Patent Description:
Normal humans have two sets of <NUM> chromosomes in every healthy, diploid cell. Under some conditions, mutations may occur on any one or more of said chromosomes, leading to chromosomal abnormalities. These abnormalities may be linked to genetic diseases, cancers and other diseases. Detection of chromosomal abnormalities may identify individuals prone to develop a specific disease or define which treatment is most recommended for a given individual. In this regard, testing for chromosomal abnormalities is very valuable.

In addition to human health, detection of chromosomal abnormalities is relevant in additional species including but not limited to insects, bacteria, plants and organic-containing mixed samples, such as soil and food stuffs, where genetically modified organism (GMO) detection due to genetic engineering may be conducted, for example for quality control. The detection of genetic mutations occurring in viruses, viroids and other non-chromosome-containing genomes is also highly pertinent.

In this context, it is relevant to detect the signatures of genome abnormalities, i.e. changes to a specific nucleic acid sequence in a biological sample, in both a qualitative and quantitative manner.

Such detections are currently done efficiently by amplifying target nucleic acids in a sample of interest. Amplification can be carried out by combining oligonucleotide primers with the sample and then subjecting the sample to amplification conditions compatible with nucleic acids quantification, such as polymerase chain reaction (PCR) conditions. These amplification methods enable to generate multiple copies of a single target nucleic acid sequence, thus reaching a detection threshold.

However, measuring the concentration of such specific nucleic acid sequences in a biological sample in a quantitative manner is more relevant, for instance when the objective is not only to detect but also to quantify a genetic disease, for example to monitor the evolution of a rare mutation.

In some cases, nucleic acids present in biological samples are damaged, typically fragmented in sequences of short length. In such populations of fragmented nucleic acids, the sequence to be amplified in a given PCR assay is sometimes randomly cut. In this scenario, amplification of this nucleic acid sequence cannot occur with PCR methods, thus leading to an underestimation of the presence of the nucleic acid sequence of interest.

<CIT> discloses an amplification-based method of quantifying specific nucleic acid sequences correcting for an amplification bias.

The present invention proposes a method to correct for such underestimation problems.

The invention is defined by the appendant claims.

The disclosure, though not part of the invention relates to a method of determining the concentration of a detected sequence (as defined further below) in non-fragmented nucleic acids, comprising correcting the measured concentration of said detected sequence in the sample comprising fragmented nucleic acids with a correction coefficient, thereby obtaining the concentration of said detected sequence in non-fragmented nucleic acids, wherein the correction coefficient is based on the length distribution (LD) and at least one parameter of the measuring method.

In some embodiments, there is provided a method of determining the concentration of a detected sequence in non-fragmented nucleic acids, comprising the following steps:.

In some embodiments, the method comprises:.

In an embodiment, the sample of fragmented nucleic acids is any combination of the following three categories:.

In an embodiment, the measuring method is an isothermal quantitative nucleic acid amplification method, preferably selected from loop mediated isothermal amplification and quantitative nucleic acid sequence-based amplification.

In another embodiment, the measuring method is a non-isothermal quantitative nucleic acid amplification method, preferably selected from quantitative Polymerase Chain Reaction, real time Polymerase Chain Reaction, digital Polymerase Chain Reaction, multiplex Polymerase Chain Reaction and multiplex digital Polymerase Chain Reaction.

In an embodiment, the parameters of said measuring method include the length of the sequence to be amplified, as further defined below. In a specific configuration of this embodiment, at least <NUM>% of nucleic acid fragments have a length shorter than the length of the sequence to be amplified. In another specific configuration of this embodiment, at most <NUM>% of nucleic acid fragments have a length shorter than the length of the sequence to be amplified.

In an embodiment, the length of the sequence to be amplified is longer than <NUM> bp and shorter than <NUM> bp, preferably longer than <NUM> bp and shorter than <NUM> bp, more preferably longer than <NUM> bp and shorter than <NUM> bp, even more preferably longer than <NUM> bp and shorter than <NUM> bp.

In an embodiment, the nucleic acid fragments length distribution (LD) is comprised in the range of from <NUM> bp to <NUM> bp, preferably from <NUM> bp to <NUM> bp, more preferably from <NUM> bp to <NUM> bp, even more preferably from <NUM> bp to <NUM> bp.

This disclosure, though not part of the invention further relates to a method of determining a function of a first concentration of a first detected sequence S<NUM> and of a second concentration of a second detected sequence S<NUM> in non-fragmented nucleic acids comprising the following steps:.

wherein said S<NUM> concentration and said S<NUM> concentration are determined in the same sample; and wherein the length of the sequence to be amplified associated with S<NUM> is different from the length of the sequence to be amplified associated with S<NUM>.

This disclosure, though not part of the invention also relates to a system configured to determine the concentration of a detected sequence in non-fragmented nucleic acids comprising:.

In a specific configuration, the module configured to measure the concentration of said detected sequence in said fragmented nucleic acids is an isothermal quantitative nucleic acid amplification module, preferably selected from loop mediated isothermal amplification module and quantitative nucleic acid sequence-based amplification module.

In an alternative configuration, the module configured to measure the concentration of said detected sequence in said fragmented nucleic acids is a non-isothermal quantitative nucleic acid amplification module, preferably selected from quantitative Polymerase Chain Reaction module, real time Polymerase Chain Reaction module, digital Polymerase Chain Reaction module, multiplex Polymerase Chain Reaction module and multiplex digital Polymerase Chain Reaction.

In an embodiment, parameters of said measure include the length of the sequence to be amplified.

The present application provides methods and systems for correcting the measured concentration of a detected sequence in a nucleic acid sample comprising fragmented nucleic acids. The methods and systems provided herein allow determination of a corrected concentration of the detected sequence that more closely approximates the true concentration of the detected sequence in non-fragmented nucleic acids. When the concentration of a detected sequence is measured by amplification of a sequence to be amplified comprising the detected sequence, fragments containing part of the sequence to be amplified but with a length shorter than the length of the sequence to be amplified will not be replicated. This failure to replicate the truncated target region (i.e., sequence to be amplified) leads to an underestimation of the concentration. The methods provided herein can be applied to correct for the resulting underestimation of the concentration of the nucleic acid in the non-fragmented nucleic acid sample.

This invention thus in some aspects provides a method of determining the concentration of a detected sequence (including determining the number of copies of said detected sequence) in non-fragmented nucleic acids comprising the following steps (hereinafter referred to as "basic method"). In some aspects, the invention provides a method of calibrating the concentration of a detected sequence (including the number of copies of said detected sequence) in a sample comprising fragmented nucleic acid molecules. Also provided are kits, software, devices, and other articles of manufacture useful for the methods described herein.

This disclosure, though not part of the invention relates to a method of determining the concentration of a detected sequence in non-fragmented nucleic acids, comprising correcting the measured concentration of said detected sequence in the sample comprising fragmented nucleic acids with a correction coefficient, thereby obtaining the concentration of said detected sequence in non-fragmented nucleic acids, wherein the correction coefficient is based on the length distribution and at least one parameter of the measuring method.

In some embodiments, there is provided a method of determining the concentration of a detected sequence (as defined further below) in non-fragmented nucleic acids, comprising the following steps:.

In some embodiments, provided herein is a method of determining the concentration of a detected sequence in non-fragmented nucleic acids comprising the following steps:.

In some embodiments, provided herein is a method of calibrating a measured concentration of a detected sequence in a sample comprising fragmented nucleic acids, such that it more closely reflects the concentration of the detected sequence in non-fragmented nucleic acids, comprising the following steps:.

In some embodiments, provided herein is a method of calibrating a measured concentration of a detected sequence in a sample comprising fragmented nucleic acids, wherein the measured concentration of the detected sequence is an underestimation of the real concentration of the detected sequence in the sample, comprising the following steps:.

In some embodiments, provided herein is a method of correcting the measured concentration of a detected sequence in a sample comprising fragmented nucleic acids, such that it more closely reflects the concentration of the detected sequence in non-fragmented nucleic acids, comprising the following steps:.

In some embodiments, provided herein is a method of correcting the measured concentration of a detected sequence in a a sample comprising fragmented nucleic acids, wherein the measured concentration of the detected sequence is an underestimation of the real concentration of the detected sequence in the sample, comprising the following steps:.

The methods of the present application in some embodiments do not comprise obtaining the sequence of the fragmented nucleic acids. In some embodiments, the methods do not comprise obtaining or predicting the genetic coordinates of the fragmented nucleic acids. In some embodiments, the method does not comprise assembly of the fragmented nucleic acids into a contiguous sequence.

The sample of fragmented nucleic acids in some embodiments is in any combination of the following three categories, as described in the "Nucleic acid sample and length distribution" subsection below:.

In some embodiments, the measuring method does not comprise sequencing the nucleic acids in the sample. In some embodiments, measuring of the concentration of the detected sequence can comprise amplifying a sequence to be amplified comprising the detected sequence. In some embodiments, the measuring method is an isothermal quantitative nucleic acid amplification method (e.g., loop-mediated isothermal amplification or quantitative nucleic acid sequence-based amplification). In some embodiments, the measuring method is a non-isothermal quantitative nucleic acid amplification method (e.g. quantitative Polymerase Chain Reaction, real time Polymerase Chain Reaction, digital Polymerase Chain Reaction, multiplex Polymerase Chain Reaction and multiplex digital Polymerase Chain Reaction).

In some embodiments according to any of the preceding methods, the detected sequence and the sequence to be amplified are the same, and the step of measuring comprises detecting incorporation of a label in nucleic acids produced during amplification (e.g., an intercalating dye, such as a fluorescent dye comprising a fluorophore), as shown in <FIG>.

In some embodiments, the detected sequence is a subset of the sequence to be amplified, and/or the step of measuring comprises detecting binding of a labeled probe to the detected sequence (e.g., a fluorescently labeled probe comprising a fluorophore), as shown in <FIG>.

The measuring method implemented in the measuring step of the basic method of the invention intrinsically uses some parameters relevant to determine the correction coefficient. In a particular embodiment, the measuring method is an amplification method using replication and an amplification mixture.

In some embodiments, the method can further comprise the step of determining the correction coefficient based on the length distribution of nucleic acids in the sample and at least one parameter of said measuring method.

In some embodiments, the at least one parameter of the measuring method can comprise length of the sequence to be amplified. In some embodiments, the at least one parameter of the measuring method only comprises the length of the sequence to be amplified.

Thus, for example, in some embodiments, provided herein is a method of determining the concentration of a detected sequence in non-fragmented nucleic acids comprising the following steps:.

In some embodiments, provided herein is a method of correcting the measured concentration of a detected sequence in a a sample comprising fragmented nucleic acids, such that it more closely reflects the concentration of the detected sequence in non-fragmented nucleic acids, comprising the following steps:.

The length of the sequence to be amplified can be denoted as La, which is actually the sum of the length of the primers (forward and reverse) plus the length of any additional base pairs located between the primers.

In an embodiment, the length of the sequence to be amplified La is equal to or longer than the length of the primers (forward and reverse), and in some embodiments is no longer than about <NUM> bp, for example no longer than about <NUM> bp, no longer than <NUM> bp, or no longer than <NUM> bp.

For replication, the primer has first to bind to a nucleic acid. Binding is optimal when all the bases of the primer are complementary to the nucleic acid (for DNA Adenine is complementary to Thymine and Guanine is complementary to Cytosine; for RNA Adenine is complementary to Uracil and Guanine is complementary to Cytosine), but binding may also be efficient if a few bases of the primer are not complementary with the nucleic acid. In other words, if the nucleic acid sequence is shortened by a few bases where the primer should bind, the replication process can still be efficient. A relevant parameter may be La, or La-n with n an integer greater than <NUM>, or r. La (r multiplied by La) with a shortening coefficient r ranging between <NUM>% and <NUM>% and with the proviso that r. La is always an integer value. In some embodiments, n is an integer from <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM>. In some embodiments, r is a value greater than <NUM> and less than <NUM>, greater than <NUM> and less than <NUM>, greater than <NUM> and less than <NUM>, greater than <NUM> and less than <NUM>, or greater than <NUM> and less than <NUM>.

With the length parameter La, the correction coefficient may be determined according to any of the embodiments described in the "Determining a correction coefficient" subsection below.

The correction coefficient may be computed similarly with La - n with n an integer greater than <NUM> (e.g., from <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM>) or r. La (r multiplied by La) with a shortening coefficient r ranging between <NUM>% and <NUM>% and with the proviso that r. La is always an integer value to take into account shortened fragments still leading to replication, as described in the "Determining a correction coefficient" subsection below.

In some embodiments, there is provided a method of determining the concentration of a detected sequence in non-fragmented nucleic acids comprising the following steps: i. determining a length distribution of nucleic acids in a sample comprising fragmented nucleic acids, wherein the fragmented nucleic acids are derived from said non-fragmented nucleic acids; ii. measuring the concentration of said detected sequence in said sample comprising fragmented nucleic acids with a measuring method; and iii. correcting the measured concentration of said detected sequence in the sample comprising fragmented nucleic acids with a correction coefficient to obtain the concentration of said detected sequence in non-fragmented nucleic acids,
wherein the correction coefficient is determined by: <MAT>.

With regards to the length distribution of nucleic acid fragments, fragments with a length shorter than La (or La - n or r. La) will not be replicated anyway. If such fragments contain a part of the sequence to be amplified, it will not be replicated and this leads to an underestimation of the concentration.

The correction coefficient described herein can also be based on the probability that the sequence to be amplified is fragmented based on sequence fragmentation bias. In some embodiments, the relative probability that a sequence to be amplified will be fragmented is known. In some embodiments, the probability that the sequence to be amplified will be fragmented depends on the source of fragmentation (e.g., naturally-occurring nucleic acid fragmentation or fragmentation by physical means such as sonication). For example, the correction coefficient can be adjusted by multiplying the correction coefficient prior to adjustment with a probability that the sequence to be amplified is fragmented based on sequence fragmentation bias. Alternatively, the probability of that the sequence to be amplified is fragmented based on sequence fragmentation bias can be accounted for by modifying the fragmentation length distribution (LD) curve.

In some embodiments, the at least one parameter of the measuring method further comprises a parameter of the amplifying step selected from the group consisting of: GC content of the sequence to be amplified; GC content of the amplification primers; length of the amplification primers; type of polymerase being used; and temperature of the amplification cycles. In some embodiments, the additional parameter of the amplifying step can be incorporated as a coefficient multiplied by the fragment length distribution (LD) curve, or as a calibration coefficient multiplied by the correction coefficient as described in the "Calibrating the correction coefficient" subsection below.

In some embodiments, the at least one parameter of the measuring method further comprises a parameter of the measuring step selected from the group consisting of: sequence of a detecting probe; photostability of a fluorophore used; chemical stability of a fluorophore used; quantum yield of a fluorophore used; and wavelength of a fluorophore used. In some embodiments, the additional parameter of the amplifying step can be incorporated as a coefficient multiplied by the fragment length distribution (LD) curve, or as a calibration coefficient multiplied by the correction coefficient as described in the "Calibrating the correction coefficient" subsection below.

In some embodiments, the correcting comprises multiplying the concentration measured in the sample comprising fragmented nucleic acids with the correction coefficient. In some embodiments, the correcting comprises multiplying the concentration measured in the sample comprising fragmented nucleic acids with the correction coefficient and an additional correction factor. In some embodiments, the additional correction factor is based on the probability that the sequence to be amplified is fragmented based on sequence fragmentation bias. In some embodiments, the additional correction factor is based on at least one parameter of the measuring method, as described above. In some embodiments, the additional correction factor is based on at least one parameter of the measuring method affecting sequence amplification (e.g., GC content of the sequence to be amplified). In some embodiments, the additional correction factor is based on at least one parameter of the measuring method affecting detection of the detected sequence (e.g., photostability or chemical stability of the fluorophore used). In some embodiments, the additional correction factor is based on an experimentally determined calibration factor, as described in the "Calibrating the correction coefficient" subsection below.

In some embodiments, the correcting is applied if at least <NUM>% of the nucleic acids in the sample have a length shorter than the length of the sequence to be amplified. In some embodiments, no more than <NUM>% of nucleic acid fragments in the sample have a length shorter than the length of the sequence to be amplified.

The present application also provides a method of determining a function of a first concentration of a first detected sequence (detected sequence Si) and of a second concentration of a second detected sequence (detected sequence S<NUM>) in the same sample of non-fragmented nucleic acids. The function may be a fraction or a ratio.

To this end, the methods described herein may further comprise:.

In some embodiments, measuring the concentration of the second detected sequence comprises amplifying a second sequence to be amplified comprising the second detected sequence. In some embodiments, the measuring method comprises a multiplex amplification step. In some embodiments, the length of the first sequence to be amplified is different from the length of the second sequence to be amplified, and a different correction coefficient is applied for the first and second detected sequences. In some embodiments, the length of the first sequence to be amplified and the second sequence to be amplified are the same. In some embodiments, other parameters of the measuring method are different between the first detected sequence and second detected sequence (e.g., parameters related to binding/detection of the probe, or any parameters affecting amplification as described in the "Determining a correction coefficient" subsection below).

In some embodiments, the first sequence to be amplified comprises a mutant allele detected sequence or a variant allele detected sequence and the second sequence to be amplified comprises a corresponding reference allele detected sequence. In some embodiments, the first sequence to be amplified comprises an insertion or deletion compared to a corresponding reference sequence comprised by the second sequence to be amplified. In some embodiments, the method further comprises a step of determining a corrected mutant allele fraction (MAF) of the first detected sequence compared to the second detected sequence. In some embodiments, the method further comprises a step of determining a corrected variant allele fraction (VAF) of the first detected sequence compared to the second detected sequence.

In some embodiments, the first sequence to be amplified is amplified from a variant nucleic acid comprising a copy number variation (CNV) and the second sequence to be amplified is amplified from a reference nucleic acid. In some embodiments, the method further comprises a step of determining a corrected copy number variation (CNVreal) of the reference sequence compared to the variant sequence.

In any of the preceding embodiments, the method can further comprise a step of calibrating the correction coefficient based on measured concentrations of the detected sequence in a first nucleic acid sample having a first fragment length distribution (LD<NUM>) and a second nucleic acid sample having a second fragment length distribution (LD<NUM>).

In some embodiments provided herein, a sample is provided which contains fragmented nucleic acids. The fragmented nucleic acids are actually fragments from original nucleic acids, i.e. non-fragmented nucleic acids, that have undergone fragmentation. Indeed, the concentration of the detected sequence in non-fragmented nucleic acids is the sought-after measure, but the available sample is actually fragmented. In some embodiments, the method does not comprise fragmenting the non-fragmented nucleic acid to generate the sample comprising fragmented nucleic acids.

The sample may contain any type of fragmented nucleic acids. In particular, the sample may be a cell-free sample, i.e. a biologic liquid in which nucleic acids have been released from cells such as saliva, blood plasma, urine or whole blood; or a cell-containing sample, i.e. a biologic sample containing essentially cells, such as a biopsy. Besides, the sample may be a naturally fragmented sample, i.e. the non-fragmented nucleic acids have been degraded naturally before sampling, i.e. in the living organism, or after sampling due to preservation treatments or storage conditions. The sample may finally be an artificially fragmented sample, i.e. non-fragmented nucleic acids are sampled and then degraded artificially for the needs of the measuring method. Further, the sample may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

Examples of cell-free naturally fragmented DNA samples are:.

Examples of naturally fragmented RNAs samples include but are not limited to messenger RNAs cleaved by ribonucleases (such as endo-and exo-nucleases). These samples may be cell-free or cell-containing samples.

Examples of artificially fragmented DNA and RNA samples are:.

The measuring method implemented in the measuring step of the basic method of the invention may be an isothermal quantitative nucleic acid amplification method or a non-isothermal quantitative nucleic acid amplification method.

Isothermal quantitative nucleic acid amplification method may be loop mediated isothermal amplification or quantitative nucleic acid sequence-based amplification. It may be combined with a reverse transcription step to allow the detection of RNA.

Non-isothermal quantitative nucleic acid amplification method may be quantitative Polymerase Chain Reaction, real time Polymerase Chain Reaction, digital Polymerase Chain Reaction, multiplex Polymerase Chain Reaction or multiplex digital Polymerase Chain Reaction. It may be combined with a reverse transcription step to allow the detection of RNA.

In some embodiments of the method, the length distribution (LD) of nucleic acid fragments is provided. In the invention, fragments refer to individual nucleic acids resulting from natural or artificial fragmentation of original non-fragmented nucleic acids. By way of illustration and not limitation, an original non-fragmented nucleic acid with a length of <NUM> base pairs (bp) may for example be fragmented in <NUM> fragments with a length of <NUM> bp, <NUM> fragments with a length of <NUM> bp and <NUM> fragments with a length of <NUM> bp, yielding a population of short chain nucleic acids. It is then possible to define a length distribution of nucleic acid fragments in this population, i.e. the number of fragments having a given length, for all possible lengths. Length distribution may be also defined with usual statistical functions (e.g., a Gaussian or Poisson distribution) or parameters like mean value and standard deviation.

Devices suitable to measure length distribution of nucleic acid fragments in a sample are for example the Tape Station <NUM> instrument or the Bioanalyzer <NUM> instrument, both from Agilent Technologies, or the LabChip GX Touch Nucleic Acid Analyzer from PerkinElmer.

With regards to the length distribution of nucleic acid fragments, fragments with a length shorter than the length of the sequence to be amplified, La (or La - n or r. La, as described above) will not be replicated. If such fragments contain a part of the sequence to be amplified, it will not be replicated and this leads to an underestimation of the concentration. The methods provided herein can be applied to correct for the resulting underestimation of the concentration of the nucleic acid in the non-fragmented nucleic acid sample.

It is likely that the correction coefficient will be small if only a few fragments have a length shorter than La (or La - n or r. Indeed, applicant notices that the correction coefficient is more relevant when at least <NUM>% of nucleic acid fragments have a length shorter than La (or La - n or r. The proportion of fragments having a length shorter than La (or La - n or r. La) may be at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%. In some embodiments, the steps of determining a correction coefficient and correcting the concentration of the detected sequence is applied when at least <NUM>% of nucleic acid fragments have a length shorter than La (or La - n or r.

Besides, it is likely that the correction coefficient will be very high if almost all fragments have a length shorter than La (or La - n or r. Indeed, applicant notices that the correction coefficient is more relevant when at most <NUM>% of nucleic acid fragments have a length shorter than La (or La - n or r. The proportion of fragments having a length shorter than La (or La - n or r. La) may be at most <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%.

In particular, the proportion of fragments having a length shorter than La (or La - n or r. La) may be in a range selected from <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%.

In another embodiment, La is longer than <NUM> bp and shorter than <NUM> bp, preferably longer than <NUM> bp and shorter than <NUM> bp, more preferably longer than <NUM> bp and shorter than <NUM> bp, even more preferably longer than <NUM> bp and shorter than <NUM> bp. In some embodiments, the length of the sequence to be amplified La is in the range <NUM>-<NUM> bp, <NUM>-<NUM> bp, <NUM>-<NUM> bp, <NUM>-<NUM> bp, <NUM>-<NUM> bp, <NUM>-<NUM> bp, <NUM>-<NUM> bp, or <NUM>-<NUM> bp.

In another embodiment, the nucleic acid fragments length distribution is comprised in the range from <NUM> bp to <NUM> bp. The nucleic acid fragments length distribution is preferably comprised in the range from <NUM> bp to <NUM> bp, preferably from <NUM> bp to <NUM> bp, more preferably from <NUM> bp to <NUM> bp, even more preferably from <NUM> bp to <NUM> bp.

In some embodiments, a correction coefficient is determined. Indeed, if an original non-fragmented nucleic acid is fragmented inside the sequence to be amplified, the measuring method used cannot detect that sequence to be amplified. This absence of detection leads to an underestimation of the concentration. This correction coefficient depends on the nucleic acid fragments length distribution, and on parameters relevant to define the probability that a copy of the sequence to be amplified is cut during fragmentation, such as the length of the sequence to be amplified, and potentially on parameters specifically associated to the measuring method.

In some embodiments, the measured concentration of the detected sequence is corrected with the correction coefficient to obtain the concentration of the detected sequence in non-fragmented nucleic acids, i.e. in the original sample.

In some embodiments, the measuring method implemented to measure the concentration of the detected sequence intrinsically uses some parameters relevant to determine the correction coefficient. In some embodiments, the measuring method is an amplification method using replication and an amplification mixture.

A parameter of particular relevance is the length of the sequence to be amplified, hereafter noted La. The length of the sequence to be amplified La is actually the sum of the length of the primers (forward and reverse) plus the length of any additional base pairs located between the primers.

In an embodiment, the length of the sequence to be amplified La is equal to or longer than the length of the primers (forward and reverse), and is shorter than <NUM> bp, preferably shorter than <NUM> bp, more preferably shorter than <NUM> bp and even more preferably shorter than <NUM> bp.

For replication, the primer has first to bind to a nucleic acid. Binding is optimal when all the bases of the primer are complementary to the nucleic acid (for DNA Adenine is complementary to Thymine and Guanine is complementary to Cytosine; for RNA Adenine is complementary to Uracil and Guanine is complementary to Cytosine), but binding may also be efficient if a few bases of the primer are not complementary with the nucleic acid. In other words, if the nucleic acid sequence is shortened by a few bases where the primer should bind, the replication process can still be efficient. A relevant parameter may be La, or La-n with n an integer from <NUM> to <NUM>, or r. La (r multiplied by La) with a shortening coefficient r ranging between <NUM>% and <NUM>% and with the proviso that r. La is always an integer value. In some embodiments, r is a value greater than <NUM> and less than <NUM>, greater than <NUM> and less than <NUM>, greater than <NUM> and less than <NUM>, or greater than <NUM> and less than <NUM>, with the proviso that r. La is always an integer value.

In some embodiments, a value for n or r can be predicted based on the predicted strength of primer binding to a sequence to be amplified that has been shortened by a few bases where the primers should bind. For example, a value for n or r can be predicted based on parameters of the sequence to be amplified and/or of the primers (e.g., GC content of the sequence to be amplified; GC content of the amplification primers; length of the amplification primers; type of polymerase being used; temperature of the amplification cycles). In some embodiments, a value for n or r can be predicted based on (a) the predicted melting temperatures (Tm) of the primers binding to the full length sequence to be amplified, (b) the predicted melting temperatures of the primers binding to the sequence to be amplified shortened by a factor of n or r as described above (i.e., La-n or r. La), and (c) the annealing temperature used in the amplification method.

In some embodiments, a value for n or r can be determined experimentally, e.g. in a calibration step as described in the "Calibrating the correction coefficient" subsection below. In some embodiments, n or r can be determined experimentally for a given sequence to be amplified and set of amplification primers for one sample, and then used to calibrate the L parameter for any sample wherein the sequence to be amplified and amplification conditions are the same.

With the length parameter La, the correction coefficient may be determined in the following manner.

Let P(X) be the probability of the event X.

Let La be the length of the sequence to be amplified (in number of base pairs).

Let f be the probability distribution of the length of nucleic acid fragments in the sample (f(i) is the probability that a fragment in the sample has a length of i base pairs).

Let <MAT> be the average length of the fragments (in number of base pairs).

Let us assume that the cut position of non-fragmented nucleic acids is random and equiprobable along the base pairs.

Let us partition the probability universe Ω in mutually exclusive events: <MAT> where <MAT>.

By application of Bayes rule P(A ∩ B) = P(A/B) P(B) <MAT> and <MAT> <MAT>.

Besides, let N be the total number of fragments in the fragmented nucleic acid sample: <MAT>.

Finally, the concentration of the detected sequence in the sample of non-fragmented nucleic acids (Creal) is obtained after multiplication of the measured concentration of the detected sequence in the sample of fragmented nucleic acids (Cmeasured) by a correction factor: <MAT> <MAT>.

This correction factor depends on the length distribution of nucleic acid fragments in the sample (f) and on a parameter of the amplification method, namely the length of the sequence to be amplified La.

The correction coefficient may be computed similarly with a La - n with n being an integer greater than <NUM> (e.g., an integer from <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM>) or r. La (r multiplied by La) with a shortening coefficient r ranging between <NUM>% and <NUM>% and with the proviso that r. La is always an integer value to take into account shortened fragments still leading to replication.

For example, in some embodiments wherein a set of amplification primers can still bind to and amplify a target region shortened by a factor of n, the correction coefficient is determined by: <MAT>.

In some embodiments wherein a set of amplification primers can still bind to and amplify a target region shortened by a shortening coefficient r, the correction coefficient is determined by: <MAT>.

Notably, it may be relevant in some cases to measure the fraction of a nucleic acid that has undergone a mutation, for instance a mutant allelic fraction (MAF). In this particular case, the first detected sequence (S<NUM>) is the mutant (mut) and the second detected sequence (S<NUM>) is the wild-type (wt). When both detected sequences are associated with sequences to be amplified of different lengths, the correction coefficients for both concentrations are different and must be considered.

It may be also relevant in some cases to measure the ratio of a variant nucleic acid (var) that has been amplified with regards to a reference nucleic acid (ref) that has been amplified (the variant being not necessarily a mutation of reference), namely a copy number variation (CNV).

In some embodiments, this method comprises the following steps.

In a first step, the concentration of S<NUM> in non-fragmented nucleic acids is determined according to the basic method described above. In this step, the correction coefficient is determined with consideration of the length of the sequence to be amplified associated with S<NUM>.

In a second step, the concentration of S<NUM> in non-fragmented nucleic acids is determined according to the basic method described above. In this step, the correction coefficient is determined with consideration of the length of the sequence to be amplified associated with S<NUM>.

In a third step, the function of the S<NUM> concentration and of the S<NUM> concentration is determined.

In this method said S<NUM> concentration and said S<NUM> concentration are determined in the same sample. To perform the measurements of the S<NUM> and S<NUM> concentrations on the same sample, multiplex PCR or multiplex digital PCR is particularly suitable.

In this method, the length of the sequence to be amplified associated with S<NUM> is different from the length of the sequence to be amplified associated with S<NUM>.

For the specific case of a mutant allelic fraction (MAF), the function is a fraction determined with following relation: <MAT> <MAT>.

A corrected variant allelic fraction (VAF) can be determined according to the same method used to determine a mutant allelic fraction (MAF).

For the specific case of a copy number variation (CNV), the function is a ratio determined with following relation: <MAT> <MAT>.

In some embodiments the method further comprises a step of calibrating the correction coefficient. In some embodiments, the calibration coefficient is calibrated based on parameters of the sequence to be amplified or parameters of the measuring method (e.g., parameters of the amplification or detection steps).

In some embodiments provided herein, the correction coefficient can also be based on the probability that the sequence to be amplified is fragmented based on sequence fragmentation bias. In some embodiments, the relative probability that a sequence to be amplified will be fragmented is known. In some embodiments, the probability that the sequence to be amplified will be fragmented depends on the source of fragmentation (e.g., naturally-occurring nucleic acid fragmentation or fragmentation by physical means such as sonication). In some embodiments, the fragmentation bias depends on the chromatin structure of the region comprising the sequence to be amplified.

In some embodiments, the fragmentation bias depends on the relative occurrence of sequence associated with fragmentation within the sequence to be amplified (e.g., a site targeted by a restriction enzyme).

For example, in some embodiments, the nucleic acid sample can comprise DNA digested by caspase-activated DNAse during apoptosis. In some embodiments, the correction coefficient can comprise a correction factor based on the probability that the sequence to be amplified is fragmented during apoptosis. In the majority of somatic tissues, apoptotic cleavage of DNA results in the formation of fragments roughly <NUM> bp. in length and multiples thereof, whereas the fragmentation pattern of the neuronal chromatin is characterized by size of ~<NUM> bp. The repeatable length corresponds to single nucleosome size (with degraded DNA linkers). Within the nucleosomal core, DNA is protected from nucleases by histones, whereas the linker is vulnerable to digestion. Thus, in some embodiments a correction factor is applied to the correction coefficient that represents the probability the sequence to be amplified is fragmented based on its positioning in a chromatin structure (e.g., in a nucleosome or linker region).

In some embodiments, sequence fragmentation bias is a positional bias. For example, in some embodiments the nucleic acid sample is an RNA sample. RNA transcripts may be preferentially cut at certain positions within the transcript, e.g. at the start and/or at the end of the transcript. Hence, this type of bias is referred to as positional bias. Thus, in some embodiments a correction factor is applied to the correction coefficient that represents the probability the sequence to be amplified is fragmented based on the position of the sequence to be amplified within an RNA transcript.

In some embodiments, the nucleic acid sample can comprise artificially fragmented nucleic acids, e.g., nucleic acids produced by mechanical shearing or enzymatic fragmentation. For nucleic acid samples comprising enzymatically fragmented nucleic acids, a correction factor can be applied to the correction coefficient that accounts for the recognition sequences or sequence preferences of the enzymes used (e.g., sequence bias of a transposase).

In some embodiments, the at least one parameter of the measuring method can further comprise a parameter of the amplifying step selected from the group consisting of: GC content of the sequence to be amplified; GC content of the amplification primers; length of the amplification primers; type of polymerase being used; and temperature of the amplification cycles. In some embodiments, the at least one parameter of the measuring method can further comprise a parameter of the measuring step selected from the group consisting of: sequence of a detecting probe; photostability of a fluorophore used; chemical stability of a fluorophore used; quantum yield of a fluorophore used; and wavelength of a fluorophore used. In some embodiments, the additional parameter of the amplifying step can be incorporated as a coefficient multiplied by the length distribution (LD) curve, or as a calibration coefficient multiplied by the correction coefficient.

In some embodiments, the effect of the additional parameters of the measuring method as described above can be determined experimentally and incorporated into the correction coefficient as an experimentally determined calibration factor.

In some embodiments the method further comprises a step of calibrating the correction coefficient based on measured concentrations of the detected sequence in a first nucleic acid sample having a first fragment length distribution (LD<NUM>) and a second nucleic acid sample having a second fragment length distribution (LD<NUM>). In some embodiments, the first nucleic acid sample is a non-fragmented sample. In some embodiments, the first nucleic acid sample comprises fragmented nucleic acids wherein less than <NUM>% of the nucleic acid fragments have a length shorter than the length of the sequence to be amplified. In some embodiments, the first nucleic acid sample is used to determine a ground-truth correction factor for the second nucleic acid sample. The relative error between the ground-truth correction factor and the predicted correction coefficient can be determined, and applied to the calculation of a corrected concentration of the detected sequence as a calibration factor.

In some embodiments, n or r can be determined based on what value of n or r yields the best fit between the predicted correction coefficient and the ground-truth correction factor, as one skilled in the art would readily understand.

In some methods according to any one of the embodiments provided herein, the method further comprises correcting the concentration with a calibration factor based on any one of the parameters described above. In some embodiments, the calibration factor can be applied as a modified dilution factor according to any one of the measuring methods provided herein.

Last, the invention relates to a system configured to determine the concentration of a detected sequence in non-fragmented nucleic acids. This system comprises the following modules.

A first a module is configured to measure the concentration of the detected sequence in a sample of fragmented nucleic acids, said fragmented nucleic acids being derived from said non-fragmented nucleic acids. This module realizes the measuring step of the basic method of the invention.

A suitable module comprises a real-time thermocycler and uses a reaction mixture including primers, intercalating fluorescent dye or fluorescent probes and polymerase enzyme in an appropriate buffer to perform the amplification reaction. Alternatively, a digital PCR platform may be used in place of a real-time thermocycler. Such a digital PCR platform is composed of a PCR reservoir (often a tube, plate or microfluidic chip) a partitioning system, a thermocycler and a fluorescence reader together with analysis software.

A second module is configured to compute a correction coefficient depending on the nucleic acid fragments length distribution of said fragmented nucleic acids and on at least one parameter of said measure, wherein the parameters of said measure include the length of the sequence to be amplified. This module is typically a computer device comprising a display screen, at least one microprocessor, a data exchange module and at least one computer-readable storage medium. Alternatively, this module may be connected to a remote server comprising at least one microprocessor, a data exchange module and at least one computer-readable storage medium.

A computer program comprising instructions which, when the program is executed by the computer or remote server, may cause the computer or remote server to automatically compute the correction coefficient.

A computer-readable storage medium comprising instructions which, when the program is executed by the computer or remote server, may be used. In an embodiment, the computer-readable storage medium is a non-transitory computer-readable storage medium.

A third module is configured to compute the concentration of the detected sequence in non-fragmented nucleic acids with the correction coefficient.

The first module may be an isothermal quantitative nucleic acid amplification module or a non-isothermal quantitative nucleic acid amplification module.

Isothermal quantitative nucleic acid amplification module typically comprises primers, intercalating fluorescent dye and a polymerase enzyme compatible with isothermal amplification in an appropriate isothermal buffer to perform the amplification reaction and a thermo-regulated fluorescent scanner equipped with compatible analysis software. Suitable modules are modules performing loop-mediated isothermal amplification, quantitative nucleic acid sequence-based amplification, signal-mediated amplification of RNA technology and strand displacement amplification.

Non-isothermal quantitative nucleic acid amplification module is typically quantitative Polymerase Chain Reaction module, real time Polymerase Chain Reaction module, digital Polymerase Chain Reaction module, multiplex Polymerase Chain Reaction module and multiplex digital Polymerase Chain Reaction module.

The second module may use the length of the sequence to be amplified as a parameter of the measure performed by the first module to compute the correction coefficient.

All these parameters may be included one by one or in combinations in the computation of the correction coefficient done by second module.

The starting sample is a <NUM> ng/µl of stock DNA (=<NUM>. 06E+<NUM> cp/µl) (Human Genomic DNA, Bio-<NUM>, Bioline, Paris, France) with an average length greater than <NUM> kbp according to the manufacturer.

The Covaris® microtube-<NUM> is used (with <NUM> to <NUM>µl ±<NUM>), which can contain up to <NUM>µg of DNA according to the manufacturer. Therefore, a dilution step of the stock solution of DNA is performed with TE: Tris-EDTA. This results in a DNA solution of <NUM> ng/µl (=<NUM>. 82E+<NUM> cp/µl), which can be sonicated in the Covaris® microtube-<NUM>.

Sonication is performed on a M220 Focused-ultrasonicator (Covaris®, Brighton, United Kingdom).

Firstly, the smallest fragment achievable by Covaris® (length distribution centered around <NUM> bp) is prepared in order to get as close as possible to the distribution of human DNA fragment lengths, i.e. close to the modes (<NUM>, <NUM> and <NUM> bp) found in human plasma.

Subsequently, length distributions centered around <NUM> bp, <NUM> bp and <NUM> bp are prepared.

All sonications were made in Covaris® microtube-<NUM> (<NUM>µl ± <NUM>) or microtube Snap-Cap (<NUM>µl ± <NUM>) which can contain up to <NUM>µg of DNA according to the manufacturer.

Grayscale data is extracted from the <NUM> TapeStation system (Agilent Technologies, Santa Clara, California, USA) electrophoresis images in order to get the base pair distribution required to calculate all the theoretical correction factors. In order to convert mass units into base pair units, the image intensity was inverted and then divided by the fragment length expected at the image pixel location.

High Sensitivity D1000 ScreenTape is used.

Primers and probes used were synthesized by Eurogentec (Eurogentec, Angers, France) and purified by high performance liquid chromatography (HPLC) in reverse phase.

The fluorophores used in the TriPlex PCR experiments are: FAM, HEX, and Cyanine (Cy5) as follows:.

Preparation template BRAF-EGFR-ALB is shown in Table <NUM>. The notation {} means Locked Nucleic Acid base.

PCR reactions were performed using Perfecta® Multiplex qPCR ToughMix® (Quanta Biosciences, Beverly, MA, USA), at a final concentration of 1X. <NUM> of fluorescein (VWR International, Fontenay-sous-Bois, France) is added in the PCR mix.

• Perfecta® qPCR Multiplex ToughMix® (1X)
• Fluorescein (<NUM>)
• Oligonucleotides BRAF V600 WT, FAM fluorophore (1X)
• Oligonucleotides EGFR L858 WT, HEX fluorophore (1X)
• Oligonucleotides ALB, Cy5 fluorophore (1X)
• Water.

The samples are obtained by dilution of the target sequences in the PCR mix so that the expected final concentration of each target sequence in the non-sonicated case is <NUM> cp/µL, as explained in Table <NUM>.

The samples are loaded in the inlet chambers of Sapphire chips (Stilla Technologies, Villejuif, France), <NUM>µL volume loaded per chamber. Three replicates are used per sonicated sample (three chambers per sample). One non-sonicated sample is loaded in triplicate in three independent chambers.

Naica™ Geode (Stilla Technologies, Villejuif, France) is programmed to partition the sample.

The PCR conditions are as follows: <NUM> for <NUM> minutes, followed by <NUM> cycles of <NUM> for <NUM> seconds and <NUM> for <NUM> seconds.

The exposure times set by default for image acquisition with the Naica™ Prism3 (Stilla Technologies, Villejuif, France) for the Blue, Green, and Red channels are <NUM>, <NUM>, and <NUM> respectively.

The predicted correction factor according to the method of the invention ("Prediction") is calculated from the fragment length distribution of the sample, experimentally measured, as shown on <FIG> for a length distribution centered around <NUM> bp, and from the length in base pairs (bp) of the sequence to be amplified.

Computing the correction factor according to the method of the invention requires the probability that the sequence to be amplified is not cut, as a function of the length of the sequence to be amplified, as shown on <FIG> for length distribution centered around <NUM> bp. The correction factor for the same conditions is shown in <FIG>.

The ground-truth correction factor ("Ground-truth") is obtained in vitro by computing the ratio between the concentration of the detected sequence experimentally measured in the non-sonicated sample versus the sonicated sample.

The relative error ("Relative error") is defined as the error of the predicted correction factor with respect to the ground-truth correction factor.

The experimental and theoretical results are compared in Table <NUM>. Predicted and ground-truth correction factors obtained with TriPlex digital PCR experiments measuring the concentration of sequences to be amplified with different sequence lengths (<NUM> bp, <NUM> bp, <NUM> bp) in sonicated samples at different fragment lengths (<NUM> bp, <NUM> bp, <NUM> bp, <NUM> bp). Each experimental measure was performed in triplicate and the displayed values are averages of the triplicate values.

It can be seen that the obtained relative error values ranging from <NUM>% to <NUM>% show that the predicted correction factors are consistently accurate and present a slight over-estimation with respect to the ground-truth correction factors.

Claim 1:
A method of determining the concentration of a detected sequence in non-fragmented nucleic acids comprising the following steps:
i. determining a length distribution (LD) of nucleic acids in a sample comprising fragmented nucleic acids, wherein the fragmented nucleic acids are derived from said non-fragmented nucleic acids;
ii. measuring the concentration of said detected sequence in said sample comprising fragmented nucleic acids with a measuring method, wherein measuring the concentration of the detected sequence comprises amplifying a sequence to be amplified comprising the detected sequence,
and
iii. correcting the measured concentration of said detected sequence in the sample comprising fragmented nucleic acids with a correction coefficient to obtain the concentration of said detected sequence in non-fragmented nucleic acids, wherein the correction coefficient is based on the length distribution (LD) and at least one parameter of the measuring method, wherein the at least one parameter of the measuring method comprises the length of the sequence to be amplified (La),
wherein the method does not comprise assembly of the fragmented nucleic acids into a contiguous sequence.