviral vectors enables the assessment of risk and therapeutic efficiency in patients.

Objectives:

Here, we describe a ddPCR-based method that can replace easily the TaqMan qPCR assay for VCN analysis, by measuring the number of pro-viral DNA copies per host cell genome in blood samples.

Methods:

VCN are determined by ddPCR, after gDNA extraction and enzymatic digestion from transduced T lymphocytes or Hematopoietic Stem Cells (HSC) and by direct lysis of colony-forming cells (CFC) derived from transduced CD34+ cells.

Results:

Firstly, we have identified key elements of sample preparation and set-up to further improve the performance characteristics of ddPCR method, resulting in an accurate analysis of VCN without the need for multiple replicates or an external calibrator, as required in qPCR methods. Secondly, we found that genomic DNA (gDNA) quantification by fluorometry allows a better prediction of the genomic copy number detected in ddPCR than by spectrophotometry. Then, we provide a new protocol to analyze VCN in blood cells but also in CFC, using a NaOH cell lysis-based approach.

Conclusion:

This multiplex ddPCR is able to analyze VCN more precisely than qPCR in all transduced hematopoietic cells, an assay useful for clinical applications.

Introduction

Gene transfer technology is one of the most promising therapeutic approaches to manufacture gene-engineered cells from autologous patient or allogeneic donor not only for ex vivo gene therapy1–3 but also for cancer treatments based on T cell receptor (TCR) or chimeric antigen receptor T cell (CAR-T) cell therapies.4,5 For many of these preclinical studies and clinical trials, due to the adverse events observed with retroviral vector,6,7 hematopoietic cells are currently stably transduced by a long terminal repeat self-inactivating (LTR) SIN lentiviral vector,8 in order to introduce the therapeutic gene into the stem cell as well as its progeny. The characterization of these gene-modified cells is based on the evaluation of transduction efficiency and, consequently, on the vector copy number (VCN) analysis in the target cells to assess the therapeutic activity of integrative vector.9–12

Previously, we have described and validated an analytical method13 to measure recombinant human immunodeficiency virus-1 (rHIV)-VCN in human colony-forming cell (CFC), providing experimental data on the transduction of hematopoietic progenitor cells, useful for clinical applications.3,14 This VCN assay is performed by quantitative polymerase chain reaction (qPCR) for the detection of proviral DNA copy numbers per host cell genome.15,16 However, this measurement is dependent on the preparation of an external calibrator including dilution and its qualification by methods like the establishment of standard curves from plasmids bearing both vector and genomic sequences.17 To overcome these limitations, droplet digital PCR (ddPCR), a novel molecular biology technique, provides an absolute quantification of target nucleic acids without the need for an external DNA calibrator or multiple replicates.18,19 This method consists of the random distribution of DNA and PCR mixtures into 20,000 nanoliter-sized droplets during the partitioning process. Following the PCR target sequence amplification in each individual droplet, the fluorescence, generated by TaqMan hydrolysis probes, is measured in two channels corresponding to wavelengths specific to the appropriate fluorophore and permits to score positive droplets. Partitions without target sequence are considered as negative. From the number of PCR-positive and PCR-negative droplets, the copy number of target DNA in the original reaction is directly determined by Poisson statistics.20,21

Based on these initial observations, we have verified the possibility to use ddPCR methods for VCN analysis on hematopoietic cells transduced by lentiviral vector. We have defined the upper and lower limits of genomic DNA (gDNA) and cell quantity per reaction, and we showed that our procedure is applicable to our all samples as a replacement for qPCR for clinical applications. Consequently, we propose here a multiplex ddPCR protocol that we can apply not only after gDNA extraction but also after direct lysis in CFC from transduced CD34+ cells.

Development of Duplex ddPCR Method

This new multiplex ddPCR method results in an accurate analysis of VCN after lentiviral transduction in blood or progenitor cells without the need for multiple replicates and of an external calibrator and, consequently, is an improvement to the previously described qPCR assay,13 which is currently used for clinical trials.3,14

Using the primers and probes15 from the HIV-Psi sequence of the lentiviral vector and human Albumin gene for host cells (reference gene), we have seen that ddPCR is very specific because false positive events are infrequent, essentially observed in the hALB detection channel and are distinguishable from true positive droplets by weak fluorescence. Whereas the ddPCR technology is based on fluorescence readings after droplet generation and PCR amplification,21 we confirm that a predigestion step is necessary to improve the amplification efficiency and minimize the intermediate fluorescence (termed “the rain”) for bulk samples. However, particular attention is required on the choice of restriction enzymes to verify that the digestion does not affect the randomized DNA repartitioning, thus increasing the variability of the ratio between the copies of two gene sequences when a duplex method is used. Concerning DNA concentration measuring,22 we have observed that quantification with a fluorescent DNA-binding dye is more predictive of the DNA copy number detected by ddPCR than UV spectrophotometry. In our established ddPCR conditions, precision and linearity criteria23 are aligned with the ISO20395:2019 recommendations. The variability for VCN detection by ddPCR was less than 5% for our method (Table S1) with a bias lower than 12.8% (supplemental information, Trueness evaluation). We have demonstrated that ddPCR detected copy numbers of each target sequence as low as 0.9 copies/μl ( Figure 1A and supplemental information). This threshold of quantification was increased to 2.9 copies/µl to obtain precise VCN values (Figure 1B). Considering that around 300 copies/µl was detected for the hALB sequence from 1 ng/µl of digested gDNA, we have chosen to routinely use this concentration, which allows to analyze VCN in the range between 0.02 copies and 20 copies.

Figure 1.

Performance of the ddPCR method. (A) Linearity of the ddPCR method using HIV-Psi and hALB target sequences. The data points represent the average result for eight replicate measurements, and the vertical error bars represent the STD. By regression linear analysis of expected target copy concentration versus measured target copy concentration, the Pearson correlation coefficient r was equal to 1 with a slope of 0.9991 (CI: 0.9872 to 1.011) for HIV-Psi sequence and r = 1 with a slope of 0.9994 (CI: 0.9908 to 1.008) for hALB. This relationship was found to be proportional between tenfold serial dilutions where the concentrations are ranged between 0.7 and 7119 copies/µl for HIV-Psi and 0.9 and 7011 copies/µl for hALB sequences (n = 8). The results were coherent with the Bio-Rad guidelines, which specify that ddPCR can detect values in the range of 1–120,000 copies per reaction or 0.05–6,000 copies/µl, respectively. (B) LOQ (limit of quantification) determination for gDNA quantity using Jurkat clone bearing a known number of copies of the integrated vector (JUC-5, with expected VCN value of 5 by TLA detection27): VCN variability related to Albumin copy concentration from serial dilutions of gDNA. Average VCN and standard deviation were calculated from 20 replicates for the samples between 1.4 and 22 hALB copies/µl and 12 replicates for others concentrations. The CV was ≤20% from 2.9 hALB copies/µl (LOQ) with an interpretable VCN value of 4.5 ± 0.8, and consequently, the minimum amount of gDNA that can be analyzed with our ddPCR condition is 0.01 ng/µl.

Alternative Method to qPCR

We applied ddPCR to evaluate VCN in hematopoietic cell lines, T lymphocytes, progenitors, and CD34+ cells transduced by green fluorescent protein (GFP) lentiviral vector and compared the results with our previous qPCR method.13 As previously described,17 qPCR is relied on the detection of fluorescence generated by the TaqMan probe degradation, due the nucleolytic activity of the DNA polymerase at each polymerization step. Fluorescence is monitored in real time, and the point at which the reaction fluorescence crosses an intensity threshold during the exponential phase of PCR amplification is called the cycle threshold (Ct). However, it is an indirect method, which is based on the estimation of the initial number of DNA copies by comparing the Ct value of a sample to an external DNA calibrator. Multiplex ddPCR has advantage to reduce the variability of VCN measurements because ddPCR results are established from positive and negative droplet quantification and not on amplification reaction efficiency, as in qPCR. In our case, we have at first observed a bias between VCN obtained by ddPCR and by qPCR equal to 1.4 ± 0.3 (n = 116) with a significant difference (p > 0.0001) between the two methods, whatever the hematopoietic stem cell type. However, we found that this difference is due to the replacement of the quencher and the predigestion steps. Indeed, it is not possible to use the carboxy tetramethylrhodamine (TAMRA) quencher in a multiplex ddPCR, so the QSY dark quencher is used instead of the TAMRA quencher results. In fact, with technological improvements, new dark quenchers can increase the precision of fluorescence detection24,25 by reducing the background noise, therefore the risk of fluorescence interference due to quencher. However, already at qPCR level, its replacement of quencher results in a significant bias for the VCN estimation (Figure S1A). If samples are treated with the same quencher and digestion steps, VCN values analyzed by ddPCR are more comparable to those determined by qPCR (Figure S1B). Thus, it is not the method itself that explains not only the VCN difference but also the process changes between the qPCR and ddPCR tests. Considering the impact of these parameters, ddPCR can easily replace qPCR to analyze VCN.

Limitations

For clinical applications of gene-modified cells, the transduction efficiency can be evaluated not only by VCN detection after gDNA extraction of bulk transduced cells but also by the frequency of VCN-positive cells. This is the case when we analyze VCN on CFCs derived from transduced CD34+ cells. The determination of the number of CFCs in a cellular product is not only an indicator of the functional capacity of the progenitor cells but also a predictor of hematopïetic reconstitution. These hematopoietic progenitors have the ability to form colonies arising from a single CD34+ cell after 2 weeks culture in a semi-solid medium. The VCN analysis in each individual colony permits a more precise estimation of the frequency of transduced cells and the distribution of VCN in each cell. However, gDNA extraction from CFC cannot be performed using a conventional column kit, due to the small quantity of cells in a colony. Indeed, in the case of harvested CFC samples, the quantity of cells varies between 1,000 and 50,000 cells depending on the type of cell progenitor cells (CFU-GM for Colony Forming Unit-Granulocyte/Macrophage and BFU-E for Burst Forming Unit-Erythroid). Hence, an alternative extraction method using a lysis solution has been also evaluated for the process of using a ddPCR method for these samples.

Optimization and Applications

Our ddPCR method can be easily applied for blood samples after gDNA extraction with a commercial kit and by adding a digestion step and a dosage of gDNA by fluorometry. However, for applications like CFC analysis, for which the number of cells is too low to perform conventional gDNA extraction, a suitable lysis protocol without extraction is required. So, the challenge was to identify lysis protocol compatible with ddPCR analysis in order to develop a direct lysis/ddPCR method adapted for small cell quantity.

Another application of ddPCR is to perform a multiplex analysis from one gDNA sample. Therefore, we have evaluated this option by adding a second primers and probes from another vector sequence. Because most of the third-generation SIN lentiviral vectors contain also the mutated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), we have designed primers and probes based on this sequence and studied the feasibility of detecting VCNs with a triplex using ddPCR analysis.

Experimental Design

PCR step conditions optimization

Using the dark quencher quencher (commercial name by thermofischer) quencher succinimidyl ester-7 (QSY-7), we determined the optimal annealing temperature of 57°C for the best separation of negative droplets for the two target genes from gDNA or plasmid DNA. We have also confirmed that reading of the ddPCR plate immediately after performing PCR results in fewer total droplets per well compared to when the plate was incubated overnight at +4°C prior to reading. Indeed, for gDNA, 15,860 ± 1,972 droplets are obtained immediately versus 17,951 ± 1,138 droplets after overnight incubation, and for plasmids, 15,659 ± 1,490 droplets were obtained immediately versus 16,963 ± 1,374 droplets after overnight incubation. These results are statistically significant by using the Mann–Whitney test with a p value < 0.0001. Consequently, it is better to read the plate after overnight leaving at +4°C because as previously described,26 the droplets shrink in size due to the condensation on well wall after PCR reaction and could be excluded due to quality metrics by the QX200 droplet reader when the plate is reading immediately.

Choice of enzyme for DNA digestion step

Two type II restriction enzymes, Hae III and Hind III, were selected from the list recommended by Bio-Rad. By using the recommendations of the restriction mapper website (www.restrictionmapper.org), we verify that our PCR amplicons cannot be cleaved by the selected enzymes. We identified 4 versus 18 gDNA fragments for hALB gene and 5 versus 19 fragments for the proviral sequence after Hind III versus Hae III digestion. The plasmid DNA bearing both HIV-Psi and hALB sequences contains seven restriction sites for Hind III and 35 for Hae III. The use of these two enzymes reduces the rain, allowing a better discrimination between positive and negative droplets without affecting their counts, whatever the DNA type (gDNA or plasmid, Figure S2A). However, because higher variability in the ratio of two target sequences was observed with Hae III than Hind III enzyme (Figure S2B), due to the larger number of restriction sites, we decided to use the Hind III enzyme for DNA digestion before VCN analysis by ddPCR.

Genomic DNA quantification by fluorometry

gDNA quantification is mainly performed by two methods: (1) U.V. absorbance at a wavelength of 260 nm, related to the concentration linearity of the Beer-Lambert law using a NanoDrop spectrophotometer or (2) by measuring the fluorescence of dye-binding nucleic acid related to the concentration linearity of a standard nucleic acid solution, using a Qubit 4 fluorometer with Broad Range (Q_BR) and High sensitivity (Q_HS) with dsDNA Assay kits. In this context, we observed that gDNA concentrations obtained with Qubit were statistically lower than the values measured with NanoDrop (Figure S3A). As explained in the Bio-Rad ddPCR manual guidelines, we can estimate the copy number of genomic hALB sequence in diploid cells from gDNA quantification and compare these results with the hALB copy number obtained by ddPCR technology. From the mass of the human genome, we calculate that there were about 120,000 copies in 400 ng of human DNA, assuming 1 copy/haploid genome. In this manner, we observe that the amount of gDNA from diploid human hematopoietic cells, quantified by Qubit, was more similar to the values obtained by ddPCR technology when compared to the values obtained using the NanoDrop technology (Figure S3B). As seen in Figure S3C, this difference was observed irrespective of the column extraction kits used or the gDNA post-extraction steps (dilution or digestion). Therefore, we decided to use the Qubit technology with the dsDNA BR kit to quantify gDNA for ddPCR analyses.

NaOH lysis for CFC

We first tested the compatibility of two direct lysis protocols with ddPCR analysis by using JUC-5 cells, a clone containing five copies checked by integration site Target Locus Amplification (TLA) analysis27 (see standards materials in Supplemental Methods and Figure S4); (1) the NaOH lysis solution without heat inactivation using the DNA extraction all reagents kit (Applied Biosystems, 4402616) versus (2) a classic Proteinase K lysis with heat activation at a temperature above 65°C previously described.13 Whereas no difference in VCN values was seen when performing the conventional qPCR analysis between the two lysis methods (data not shown), we have observed a VCN underestimation with proteinase K lysis compared to NaOH lysis by ddPCR with VCN = 3 ± 0.4 (n = 39) versus 4.8 ± 0.4 (n = 36) ( Figure 2A). We investigated this discrepancy and found a lower detection of HIV-Psi and hALB copy number (data not shown) and consequently on VCN, after proteinase K lysis. This may be due to (1) the application of the technical procedure where cell transfer into 96-wells plate for heat inactivation can have an impact on the random distribution into the droplets and/or (2) a PCR inhibition or DNA accessibility issue because amplification was not observed without lysis dilution in qPCR or ddPCR irrespective of the mode of lysis. Following that, we have determined that VCN can be analyzed between a 1/5 and 1/20 dilution of NaOH lysate from 1,000 to 50,000 cells (Figure 2B). In this condition, we have found that the hALB copy detected by ddPCR comprises between 1.3 and 500 copies/µl, and the limit of quantification can be determined at 2.5 copies/µl to provide a mean VCN of 4.9 copies with a CV ≤ 20% (n = 20) (Figure 2C). This limit of VCN quantification was comparable to the LOQ value obtained with serial dilutions of digested gDNA from the column extraction kit. Additionally, these lysis conditions were then applied to CFC from transduced CD34+ cells (Figure 2D-E). From the lysed samples of CFU-GM and BFU-E, we observe a correlation between the Ct of qPCR and the ddPCR copy quantities for both hALB and HIV-Psi sequences, demonstrating the feasibility to analyze both sequences. Moreover, the distribution of VCN+ CFC was quite similar with qPCR or ddPCR analysis (Table S2). All these results support the use of NaOH lysis to analyze VCN in CFC.

Figure 2.

Optimization of direct lysis associated with ddPCR analysis. (A) VCN analysis after NaOH lysis or Proteinase K lysis treatment. Serial quantities of 500–50,000 JUC-5 cells were lysed by NaOH lysis solution or proteinase K and analyzed by ddPCR. Average VCN and standard deviation were calculated from nine different cell extractions of two replicates. (B) hALB copy analysis after several dilutions of NaOH lysis. Serial quantities of 500–50,000 JUC-5 cells were lysed by NaOH lysis solution (six different cell extractions) and diluted with H20 to 1/5, 1/10, and 1/20 before ddPCR analysis. As expected, the hALB sequence copy proportionality was equal to, respectively, 2 between dilution 1/5 and 1/10 and dilution 1/20. (C) LOQ determination after cell quantity: VCN variability related to Albumin copy concentration. Serial quantities of JUC-5 cells were lysed by NaOH lysis solution and analyzed by ddPCR. VCN mean and standard deviation were calculated from 20 replicates for the samples between 1.3 and 88 hALB copies/µl and 14 replicates for others concentrations. Correlation between qPCR Ct and ddPCR copy quantity for CFU-GM (D) and BFU-E (E): each progenitor was lysed by NaOH lysis solution, and HIV-Psi and hALB sequences were analyzed by qPCR or ddPCR using QSY quencher. Using linear regression analysis, the Pearson correlation was equal to –0.8976 for hALB (n = 78) sequence and –0.9773 for HIV-Psi (n = 58) sequence from CFU-GM and –0.9913 for hALB (n = 68) and –0.984 for HIV-Psi (n = 61) sequence from BFU-E.

Multiplex ddPCR

For simultaneous analysis of three targets in ddPCR, we used probes conjugated to FAM for HIV-Psi and WPRE sequences and to VIC for Albumin, all associated with QSY-7 quencher. The concentration of WPRE probes has been adapted to separate HIV-Psi positive droplets, WPRE positive droplets, and double positive droplets ( Figure 3A). We found similar VCN results from same samples whatever the multiplex ddPCR and the sequence used (Figure 3B). These results confirm the use of the multiplex ddPCR from one DNA sample when used with our conditions.

Figure 3.

Multiplex ddPCR. (A) Positive control represented in 2D plots of fluorescence droplets from a triplex experiment with WPRE sequence addition. QSY/FAM WPRE and QSY/FAM HIV-Psi had readings in channel 1 and QSY/VIC hALB in channel 2. (B) VCN comparison between HIV-Psi and WPRE sequence after duplex or triplex ddPCR. VCN from gDNA extracted by column kit or direct lysis without extraction was analyzed by using WPRE/hALB sequences and was compared with HIV-Psi/hALB sequences and is similar. In duplex ddPCR, VCN recovery between HIV-Psi and WPRE was equal to 97 ± 4% (n = 16) after extraction by column lysis and was equal to 99 ± 12% (n = 28) after direct lysis (NaOH lysis). In triplex ddPCR, the VCN recovery between HIV-Psi and WPRE was equal to 97 ± 3% (n = 16) after extraction by column lysis and was equal to 98 ± 6% (n = 28) after direct lysis.

Conclusion

Measuring the number of vector copies in target cells is an important parameter to define the best conditions of lentiviral transduction efficiency of T lymphocytes or CD34+ cells in the perspective of experimental or clinical applications. Here, we have identified key elements of sample preparation and set-up to further improve the performance characteristics of ddPCR method, resulting in an accurate analysis of VCN without the need for multiple replicates or an external calibrator, as required in qPCR methods. This method consists of duplex detection of HIV-Psi sequences normalized to hALB (reference gene) or a triplex detection of HIV-Psi and WPRE normalized to hALB performed after a classical gDNA extraction or an adapted direct lysis protocol for low number of cells.

Materials

• Nuclease-free water

• Genomic DNA extraction kit

• ○ NucleoSpin Blood Mini kit (Macherey-Nagel, 740951)

• ○ DNA extraction all reagents kit (Applied Biosystems, 4402616)

Qubit™ dsDNA BR (Broad Range) assay Kit Q32850

10× restriction enzyme buffer

Restriction enzyme for the fragmentation of the template >66 ng per reaction HindIII (NEB, cat. no. R3104S) is recommended by Bio-Rad

Primers and probes (20µM)

○ For hALB (M12523.1)

Probe: 5′-CCTGTCATGCCCACACAAATCTCTCC-3′ (5’VIC/3'QSY 7)

Forward primer: 5′-GCTGTCATCTCTTGTGGGCTGT-3′

Reverse primer: 5′-ACTCATGGGAGCTGCTGGTTC-3′

○  For HIV-PSI

Probe: 5′-CGCACGGCAAGAGGCGAGG-3′ (5’FAM/3'QSY 7)

Forward primer: 5′-CAGGACTCGGCTTGCTGAAG-3′

Reverse primer: 5′-TCCCCCGCTTAATACTGACG-3′

○  For WPRE

Probe: 5′-TGTTTGCTGACGCAACCCCCACT-3′ (5’FAM/3'QSY 7)

Forward primer: 5′-AGGAGTTGTGGCCCGTTGT-3′

Reverse primer: 5′-TGACAGGTGGTGGCAATGC-3′

2× ddPCR super mix (no dUTP) (Bio-Rad, cat. no. 1863024)

DG32™ Automated Droplet Generator Cartridges (Bio-Rad, cat. no. 1864108)

ddPCR 96-well plates (Biorad, cat. no. 12001925)

Pipet Tips Auto DG System (Biorad, cat. no. 1864121)

Pipet Tips waste bins (Biorad, cat. no. 1864125)

PCR Plate Heat Seal, foil, pierceable (Bio-Rad, cat. no. 1814040)

Automated Droplet Generation Oil for probes (Bio-Rad, cat. no. 1864110)

ddPCR™ Droplet Reader Oil (Bio-Rad, cat. no. 1863004)

Equipment

Qubit™ 4 Fluorometer (ThermoFisher Scientific, cat. no. Q33226)

NanoDrop ND-8000 Spectrophotometer (Thermo Fisher Scientific)

QX200 Automated Droplet Generator (Bio-Rad, cat. no. 1864101)

Heat sealer (Bio-Rad, cat. no. 1864000)

QX200 Droplet Reader (Bio-Rad, cat. no. 1864003)

QuantaSoft Analysis Pro Software (Bio-Rad)

Thermal cycler UNO96 (VWR, cat. no. 73262549)

Procedures

Preparation of genomic DNA from bulk cells

Nucleic acid purification:

gDNA from cell lines, bulk CD34+, or bulk T lymphocytes (cell pellet > 1E6 cells) is purified with column extraction kits, the QIAmp DNA blood Mini kit (Qiagen, 51104) or the NucleoSpin Blood Mini kit (Macherey-Nagel, 740951), following manufacturer instructions. The DNA elution is in a volume of 100 µl of nuclease free water.

• Nucleic acid quantification:

The Qubit™ dsDNA BR assay is specific for double-stranded DNA (dsDNA) and is used for fluorometry measurement. This method can accurately measure initial sample concentration from 100 pg/μl to 1,000 ng/μl.

○ Dilute the Qubit™ reagent 1:200 in Qubit™ buffer to prepare the working solution sufficient to accommodate all standards and samples.

○ Calibrate the assay by using two DNA standards. For each DNA standard, add 10 μl of the standard to 190 μl of working solution in a Qubit™ Assay Tube to a final volume of 200 μl.

○ Measure the purified DNA. For each sample, add 1–2 μl of the purified DNA to the working solution to a final volume of 200 μl in a Qubit™ Assay Tube.

○ Vortex tubes for 2–3 s, incubate at room temperature for 2 min.

○ Read standard 1, then standard 2 using the Qubit™ 4 fluorometer.

○ Insert sample tubes one by one to read the sample concentrations.

The DNA quantification was, respectively, ranged between 2 and 1000 ng with BR dsDNA kit.

Nucleic acid digestion:

Purified gDNA (500 ng) was digested with HindIII-HF (NEB) enzymes with a concentration of 0.1 U/µl in a total reaction mixture of 50 µl at 37°C for 1 h.

Digested DNA samples are diluted with water to obtain a DNA concentration of 2.5 ng/µl.

Preparation of genomic DNA from individual CFC

After harvesting each colony in 96-well plate, gDNA from CFC was extracted with a single-step NaOH lysis, using the DNA extraction all reagents kit.

Add 20 µl by well of lysis solution and mix your sample

Incubate for 3 min

Add 20 µl by well of the DNA stabilizing solution

Store at –20°C

After thawing, take 10 µl of sample and dilute in nuclease free water to 1/10° for CFU-GM and 1/20° for BFU-E

NB: This direct NaoH lysis can be applied for a cell quantity ranging between 500 and 50 000 cells.

Multiplex ddPCR

• Preparation of PCR reaction:

  Each 25 µl reactions contains:

• Droplet generation and PCR amplification:

  The sample mixture (20 µl) was transferred to a DG32 cartridge and placed into the QX200 auto droplet generator where sample droplets were then generated and transferred onto a 96-well PCR plate.

• Droplet reading

  After thermal cycling, plates were stored at +4°C, before their transfer to the QX200 droplet reader (Bio-Rad) to read the droplet fluorescence using Quantasoft software.

• Interpretation of results

Authorship Contribution Statement: The authors confirm the contribution to the paper as follows: study design by A.P. and S.C., study execution by A.P., J.B., E.N., and P.S., data analysis by A.P., B.K., N.A., S.M., and S.C., manuscript drafting by A.P., S.M., N.A., C.R., S.F., and S.C.

Authors’ Disclosure Statements: The authors declare no commercial or financial conflict of interest. A.P., J.B., E.N., B.K., S.F., C.R., N.A., and S.C. are employees in the Innovation, Analytical, and Process Development Business Unit of Yposkesi, a viral vector contract manufacturing organization where the development of this test was performed to implement as part of the standard quality control analysis to be offered to our gene therapy product developer clients.

Funding Statement: This work did not receive any grant.