Normalisation prior to sequencing

Abstract

The invention relates to a method for normalising the amount of nucleic acid across a plurality of samples, characterised in that it comprises the following steps: - providing a plurality of samples, each having a volume V comprising nucleic acid; - collecting a known volume VCO of each sample; - based on said collected volumes VCO, carrying out a first sequencing operation that makes it possible to obtain, for each of the volumes VCO, a number of reads; - depending on the number of reads for each volume VCO, calculating a volume of sample to be collected VEP for each sample, such that each VEP corresponds to an identical theoretical number of reads NLT.

Description

Normalization before sequencing

[0001] The present invention relates to the technical field of sequencing, and more specifically to the technical field of next-generation sequencing, sometimes called "next-generation sequencing" or "NGS".

[0002] For the purposes of this description, an interval described by the expression "between [...] and [...]" includes the bounds. For example, the interval "between 1 and 2" includes the two values ​​"1" and "2" as well as all values ​​that are both strictly greater than 1 and strictly less than 2.

[0003] In this description, the term "approximately" preceding a numeric value means that the value can be changed by plus or minus 10%. In the specific case of a numeric value acting as an interval boundary, the term "approximately" means that the lower boundary can be reduced by 10% or the upper boundary can be increased by 10%. It is also possible to omit the term "approximately" preceding a numeric value.

[0004] Next-generation sequencing (NGS) is a revolutionary method for reading nucleic acid sequences, particularly DNA, much faster and at a lower cost than traditional sequencing methods. This technology has radically transformed genomics, molecular biology, and various areas of biomedical research. Its impact on research and medicine is immense, paving the way for innovative discoveries and applications in numerous fields of science and health.

[0005] NGS sequencing relies on interdependent steps, from sample preparation to bioinformatic analysis and interpretation of results.

[0006] The NGS process therefore comprises several key steps, each playing a crucial role in generating accurate and detailed sequencing data.

[0007] The first step in NGS is library preparation. During this step, the target DNA or RNA is extracted from a sample (blood, saliva, etc.) and fragmented into small pieces. During this process, Unique Dual Indexing (UDI) sequences are attached to the fragments to link each fragment to the source sample.

[0008] After library preparation, the sample undergoes purification and amplification by PCR or in situ amplification to increase the amount of target genetic material. This step is crucial to ensure that sufficient genetic material is available for sequencing.

[0009] Next, the prepared nucleic acid sample, specifically DNA, is loaded onto a next-generation sequencer. The actual sequencing then begins, using various technologies such as sequencing by synthesis (SBS), ligation sequencing, or other methods based on nanopores or semiconductors.

[0010] Each technology has its own unique mechanisms for reading DNA sequences. For example, in SBS, fluorescent nucleotides are added sequentially and their incorporation is detected by light signals.

[0011] After sequencing, the raw data undergoes a bioinformatics processing procedure. This includes aligning the obtained sequences with known genomic references and assembling these sequences to reconstruct the original sequence. This step is crucial for correctly interpreting NGS data. The data are also analyzed to detect genetic variations such as point mutations, insertions, deletions, and chromosomal rearrangements.

[0012] Finally, sequencing results are interpreted in clinical or research contexts. In biomedical research, NGS is used to understand the genetic basis of diseases, discover new biomarkers, and develop personalized therapies. Clinically, it aids in the diagnosis and treatment of various conditions, including genetic diseases, cancers, and infections.

[0013] UDI sequences are essential in next-generation sequencing (NGS) to improve data accuracy and reliability. This technique involves assigning two unique indices to each sample, thus reducing the risk of misassignment and cross-contamination.

[0014] During library preparation, the DNA or RNA fragments of each sample are labeled with these indices, enabling specific and precise identification during sequencing. This doubly indexed method is particularly important during multiplexing, where many samples are mixed and sequenced simultaneously, as it minimizes the risk of incorrect sequence assignment, thus improving the quality and reliability of genomic sequencing data.

[0015] In the context of NGS, a "read" is a sequence of nucleotide bases that is read.

[0016] The number of reads is the total number of individual reads obtained during sequencing. It influences the accuracy and resolution of the results. A high number of reads generally improves the quality of the results.

[0017] After purification and amplification, the concentrations of nucleic acid, particularly DNA, are not the same for the different samples treated.

[0018] These samples are nevertheless intended to be mixed and sequenced in a single step.

[0019] As a result, one of the crucial steps in the NGS process that conditions the final results lies in introducing the same amount of genetic material into the sequencer for each sample taken.

[0020] To do this, a number of techniques are commonly used, such as, for example, fluorescence assay or real-time PCR assay.

[0021] These dosages are complex to implement, they are costly, they require the ordering of specific equipment, and they also lengthen the overall handling time.

[0022] In earlier art, a number of steps are revealed.

[0023] Application WO18136526, filed on behalf of COUNSYL INC., discloses a method for preparing libraries of improved capture probes. This application addresses several aspects of sequencing, including sequencing depth and performance-based probe selection.

[0024] Application WO23034090 filed on behalf of NATERA, INC., discloses a method for non-invasive prenatal testing. This method includes cell-free DNA extraction from a pregnant woman's blood sample, amplification, and sequencing to determine the ploidy state of chromosomes of interest.

[0025] In application WO19170773 on behalf of CANCER RESEARCH TECHNOLOGY LIMITED, a computerized method for the detection of circulating tumor DNA from a sample taken from a patient is disclosed.

[0026] In application WO22197864 filed on behalf of NATERA, INC., a method for detecting transplant rejection is disclosed. The amount of donor DNA present in the recipient is one of the parameters considered to determine if there is a problem with transplant rejection in the recipient.

[0027] Applications WO22182878 and WO21243045, in the name of the same applicant, contain similar disclosures.

[0028] In application US20230399679 on behalf of GTSEEK, LLC., a method using ligand-modified primers to normalize DNA concentrations is disclosed.

[0029] In the publication Bruinsma, S., Burgess, J., Schlingman, D., Czyz, A., Morrell, N., Ballenger, C., ... Gormley, NA (2018). Bead-linked transposomes enable a normalization-free workflow for NGS library preparation. BMC Genomics, 19, 722. https: / / doi.org / 10.1186 / s12864-018-5096-9, a method for preparing libraries for next-generation sequencing is disclosed. This method uses bead-linked transposomes to capture a fixed amount of DNA, allowing for the direct preparation of libraries from blood and saliva samples. It is presented as being efficient, even with small amounts of DNA. It eliminates the need for DNA quantification prior to sequencing.

[0030] Other documents can also be related as technical background.

[0031] Thus, in US patent application US20160046987 filed on behalf of Abbott Molecular Inc., a method for normalizing DNA libraries intended for sequencing is disclosed. A capture substrate (often magnetic and functionalized) is presented as being capable of binding a precise amount of DNA. The method relies on the passive capture of a fixed amount of DNA by saturating a substrate. This assumes that all samples bind uniformly, which is rarely the case in reality. Furthermore, the samples are normalized "blindly" by saturation capture, which does not allow for fine-tuning. This method is therefore not entirely satisfactory and, in any case, less efficient than the one presented in this description.

[0032] In US patent application US2024132942 filed on behalf of Watchmaker Genomics, Inc., a method for normalizing sequencing libraries is disclosed, using DNA-binding proteins such as certain CRISPR proteins. These proteins are described as being able to bind to specific adaptor sequences on nucleic acids, allowing for the extraction of standardized amounts of DNA fragments between multiple samples.

[0033] Furthermore, in application CA3097745 filed on behalf of BIOFIRE DIAGNOSTICS, LLC., a method and kit for standardizing and quantifying genetic sequencing data are disclosed. An internal standard is added to the sample before any preparation. This is intended to enable quantification of absolute quality.

[0034] Next, in US patent application US20150292001 filed on behalf of THE UNIVERSITY OF TOLEDO, a nucleic acid sequencing method is disclosed, aimed at improving the accuracy and reproducibility of measurements of the quantities of nucleic acids present in different samples. The method relies on the addition of competing internal controls.

[0035] Finally, in application US20080318233 on behalf of Travis C. Glenn et al., a method for labeling and normalizing DNA fragments for sequencing is disclosed.

[0036] Surprisingly, the plaintiff highlighted that a new method for normalizing the amount of nucleic acid before sequencing could be used.

[0037] Thus, the invention relates to a method for standardizing the quantity of nucleic acid between at least two samples, characterized in that it comprises the following steps:

[0038] - have at least 2 samples, each with a volume V comprising nucleic acid;

[0039] - take a known volume VCO from each of at least 2 samples;

[0040] - from the said VCO volumes taken, proceed with a first sequencing allowing the obtaining, for each of the VCO volumes, of a number of reads;

[0041] - depending on the number of readings of each volume VCO, calculate a sample volume to be taken VEP for each sample such that each VEP corresponds to an identical theoretical number of readings NLT.

[0042] The invention relates to a method for normalizing the quantity of nucleic acid between x samples, with x ≥ 2, characterized in that it comprises the following steps:

[0043] - to have samples each having a volume V comprising nucleic acid;

[0044] - take a known volume VCO from each of at least 2 samples;

[0045] - from the said VCO volumes taken, proceed with a first sequencing allowing the obtaining, for each of the VCO volumes, of a number of reads;

[0046] - depending on the number of readings of each volume VCO, calculate a sample volume to be taken VEP for each sample such that each VEP corresponds to an identical theoretical number of readings NLT.

[0047] In one embodiment, the nucleic acid is chosen from the group consisting of ribonucleic acid (RNA) and in particular messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small interference RNA (siRNA), microRNA (miRNA) and / or long non-coding RNA (lncRNA), deoxyribonucleic acid (DNA) and in particular chromosomal DNA (chDNA), mitochondrial DNA (mDNA), plasmid DNA (pDNA), circular DNA (cDNA), linear DNA (lDNA) and / or recombinant DNA (rDNA), and mixtures thereof.

[0048] In one embodiment, the nucleic acid is chosen from the group consisting of ribonucleic acid (RNA) and in particular messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small interference RNA (siRNA), microRNA (miRNA) and / or long non-coding RNA (lncRNA).

[0049] In one embodiment, the nucleic acid is chosen from the group of bacterial RNAs.

[0050] In one embodiment, the nucleic acid is chosen from the group consisting of deoxyribonucleic acid (DNA) and in particular chromosomal DNA (chDNA), mitochondrial DNA (mDNA), plasmid DNA (pDNA), circular DNA (cDNA), linear DNA (lDNA) and / or recombinant DNA (rDNA).

[0051] In a preferred embodiment, the nucleic acid is circular DNA (cDNA).

[0052] In a preferred embodiment, nucleic acid is DNA.

[0053] In a preferred embodiment, the nucleic acid is DNA complementary to RNA.

[0054] In a preferred embodiment, the nucleic acid is DNA complementary to RNA selected from the group consisting of mRNA, tRNA, rRNA, snRNA, siRNA, miRNA, and lncRNA.

[0055] In a preferred embodiment, the nucleic acid is DNA complementary to bacterial RNA,

[0056] In a preferred embodiment, the nucleic acid is DNA complementary to miRNA, preferably salivary.

[0057] In a preferred embodiment, the samples comprise purified nucleic acid. In a preferred embodiment, the samples comprise amplified nucleic acid. In a particularly preferred embodiment, the samples comprise both purified and amplified nucleic acid.

[0058] In one embodiment, each sample comprises nucleic acid labeled with an identification means. This identification means allows each nucleic acid fragment to be linked to its original sample. In a preferred embodiment, this identification means is labeled with UDI sequences.

[0059] In one embodiment, the number of samples is between 2 and approximately 1000, preferably between 2 and approximately 950, preferably between 2 and approximately 900, preferably between 2 and approximately 850, preferably between 2 and approximately 800, preferably between 2 and approximately 750, preferably between 2 and approximately 700, preferably between 2 and approximately 650, preferably between 2 and approximately 600, preferably between 2 and approximately 550, preferably between 2 and approximately 500, preferably between 2 and approximately 450, preferably between 2 and approximately 400, preferably between 2 and approximately 350, preferably between approximately 5 and approximately 350, preferably between approximately 20 and approximately 350, preferably between approximately 50 and approximately 300, preferably between approximately 50 and approximately 200, preferably between approximately 50 and approximately 100,preferably between approximately 75 and approximately 125, preferably between approximately 90 and approximately 110, preferably between approximately 95 and approximately 105.

[0060] In one embodiment, the VCO volumes are mixed between the step of collecting said VCO volumes and the step of performing said first sequencing.

[0061] In one embodiment, this mixing step is carried out by means of vortexing followed by centrifugation.

[0062] In one embodiment, said known volume VCO is between approximately 0.5 µl and approximately 2.5 µl, preferably between approximately 0.6 µl and approximately 2.4 µl, preferably between approximately 0.7 µl and approximately 2.3 µl, preferably between approximately 0.8 µl and approximately 2.2 µl, preferably between approximately 0.9 µl and approximately 2.1 µl, preferably between approximately 1.0 µl and approximately 2.0 µl, preferably between approximately 1.1 µl and approximately 1.9 µl, preferably between approximately 1.2 µl and approximately 1.8 µl, preferably between approximately 1.3 µl and approximately 1.7 µl, preferably between approximately 1.4 µl and approximately 1.6 µl. µl, preferably about 1.5 µl.

[0063] In one embodiment, the VCO volume is the same for each sample. This embodiment is generally preferred because it simplifies calculations.

[0064] In one embodiment, the VCO volume differs for at least some of the samples. This embodiment can be useful when the sample volumes are very different, and obviously, additional calculation steps are required in this case.

[0065] Sequencing depth in the context of NGS is an important concept for understanding the quantity and quality of genetic sequencing data obtained.

[0066] It can be expressed in several ways.

[0067] First, it can be expressed as "X", representing the average number of times a particular nucleotide base in the genome is sequenced. Therefore, for example, a sequencing depth of 30X means that, on average, each base in the genome has been sequenced 30 times.

[0068] This method of presenting sequencing depth is particularly useful when sequencing a domain of known size, such as the entire human genome. Indeed, the entire human genome is known to be approximately 3.2 billion nucleotide pairs in size. Therefore, it is easy to express the sequencing depth as "X".

[0069] Sequencing depth can also be expressed as the number of reads. This is particularly useful when the size of the genetic material being studied is unknown, difficult to quantify, and / or variable. This is the case, for example, when sequencing DNA from an miRNA sample.

[0070] In one embodiment, said first sequencing has a sequencing depth greater than 0.5X.

[0071] In one embodiment, said first sequencing has a sequencing depth greater than 1000 reads, preferably greater than 10000 reads.

[0072] In one embodiment, said first sequencing is carried out over a period of time between approximately 2 hours and approximately 18 hours, preferably between approximately 5 hours and approximately 15 hours, preferably between approximately 7 hours and approximately 13 hours, preferably between approximately 8 hours and approximately 12 hours, preferably approximately 10 hours.

[0073] In one embodiment, said VEP volume is between approximately 0.5 µl and approximately 7.5 µl, preferably between approximately 0.6 µl and approximately 7.2 µl, preferably between approximately 0.7 µl and approximately 6.9 µl, preferably between approximately 0.8 µl and approximately 6.6 µl, preferably between approximately 0.9 µl and approximately 6.3 µl, preferably between approximately 0.9 µl and approximately 6.0 µl, preferably between approximately 0.9 µl and approximately 5.7 µl, preferably between approximately 0.9 µl and approximately 5.4 µl, preferably between approximately 0.9 µl and approximately 5.1 µl, preferably between approximately 0.9 µl and approximately 4.8 µl. Obviously, this volume varies depending on the samples.

[0074] In one embodiment, said number of theoretical NLT reads is between about 1,000,000 reads and about 1,500,000 reads, preferably between about 1,000,000 reads and 1,300,000 reads, preferably between about 1,100,000 reads and 1,200,000 reads.

[0075] In one embodiment, for each sample, the sum of the volumes VCO and VEP is less than 0.95 times the volume V, preferably less than 0.90 times the volume V, preferably less than 0.85 times the volume V, preferably less than 0.80 times the volume V.

[0076] The invention also relates to a sequencing method, characterized in that it comprises:

[0077] - the implementation of a method for standardizing the quantity of nucleic acid between at least two samples according to the invention;

[0078] - the taking, for each at least 2 samples, of said VEP volume;

[0079] - the performance of a second sequencing.

[0080] The invention also relates to a sequencing method, characterized in that it comprises:

[0081] - the implementation of a method for normalizing the quantity of nucleic acid between x samples, with x ≥ 2, according to the invention;

[0082] - the taking, for each at least 2 samples, of said VEP volume;

[0083] - the performance of a second sequencing.

[0084] In one embodiment, said second sequencing has a sequencing depth of between about 15X and about 45X, preferably between about 20X and about 40X, preferably between about 25X and about 35X, preferably about 30X.

[0085] In one embodiment, said second sequencing has a sequencing depth of between about 10 million reads and about 40 million reads, preferably between about 13 million reads and about 30 million reads.

[0086] In one embodiment, said second sequencing is carried out over a period of time between approximately 3 hours and approximately 23 hours, preferably between approximately 8 hours and approximately 18 hours, preferably between approximately 10 hours and approximately 16 hours, preferably between approximately 12 hours and approximately 14 hours, preferably approximately 13 hours.

[0087] In one embodiment, the samples are mixed between the step of collecting said VEP volumes and the step of performing said first sequencing.

[0088] In one embodiment, this mixing step is carried out by means of vortexing followed by centrifugation.

[0089] The invention also relates to a method for processing several series of samples, characterized in that it comprises the following steps:

[0090] - the implementation of a method for standardizing the quantity of nucleic acid between at least two samples from a first series of samples according to the invention;

[0091] - the taking, for each at least 2 samples, of said VEP volume;

[0092] - the performance of a second sequencing;

[0093] characterized in that the performance of said second sequencing takes place simultaneously, and preferably on the same sequencing plate, with the performance of a first sequencing of a method of normalizing the quantity of nucleic acid between at least two samples according to the invention, said other samples belonging to another series of samples.

[0094] The invention also relates to a method for processing several series of samples, including a first series of samples and at least one other series of samples, characterized in that it comprises the following steps:

[0095] - the implementation of a method for normalizing the quantity of nucleic acid between x samples, with x ≥ 2, of said first series of samples according to the invention;

[0096] - the taking, for each at least 2 samples from the said first series of samples, of the said VEP volume;

[0097] - the performance of a second sequencing;

[0098] characterized in that the performance of said second sequencing takes place simultaneously, and preferably on the same sequencing plate, with the performance of a first sequencing of a method of normalizing the quantity of nucleic acid between y samples, with y ≥ 2, belonging to at least one other series of samples according to the invention.

[0099] In one embodiment, x is equal to y. In another embodiment, x is strictly greater than y. In another embodiment, x is strictly less than y.

[0100] The invention also relates to a method for processing several series of samples, including a first series of samples and at least one other series of samples, characterized in that it comprises the following steps:

[0101] - the implementation of a method for standardizing the quantity of nucleic acid between at least 2 samples, from said first series of samples according to the invention;

[0102] - the taking, for each at least 2 samples from the said first series of samples, of the said VEP volume;

[0103] - the performance of a second sequencing;

[0104] characterized in that the performance of said second sequencing takes place simultaneously, and preferably on the same sequencing plate, with the performance of a first sequencing of a method of normalizing the quantity of nucleic acid between at least 2 samples, belonging to at least one other series of samples according to the invention.

[0105] In these embodiments, it is important to adjust the quantities of nucleic acids according to the target number of reads. For example, if the target number of reads for the first sequencing is 10,000 and the target number of reads for the second sequencing is 20 million, then the quantity of nucleic acid in the samples intended for the first sequencing should be equal to 0.0005 times (10,000 / 20,000,000) the quantity of nucleic acid in the samples intended for the second sequencing.

[0106] In one embodiment, the ratio of the quantity of nucleic acid in the first sample to the quantity of nucleic acid in the second sample is between 0.0000232 and 0.00232, preferably between 0.00005 and 0.0015, preferably between 0.0001 and 0.001, preferably between 0.0001 and 0.0007, preferably between 0.0001 and 0.0006, preferably between 0.0001 and 0.0005, preferably between 0.0001 and 0.0003, preferably 0.000232.

[0107] This embodiment with simultaneous steps is particularly interesting; it is distinguished by the possibility of simultaneously managing two distinct processes: the quantification of nucleic acid from several samples of one series and the sequencing of several other samples from another series.

[0108] This approach allows for remarkable resource optimization. In particular, it does not require an increase in the time allocated to the sequencing machine, thus preserving the operational efficiency of the process.

[0109] Furthermore, the use of consumables, and in particular sequencing plates, remains unchanged compared to conventional sequencing, which is a considerable advantage from an economic point of view.

[0110] Next, improved management of laboratory workflows is observed, allowing for more complete and diversified analysis of samples without requiring additional equipment.

[0111] Furthermore, the risk of error is limited, because the sample handling steps are also limited.

[0112] In one embodiment, the first and / or second sequencing is an NGS sequencing.

[0113] In one embodiment, the first and / or second sequencing is an NGS sequencing performed on a sequencing plate.

[0114] In one embodiment, the first and second sequencing is an NGS sequencing.

[0115] In one embodiment, the first and second sequencing is an NGS sequencing performed on a sequencing plate.

[0116] In one embodiment, said sequencing plate is chosen from the group consisting of plates using flow cells, semiconductor chips, nanopore flow cells, microtiter plates, bead-based plates.

[0117] Preferably, said sequencing plate is a plate using flow cells.

[0118] In one embodiment, the second sequencing results in a number of reads of between about 6 million and about 28 million, preferably between about 7 million and about 26 million, preferably between about 8 million and about 25 million, preferably between about 9 million and about 24 million, preferably between about 10 million and about 23 million, preferably between about 11 million and about 22 million, preferably between about 12 million and about 21 million, preferably between about 13 million and about 20 million.

[0119] By using partial or complete sequencing to adjust the amounts of nucleic acid taken, the method according to the invention offers a large number of advantages.

[0120] Since the method used is the same as for subsequent sequencing, no bias is introduced between techniques, as can occur, for example, when using dye for quantification, and the result is very consistent across different samples. Furthermore, this method does not require any additional equipment for quantification.

[0121] Description of the figure

[0122] Figure 1 shows a graphical representation corresponding to Table 1. It is immediately deducible that when the first sequencing assay according to the invention is used, the number of subsequent readings during complete sequencing is much more homogeneous, which improves the quality of the results. In Figure 1, the graphical representation corresponding to the fluorimetric assay is on the right, and the graphical representation corresponding to the first sequencing assay according to the invention is on the left.

[0123] Example 1: Comparison between a method according to the invention and a method according to the prior art

[0124] 1.1: Fluorescence spectrometry assay before sequencing and then sequencing

[0125] We have 96 samples, corresponding to 96 DNA libraries.

[0126] The following steps are completed.

[0127] Step 1: Dilute the Qubit® dsDNA HS Reagent (ThermoScientific product, part number Q32854, used for the precise quantification of double-stranded DNA) 1:200 (=1X) in the Qubit® dsDNA HS Buffer (specific buffer solution for use with the Qubit® dsDNA HS reagent). Prepare 200 μL per sample to be analyzed, including 3 additional volumes (2 standards and 1 control DNA).

[0128] Step 2: Dispense the mixture (Qubit® dsDNA Reagent + Qubit® dsDNA Buffer) into the plate containing the 96 samples: 190 µl for the standards, in A1 and B1 (specific locations on the plate) 199 µl for the control (in C1) and the samples to be tested.

[0129] Step 3: Add the DNA: 10 µl of standard DNA, in A1 (0 ng / µl) and B1 (10 or 100 ng / µl depending on the kit used, which indicates the concentration of DNA in these standard samples) 1 µl of control DNA (in C1) or DNA from the samples (the samples to be tested).

[0130] Step 4: Analyze the 96 wells with a Fluorimeter (Spark M10, TECAN, an instrument used to measure fluorescence) and retrieve the concentrations of each sample / library.

[0131] Step 5: Perform equimolar calculations for each preparation based on the molarities.

[0132] Step 6: Make the equimolar mixture (96 to 1 tube).

[0133] As immediately deducible from the above, it should be noted that the pre-sequencing fluorescence spectrometry assay method requires expensive special reagents needed only for said assay, notably those sold under the Qubit® brand.

[0134] In addition, specific equipment is also required: spectrofluorimeter, spectrofluorimeter plate (n=96), single-channel pipettes, multi-channel pipettes, disposable tips.

[0135] Moreover, in practice, the procedure takes longer (an additional day).

[0136] In addition, this requires individual assays of each of the samples (n=96), as well as numerous calculations for equimolar pooling (mixing of all libraries with previously calculated volumes).

[0137] Finally, it is important to recognize that, although fluorimetric assays are relatively reliable, they can be affected by various factors. Errors such as incorrectly sized libraries, improper preparation, the presence of primer dimers, or technical problems with Qubit® reagents can skew the results.

[0138] Next, the sequencing steps are as follows.

[0139] Step 1: Assay dsDNA High Sensitivity Kit DeNovix (a DeNovix brand kit for double-strand DNA quantification) + TapeStation, Agilent (an Agilent brand instrument used for analyzing the quality and size of nucleic acid samples) of the equimolar mixture.

[0140] Step 2: Sequencing NovaSeq6000 (a next-generation sequencer produced by Illumina, used for high-throughput sequencing of DNA and RNA, here specified with a sample load at a concentration of 1.6nM) or other ILLUMINA sequencer (a leading brand in genetic sequencing technologies) depending on the number of samples (sequencer selection may depend on the volume and nature of the samples to be analyzed).

[0141] 1.2: Assay by first sequencing according to the invention followed by second sequencing

[0142] Step 1: Equivolume mixing: 1.5µL / library.

[0143] Step 2: dsDNA High Sensitivity Kit Assay. DeNovix (ref: KIT-DSDNA-HIGH-2) of the mixture (1 tube) (Kit for the precise measurement of double-stranded DNA, by DeNovix): Prepare the mixture per sample: 198 µL AccuClear Buffer (a buffer for biochemical reactions, part of the AccuClear kit); 2 µL AccuClear Dye 100X (a dye for DNA detection, 100 times concentrated, also part of the AccuClear kit). For 2 standards + the "sample" tube(s) + 1 control: Dispense the buffer + dye mixture: Put 190 µL of the mixture into the "standard" tubes, add 10 µL of the 0 ng / μL and 25 ng / μL standards (DNA concentration standard for calibration); Put 199 µL of the mixture into the "sample" tube(s), add 1 µL of Vortex the tubes for 2 or 3 seconds and centrifuge (mix and separate the components by centrifugal force). Incubate for 5 minutes at room temperature in the dark (allow to rest).Analyze the pool using the Denovix device (measure the concentration of DNA in the sample with the Denovix device).

[0144] Step 3: Loading NextSeq2000 at 750pM: using the dosing data from the Pool.

[0145] Step 4: Perform equimolar calculations for each preparation based on the number of reads obtained per sample (calculate the quantity of each sample to be used for sequencing, based on the number of sequences read).

[0146] Step 5: Make the equimolar mixture (96 to 1 tube).

[0147] The NGS first sequencing assay method according to the invention has several significant advantages over the fluorescence spectrometry assay method.

[0148] This technique does not require the use of specific reagents, which simplifies the process and reduces costs.

[0149] The process involves equivolume sampling of libraries, followed by sequencing and mixing of libraries according to volumes based on the number of reads obtained.

[0150] The crucial aspect of this technique lies in the fact that the mixing method is identical to that used to obtain the final results, thus eliminating potential bias. The only significant variation comes from the pipetting precision. An additional advantage of this method is the ability to reliably identify non-viable samples during this initial sequencing, thanks to the use of the same technology.

[0151] Next, the sequencing steps are as follows.

[0152] Step 1: Assay dsDNA High Sensitivity Kit DeNovix (a DeNovix brand kit for double-strand DNA quantification) + TapeStation, Agilent (an Agilent brand instrument used for analyzing the quality and size of nucleic acid samples) of the equimolar mixture.

[0153] Step 2: Sequencing NovaSeq6000 (a next-generation sequencer produced by Illumina, used for high-throughput sequencing of DNA and RNA, here specified with a sample load at a concentration of 1.6nM) or other ILLUMINA sequencer (a leading brand in genetic sequencing technologies) depending on the number of samples (sequencer selection may depend on the volume and nature of the samples to be analyzed).

[0154] 1.3 Results and general conclusion

[0155] The results are given in the table below, which shows the number of readings for each sample during the second sequencing, following a normalization of the quantities of nucleic acid by first sequencing (according to the invention) or by fluorimetric assay.

[0156]

[0157] A graphical representation of this table is given in.

[0158] It can be immediately deduced that when the first sequencing assay according to the invention is used, the subsequent number of readings obtained by the second sequencing is much more homogeneous, which improves the quality of the results.

[0159] Therefore, it is proven that the method of normalizing nucleic acid quantities by first sequencing according to the invention is superior to the method used in the prior art.

[0160] Example 2: Example of normalization of nucleic acid quantity by first sequencing

[0161] This example illustrates the calculations performed after the first sequencing.

[0162] The results are given in the table below.

[0163] WellSampleIdentificationUDI SequencePre-sequencing Reads% First SequencingVCO (µl)VEP (µl)A155220810203934CGTTAGGATT-GTGTAAGGAT15897921.4176962381.51.10214019B155220810203981TTCCATTACG-AGTCCTTGCG11284641.0063072191.51.552706738C155220810201024TAGCGGTAAC-TAACGCCAGC14094751.2568986411.51.243139223D15522081020 0877GTAGCCAGGA-CCTCTAAGTA18902931,6856678571,50,92693231E155220810200795AGGATACTCT-GCATCGCCAT12420941,107636716 1,51,410661074F155220810200535TATCCTCCAG-CGCACCAATG13632321,2156614681,51,285308485G155220810200435TAAGTCGTTC-T ATATCTCGC18144231,6180108221,50,965691934H155220810201052TCCGGATTGA-ATAGCAGTCA23008232,0517577841,50,761542134A 255220810202584ACGTCTTGTT-AGCGGACGTT17268011,5398739461,51,014693445B255220810202579ATGAAGTGCG-GACATGTGCG135168 61,2053653291,51,296287493C255220510105887CGATCACTGC-TCAGGTGTGC764630,0681858431,522,91531402D255220810202651CC TATCGGAA-CTCTGGACAA12751391,1371045791,51,374104044E255220810203459CAGAGAGCTT-CAGGAAGGCT16402281,4626725161,51,068250058F255220810204510GCAACTTGCG-TGGCGTAAGG13017841,1608652451,51,345978792G255220810204540TATGGAGGAC-GTACGTATTC10490840,9355201431,51,670193861H255220810200998TGAGATCAGA-ACGGTGCCAA11469751,0228143951,51,527647644A355220510106832TCAGCCTATT-GCCACCTAAT16310191,4544603951,51,074281573B355220510106880GTTGTGAGCG-TTCTTGATCG10019310,8934714791,51,74879673C355220810203893TCAGTAACAC-AGAAGACAGC8826900,7871383751,51,985038526D355220810200882AAGGCTCAGA-CAGACATGTA14248971,2706512021,51,229684431E355220810200873GTGTGGTGGT-TCAACTGGTT10057330,896861911,51,742185706F355220510105890CCGAGCTTAG-AGCTTACCGG7622860,6797681671,52,298577773G355220510105987ATCACGCTTC-CTATTCGAAC7115130,6344913691,52,462602449H355220810204562TAGCTATGCA-GAGCCTGACA15265261,3612788131,51,147817762A455220510105397TGTTCCTCAT-AACACTGTTG13395431,1945368141,51,30803838B455220510105986CATACCTTCT-TTCCTCTCTT10057350,8968636941,51,742182241C455220510105975GCCTTCAATG-GCTACAACCG12386601,1045744481,51,414571921D455220810201017CTTGACCAGC-CACTTCAGGC17222371,5358040011,51,017382425E455220810204501CTACACACAA-AGTGTCGTAA6951 360,6198871881,52,520619931F455220810204028TAGGCTGAAT-GATCTAGGCG13583511,2113088391,51,289927019G455220810202395TCGGAGTC CT-CATCCAGATT15254611,36032911,51,14861911H455220810204207AACATCGCGG-TGTACCGTCG15142661,3503459641,51,157110875A5552208 10204123GTTGTCTTAC-GCAATACTAC11260991,0041982321,51,555967687B555220810203975GTGGCAACTA-ATCCGCTGGA11338571,0111164251,51 ,54532155C555220510106934GAGCAGGCAT-GAGGTGGTTG13180471,1753677671,51,32937115D555220810204621AACGGCACCT-CGCCTAAGCT11842721,0560739761,51,479536505E555220810200446AGTAACCTTG-AAGGAACCGG5998620,5349266451,52,920961248F555220810203818TCTCATAAG C-GTTCAATGTC9844600,8778917231,51,779832249G555220810200723TGCTTGCCAA-TCGCCTCTAA6147060,5481637751,52,850425498H5552208 10200374CGGTTCCTGT-GTTCGGCTTG15680461,3983042531,51,117424907A655220810204443CCAAGTAGAT-CCGTTCTCCT8523360,7600702111,52,055731139B655220510105852AAGGTTGGCG-GTCTAACAGG8743890,7797359631,52,003883462C655220510105382TGCTCTGGTC-AACGTAGAAC45638 60,4069819921,53,839236208D655220510105396ACTGTAACGA-TGTGGACTTA15774451,4066858061,51,110766877E655220510105486GATTCCAG GT-ATGGTTCTTG4331980,3863041041,54,044740872F655220810204341TTCACCAGAT-GTCATCAACT13295621,1856362621,51,317857803G655220810202626ACTTCCAAGG-CAAGAGTAGG13340231,1896143571,51,31345086H655220510106439CCGAATATTC-TGGATTGTTC1688830,1506013331,5 10,3750742A755220510106438CTCTTATCCA-ACCAACAGAA2359610,2104181061,57,425691772B755220810201051TCACAGCGGT-CCTGACGATG9303 660,8296534251,51,883316519C755220510106460CCTCTGTCGT-CTTCATGCAT892820,079617181,519,62516136D755220810204145TCTGTTCTCG -GCGATTCACG3623750,3231477291,54,835249828E755220510107032GATACTTCAC-AAGGCTGCTC14475861,2908841071,51,21041075F75522051 0106930AGTGCTGATA-TGGTAATCGA15247131,3596620711,51,149182604G755220510106927ATCCTTCGGT-AATTGGACTG15712191,4011337741,51,115168322H755220510107048GACAACGATT-CCAAGCCTCT18840881,6801345511,50,929985041A855220510107043GAACCGGTAG-TGAGAGCCTG14467431,2901323621,51,211116042B855220510106957AGCAATGAGC-GAGAGCGAAC10719390,9559010781,51,634583364C855220510106860CAAGACTCCA-ATTAGTCCGA14734711,3139670431,51,189147025D855220510106846ACCGTGTAGG-GAAGATCTCG14637261,3052769441,51,19706397E855220810202557AGGCACAGGT-GTCCGGTTAT10766160,9600717911,51,62748246F855220810200764CGACAGATCG-TAACTACACG6445330,5747619871,52,718516595G855220510105990ACGCGACAAC-ACCTATGTTC14716351,3123297911,51,190630595H855220810201069ACTTGCGTTA-CGATGTTAGA14517381,2945866511,51,206948951A955220810202599CACCACTCAT-TGCCACCGTT5850420,5217109171,52,994953621B955220810202562CTTCGTAACT-ACACCGTCCT11162400,9954064731,51,569710507C955220810204081CAGTATTCGG-CAGGTCACAG10527170,9387598691,51,664429905D955220510105991CAGTCTGGAC-TTGTTACAGC10200380,9096183871,51,717753315E955220810202362_55220810204554TACCGTTCTA-GACGTCCGTA10171460,9070394471,51,722637317F955220810202532GTGTCCACAG-GCTCCTTAGG8190910,7304239981,52,139168488G955220810204026TTACGACTGT-CTGGCCTTAT8045640 ,7174695531.52 AGATGCCTTC9026930,8049760411,51,94105156B1055220510106929AGCTCAGGAA-GCAGTAGAGA17405601,5521435281,51,006672368C1055220510 107053GATAGGCGGT-TTGTTCGGTT14559511,2983435911,51,203456474D1055220510106858AGTAGGAAGT-GTGGCGAGAT10404420,9278136441,51,6 84066633E1055220510106913CATGTTGTAG-CGTTGCATGG11785461,0509678181,51,486724876F1055220810204538CACATTCTTC-TACACCATTC6628700,5911139981,52,643314159G1055220810202182GCAGCTCGTA-ACGGCATATA6057000,5401326781,52,892807753H1055220810201057GTTCAGACG G-ATCTATCGAG12414891,1070972081,51,411348515A1155220810200934TCCTGGAAGT-CTCTTGTGTT9149070,8158678691,51,91513854B11552208 10204139GCATTGTTAG-ACCGATTGCG7407210,6605375891,52,365497476C1155220510106956GACCTACAGC-TCTACGCAAC15220461,3572837751,51,151196256D1155220810204417CACCGACGTA-GATCACTCTA8610900,7678765861,52,034832197E1155220810204377CTCTCACCTT-GCTGCGTCT T12018031,0717072371,51,457954137F1155220810200786CTCGTTCATT-TCGAGAAGTT14592421,3012783391,51,200742342G11552208102 04405TGGTGGCAAG-CTCAGTTGCG9445530,8423046751,51,855029476H1155220810200631GATTGCTTGA-ATTGCGGAGC17049211,5203624671, 51.027715452A1255220810200761CCGTTAAGGT-CGGAAGTTAC15728951.4026283461.51.113980054B1255220810203382TGCTGAGAGG-TAGTCG TGAG13897351,2392955061,51,260796955C1255220810204151TTGTCACTTG-GCCGTTGGTT11651751,0390442361,51,503785832D12552208 10203873GCTGTTATGT-TACAGGCAGG5413830,4827780251,53,236477053E1255220510107017GCAGCAGTTG-CTGCAGCGTA12930751,15309899 Table 2: Normalization by first sequencing according to the invention,

[0164] It should be noted that normalization allows for very strong adjustment of the volumes to be taken VEP, which vary, in this series, from 0.75 µl (sample 55220810204003, well H12) to 19.62 µl (sample 55220510106460, well C7).

Claims

1. A method for normalizing the quantity of nucleic acid between at least 2 samples, characterized in that it comprises the following steps: - having at least 2 samples, each having a volume V containing nucleic acid; - taking a known volume VCO from each of at least 2 samples; - from said volumes VCO taken, proceeding with a first sequencing allowing the obtaining, for each of the volumes VCO, of a number of reads; - depending on the number of reads of each volume VCO, calculating a sample volume to be taken VEP for each sample such that each VEP corresponds to an identical theoretical number of reads NLT.

2. Method according to claim 1, characterized in that the nucleic acid is selected from the group consisting of ribonucleic acid (RNA) and in particular messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small interference RNA (siRNA), microRNA (miRNA) and / or long non-coding RNA (lncRNA), deoxyribonucleic acid (DNA) and in particular chromosomal DNA (chDNA), mitochondrial DNA (mDNA), plasmid DNA (pDNA), circular DNA (cDNA), linear DNA (lDNA) and / or recombinant DNA (rDNA), and mixtures thereof.

3. A method according to any one of the preceding claims, characterized in that the number of samples is between 2 and 1000, preferably between 2 and 950, preferably between 2 and 900, preferably between 2 and 850, preferably between 2 and 800, preferably between 2 and 750, preferably between 2 and 700, preferably between 2 and 650, preferably between 2 and 600, preferably between 2 and 550, preferably between 2 and 500, preferably between 2 and 450, preferably between 2 and 400, preferably between 2 and 350, preferably between 5 and 350, preferably between 20 and 350, preferably between 50 and 300, preferably between 50 and 200, of preferably between 50 and 100, preferably between 75 and 125, preferably between 90 and 110, preferably between 95 and 105.

4. A method according to any one of the preceding claims, characterized in that said known volume VCO is between 0.5 µl and 2.5 µl, preferably between 0.6 µl and 2.4 µl, preferably between 0.7 µl and 2.3 µl, preferably between 0.8 µl and 2.2 µl, preferably between 0.9 µl and 2.1 µl, preferably between 1.0 µl and 2.0 µl, preferably between 1.1 µl and 1.9 µl, preferably between 1.2 µl and 1.8 µl, preferably between 1.3 µl and 1.7 µl, preferably between 1.4 µl and 1.6 µl, preferably of 1.5 µl.

5. Method according to any one of the preceding claims, characterized in that said VCO volume is the same for each sample.

6. Method according to any one of the preceding claims, characterized in that said VEP volume is between 0.5 µl and 7.5 µl, preferably between 0.6 µl and 7.2 µl, preferably between 0.7 µl and 6.9 µl, preferably between 0.8 µl and 6.6 µl, preferably between 0.9 µl and 6.3 µl, preferably between 0.9 µl and 6.0 µl, preferably between 0.9 µl and 5.7 µl, preferably between 0.9 µl and 5.4 µl, preferably between 0.9 µl and 5.1 µl, preferably between 0.9 µl and 4.8 µl.

7. Sequencing method, characterized in that it comprises: - carrying out a method for normalizing the quantity of nucleic acid between at least two samples according to any one of the preceding claims; - taking, for each of at least 2 samples, said VEP volume; - carrying out a second sequencing.

8. Method for processing several series of samples, including a first series of samples and at least one other series of samples, characterized in that it comprises the following steps: - carrying out a method for normalizing the quantity of nucleic acid between x samples, with x ≥ 2, of said first series of samples according to any one of claims 1 to 6; - taking, for each at least 2 samples of said first series of samples, of said volume VEP; - carrying out a second sequencing; characterized in that the carrying out of said second sequencing takes place simultaneously, and preferably on the same sequencing plate, with the carrying out of a first sequencing of a method for normalizing the quantity of nucleic acid between y samples, with y ≥ 2, belonging to at least one other series of samples according to any one of claims 1 to 6.

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