Team:Patras/Engineering
Nanopore and NGS analysis
Our novel CYP2D6 and CYP2C19 variants were obtained from 103 samples after performing targeted resequencing using MinION from Oxford Nanopore Technologies, all part of a previous research project within the Laboratory of Pharmacogenomics and Individualized Therapy.
Oxford Nanopore MinION sequencer is an exciting third-generation sequencing paradigm. Third-generation sequencing is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis or performing targeted screening to one that promises high quality de novo assembly and structural variation (insertions, deletions, duplications) detection for human-sized genomes. MinION is a portable, real-time, long-read, low-cost device capable of sequencing many gigabases in a single sequencing experiment, producing several GB of data per experiment.
MinION is also using the innovative technology of nanopores. Specifically, It contains an array of tiny holes called nanopores, embedded in a membrane, across which an electrical current is passed, and single-stranded DNA or RNA can be streamed through the pores and sequenced in real-time. This makes nanopore sequencing unique; it is the only sequencing technology that enables direct, real-time analysis of short to ultra-long fragments of DNA or RNA in fully scalable formats. MinION comes with a flow cell containing the nanopores and the sensing chemistry used in nanopore sequencing. It also contains the circuitry required to convert the current flow through the pore into an electrical signal that is used to sequence DNA. The flow cell sensor array has 2048 wells and typically contains over 1,000 nanopores. These wells are connected to electrical channels and can only record up to 512 channels at any one time.
Before sequencing, a software must be installed and a library must be prepared according to the given instructions from the MinION . MinKNOW is a software for testing hardware, checking flow cells, data acquisition, real-time analysis and feedback, basecalling, data streaming, and device control. MinKNOW takes the raw data and converts it into reads by recognizing the distinctive change in current that occurs when a DNA strand enters and leaves the pore. MinKNOW then basecalls the reads and writes out the data into .fast5 or FASTQ files.
The pipeline followed is presented below:
Guppy was used to run basecalling and generate the FASTQ files. Proceeding to alignment, GRCh37/hg19 was used as a reference genome resulting in SAM/BAM files. Finally, the Nanopolish tool was used for variant calling, and final VCF files were obtained. At this analysis step, it was noticed that among the already reported variants according to the genomic databases, some single nucleotide variants are not included in these databases and are supported by acceptable quality scores. To determine the potential pathogenicity of the assessed novel variants, in silico scores were used to predict whether an amino acid substitution leads to a protein damaging effect. Specifically, SIFT, Clinpred, and DANN scores were used to indicate a potentially damaging effect of variants, resulting in the novel ones presented in the variants section.
| CYP2C19 Variants | CYP2D6 Variants |
|---|---|
| p.L15P | p.L61S |
| p.R26* | p.F112S |
| p.E274G | p.S476I |
| p.N286D | |
| p.D293G | |
| p.T304A | |
| p.I327V | |
| p.L380P | |
| p.L413M | |
| p.F487S | |
From the presented variants, the p.R26* on CYP2C19, did not proceed to functional characterization because the mutation resulted in a stop codon an in a non-functional oligopeptide has not any function.
Wet Lab
- All
- Learn from the existing
- Cloning of the pENTR/D-TOPO vector
- Site-directed mutagenesis
- TA Cloning
- Transfection
Learn from the existing
For the evaluation of the enzyme activity with in vitro assays, is usually used a heterologous expression system that provides the advantage of high reproducible conditions without patients undergoing invasive procedures and eliminating the risk of developing adverse drug reactions. According to literature, specifically for the functional characterization of the differential effects of CYP variants due to single nucleotide variants, there are various expression systems such as E. coli, bacteria, yeast, baculoviruses, and mammalian cell lines that are used in research.
Exogenous DNA delivery into animal cells is a widely used process for the expression of functional recombinant proteins. However, when applied in mammalian cells, we face the problem of low protein expression and consequently the increasing costs. Thus, for achieving a successful transfection in mammalian cell lines, Lipofectamine 3000 was used.
Additionally, 293FT cells were selected because they allow very high levels of protein to be expressed from vectors containing the SV40 origin, compared to other cell lines, COS-7, HepG2, and 293T cells.
pcDNA3.4 TOPO vector was used as it contains a full-length CMV promoter and other expression elements that allow for higher expression than other pcDNA-based expression constructs, making it suitable to achieve higher levels of CYP expression in 293FT cells.
In the exhibition of CYP catalytic activity, the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) plays a vital role. CPR provides the essential first electron, whereas the second electron may come directly from CPR or via cytochrome b5 (CYB). According to several studies, CYP activities were increased when using a co-expression system of CPR and CYP compared to the expression of CYP alone. However, the mechanism underlying the benefits of CYP and CPR plasmid co-expression in mammalian cells is not yet found.
In addition to CPR, electron transfer via CYB also plays a role in improving the enzymatic activities of several CYP isoforms.
Therefore, the co-expression of CYP, CPR, and CYB is supported for achieving optimal conditions for the evaluation of enzymatic activity in vitro and, due to its advantages, was selected for our experiments.
Design: cDNAs Amplification with TOPO sites
In our experiments, we used a pENTR/D-TOPO system. The vector is linearized with two DNA topoisomerases I on each 5’ and 3’ ends. The enzyme DNA topoisomerase I acts as both a restriction enzyme and a ligase; thus, no ligase or any restriction enzyme is needed to be added for this cloning procedure. In this system, PCR products are directionally cloned by adding four bases to the forward primer (CACC). The overhang in the cloning vector (GTGG) invades the 5′ end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation. Inserts can be cloned in the correct orientation with efficiencies equal to or greater than 90%. The Forward primers of each gene of interest are designed with a TOPO site (CACC) at the 5’ end of their sequence for directional cloning of the blunt-end PCR product into a pENTR/D-TOPO vector as presented in the table below.
| # | Name | Type | Description | Length |
|---|---|---|---|---|
| 1 | BBa_K3839028 | Primer | CYP2D6 TOPO site Forward Primer | 20 |
| 2 | BBa_K3839029 | Primer | CYP2D6 TOPO site Reverse Primer | 22 |
| 3 | BBa_K3839030 | Primer | CYP2C19_201 TOPO site Forward Primer | 26 |
| 4 | BBa_K3839031 | Primer | CYP2C19_201 TOPO site Reverse Primer | 30 |
| 5 | BBa_K3839032 | Primer | POR-218 TOPO site Forward Primer | 23 |
| 6 | BBa_K3839033 | Primer | POR-218 TOPO site Reverse Primer | 20 |
| 7 | BBa_K3839034 | Primer | CYB5A_202 TOPO site Forward Primer | 19 |
| 8 | BBa_K3839036 | Primer | CYB5A_202 TOPO site Reverse Primer | 23 | 9 | BBa_K3839035 | Coding | pENTR/D-TOPO | 2580 |
The initial wild-type templates for CYP2D6, CYP2C19, CPR, and cytochrome b5 cDNAs were purchased from IDT as gBlocksTM Gene Fragments. We amplified these cDNAs using a high-fidelity Q5 polymerase.
In our first experiments, we chose the Standard PCR for the amplification of the cDNA fragments. However, following this protocol resulted in a not satisfactory amount of PCR products. Consequently, we performed a Touch-down PCR to achieve high specificity and efficiency of the amplification.
The optimized conditions of the Touch-down PCR are shown in the table below.
| Component | 50μl Reaction | Final Concentration |
|---|---|---|
| 5x Q5 | ||
| Reaction Buffer | 10 μl | 1x |
| 10 mM dNTPs | 1 μl | 200 μM |
| 10 µM Forward Primer | 2.5 µl | 0.5 μM |
| 10 µM Reverse Primer | 2.5 µl | 0.5 μM |
| Template DNA | variable | < 1,000 ng |
| Q5 High-Fidelity DNA Polymerase | 0.5 µl | 0.02 U/µl |
| 5x Q5 High GC Enhancer (optional) | (10 µl) | (1x) | Nuclease-Free Water | to 50 µl |
| Step | Temp | Time |
|---|---|---|
| Initial Denaturation | 95°C | 10 minutes |
| Denaturation | 94°C | 30 seconds |
| Touchdown | 62-54°C* | 30 seconds |
| 72°C | 30 seconds | |
| Final Extension | 72°C | 10 minutes |
| Hold | 4 | infinity |
| Temp | Cycles |
|---|---|
| 62°C | 5 cycles |
| 60°C | 5 cycles |
| 58°C | 5 cycles |
| 56°C | 5 cycles |
| 54°C | 5 cycles |
Build: Cloning of the pENTR/D-TOPO vector
All the pENTR/D-TOPO vectors cloned with the wild-type CYP2D6, CYP2C19, CPR, and cytochrome b5 cDNAs (BBa_K3839038, BBa_K3839039, BBa_K3839040, BBa_K3839041), were transferred into NEB 5-Alpha High-Efficiency Competent E. coli cells according to the transformation protocol. For the clone selection, the antibiotic Kanamycin was used in the plates. After the incubation, single colonies were selected and cultured in liquid SOB Medium overnight. Finally, the plasmid DNAs were extracted and purified with Monarch® Plasmid Miniprep Kit.
You can preview the vector map on our Parts page.
Design: Site-directed Mutagenesis
The plasmids with the wild-type CYP2D6 cDNA and the plasmids containing the wild-type CYP2C19 cDNA were used each as a template to generate the novel enzymatic allelic variant constructs, using a Q5® Site-Directed Mutagenesis Kit. The primers for the mutagenesis of the variants CYP2D6 and CYP2C19 cDNAs are shown in the table below.
All the studied variants are substitutions, and for that reason, the mutagenic sites were placed onto the forward primer as a miss-match. The substitution was created by incorporating the desired nucleotide change in the center of the forward primer, including at least ten (10) complementary nucleotides on the 3´side of the mutation(s). The reverse primer was designed so that the 5´ ends of the two primers anneal back-to-back.
Subsequently, were designed 12 pairs of primers.
| # | Name | Type | Description | Length |
|---|---|---|---|---|
| 1 | BBa_K3839000 | Primer | Forward mutagenic primer CYP2D6 L61S | 21 |
| 2 | BBa_K3839001 | Primer | Reverse primer CYP2D6 L61S | 27 |
| 3 | BBa_K3839002 | Primer | Forward mutagenic primer CYP2D6 F112S | 21 |
| 4 | BBa_K3839003 | Primer | Reverse primer F112S | 19 |
| 5 | BBa_K3839004 | Primer | Forward mutagenic primer S476I | 21 |
| 6 | BBa_K3839005 | Primer | Reverse primer CYP2D6 S476I | 19 |
| 7 | BBa_K3839006 | Primer | Forward mutagenic primer L15P | 23 |
| 8 | BBa_K3839007 | Primer | Reverse primer CYP2C19 L15P | 24 |
| 9 | BBa_K3839008 | Primer | Forward mutagenic primer CYP2C19 E274G | 21 |
| 10 | BBa_K3839009 | Primer | Reverse primer CYP2C19 E274G | 18 |
| 11 | BBa_K3839010 | Primer | Forward mutagenic primer CYP2C19 N286D | 24 |
| 12 | BBa_K3839011 | Primer | Reverse primer CYP2C19 N286D | 20 |
| 13 | BBa_K3839012 | Primer | Forward mutagenic primer CYP2C19 D293G | 21 |
| 14 | BBa_K3839013 | Primer | Reverse primer CYP2C19 D293G | 27 |
| 15 | BBa_K3839014 | Primer | Forward mutagenic primer CYP2C19 T304A | 21 |
| 16 | BBa_K3839015 | Primer | Reverse primer CYP2C19 T304A | 18 |
| 17 | BBa_K3839016 | Primer | Forward mutagenic primer CYP2C19 I327V | 21 |
| 18 | BBa_K3839017 | Primer | Reverse primer CYP2C19 I327V | 19 |
| 19 | BBa_K3839018 | Primer | Forward mutagenic primer CYP2C19 L380P | 21 |
| 20 | BBa_K3839019 | Primer | Reverse primer CYP2C19 L380P | 21 |
| 21 | BBa_K3839020 | Primer | Forward mutagenic primer CYP2C19 L413M | 21 |
| 22 | BBa_K3839021 | Primer | Reverse primer CYP2C19 L413M | 21 |
| 23 | BBa_K3839022 | Primer | Forward mutagenic primer CYP2C19 F487S | 21 |
| 24 | BBa_K3839023 | Primer | Reverse primer CYP2C19 F487S | 21 |
Build: Cloning of the obtained variant vectors
The following procedures of transformation and plasmid extraction were performed for each designed vector separately.
After receiving the vectors containing the genes’ variants, High-Efficiency E.coli cells (50 μL of NEB 5-Alpha competent cells) were transformed with 1 μg of designed plasmids. The mixture was allowed to stand on ice for 30 minutes, followed by heat shock at 42 oC for 30sec, placed on ice for 5 minutes, added 950 μL of S.O.C. Medium to the resultant mix, and cultured with shaking vigorously (250 rpm) at 37 oC for 30 min. After that, 100 mL of the culture solution was spread on a kanamycin-containing SOB agar plate and cultured at 37 oC for 16 h. Single colonies were selected and transplanted into 5.0 mL of kanamycin-containing liquid medium (SOB) and cultured at 37 oC while shaking at 250 rpm for 18 h.
After that, plasmids from twelve colonies were subsequently extracted and purified using the Monarch® Plasmid Miniprep Kit. 5mL of bacterial cultures was centrifuged at 13,000 RPM, and the pellets were resuspended in 200μl of resuspension buffer. After lysis and two stages of washing buffers, the extracted plasmids were diluted in 30μl of elution buffer.
Digestion of the pENTR/D-TOPO vectors + mutated inserts
In order to remove the desired sequence containing the gene variants, digestion with two restriction enzymes, NotI and AscI, was performed. 5-6 ng of the pENTR/D-TOPO vector subcloned with the CYP2D6 and CYP2C19 variants were digested with 1μl of each enzyme (10,000 units/ml) at a final volume of 20μl, at 37oC for 30 minutes, following the digestion protocol and conditions of each enzyme.
Test: Electrophoresis
All the digestion mixture was run at a low-melting agarose gel of 0,8%, and the DNA band of interest was removed from the gel with cutting. The low-melting gel provides us with greater sieving properties and higher clarity than standard melting temperature agarose.
Purification of the extracted DNA band was performed using a Gel extraction kit.
Building the mammalian expression vector
The next step was to subclone the DNA fragments with the designed gene variants into a pcDNA3.4 vector with TOPO TA cloning sites. The pcDNA 3.4 TOPO plasmid is a constitutive mammalian expression vector designed to deliver high levels of transgene expression.
DNA Pol I, Large (Klenow) fragment without the 3’- 5’ exonuclease activity, was used to generate blunt-ends with A and T overhangs on the DNA fragments, setting the conditions for achieving the directional TOPO cloning with the vector.
Wild-type and variant CYP2C19 cDNAs subcloned into the pcDNA3.4 vector. Plasmids carrying CPR or cytochrome b5 cDNAs were also subcloned into the pcDNA3.4 vector.
Competent E. coli cells were transformed with the final vectors subcloned with the DNA fragments, according to the given protocol. After incubation for 24 hours and the colony selection, the E.coli cells were cultured for 16 hours in SOB medium containing Ampicillin. Finally, the plasmid DNA was extracted and purified at a final volume of 30μL.
Test: Electrophoresis
To verify the integrity of the produced subcloned vectors, electrophoresis on agarose gel 1% was run.
Prepartion
Before the transfection of the eukaryotic cells, 5ng of the mammalian expression vectors were prepared in a solution of H2O, CH3COONa, and absolute ethanol to purify and precipitate the DNA. This solution was stored at -20°C overnight.
Transfection
293FT cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at 37°C under 5% CO2.
24 h before transfection, the cells were plated at a density of 2*106 cells/100-mm dish. Subsequently, Opti-MEM medium (Invitrogen) was added to the culture medium.
After 24 hours of incubation, cells were transfected with the designed plasmid encoding the wild type and variant CYP2D6 and CYP2C19 cDNAs, CPR cDNA, and Cytochrome B5 cDNA using Lipofectamine 3000, at concentrations of 7,5μl/1x106 cells. 3-5 ng of the designed vectors were transfected into the cells.
For selecting the transfected cells, Neomycin (G418) was utilized at a concentration of 800μg/mL of full medium.
After 6 h of incubation at 37°C, the culture medium was replaced with 10% FBS-DMEM. The incubation continued for 16 hours at 37°C, followed by dispension of medium and washing with PBS, the 293FT cells were scraped off.
The cells were centrifuged at 1500g for 5 minutes and resuspended in a homogenization buffer [10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 10% glycerol]. Microsomal fractions were prepared by differential centrifugation at 9000g for 20 minutes so as the components of the cell were separated according to their density, followed by centrifugation of the resulting supernatant at 105,000g for 60 minutes.
The microsomal pellet was resuspended in 50 mM Tris-HCl (pH 7.4) containing 1 mM EDTA, 20% glycerol, 150 mM KCl, and Protease Inhibitor Cocktail for Use with Mammalian Cell and Tissue Extract and stored at –80°C.
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