Method for detecting the presence of a target species by repeated real-time sequencing - Patents.com

Abstract

The present invention relates to metagenomic analysis of a sample to detect the presence of a species of interest in the sample. The analysis is performed iteratively. During each iteration, sequences corresponding to each species of interest are identified and counted. The iterations stop when the presence of the species of interest is confirmed or a maximum number of iterations have been performed. Detection of the species of interest is followed by a more accurate characterization of the genome of the species of interest. The characterization performs supplemental iterations.

Description

[Background technology]

[0001] The technical field of the present invention is the identification of biological species of interest by metagenomic analysis using real-time sequencing techniques.

[0002] Sequencing technologies, called "real-time" technologies, have recently emerged. These platforms allow immediate or short-delayed access to the read sequences. Moreover, they allow access to the entire nucleic acid sequence present in the analyzed sample, since the amount of read sequences is no longer limited by the need for the movement of nucleic acids on a support. An example of a technology is the nanopore sequencing of DNA, implemented by Oxford Nanopore Technologies' MinION and GridION sequencing platforms.

[0003] Nanopore sequencing of DNA is based on the passage of a molecule containing an oligonucleotide strand through a nanopore that forms a channel. As the molecule passes through the channel, a potential difference that depends on the nature of each base that forms the strand can be measured on both sides of the channel. The potential difference makes it possible to distinguish between the five standard bases of DNA or RNA (G, C, T or U). Thus, while passing through the nanopore, the oligonucleotide strand induces a temporal sequence of potential differences, on the basis of which the order of the bases that form the strand is determined.

[0004] WO2021 / 013900 describes a method for detecting a species of interest, more specifically bacteria, in a sample. The sample is mixed with a known amount of a control species beforehand. After sequencing, the number of sequences corresponding to the control species is used to quantify the concentration of the species of interest or to determine the minimum detectable concentration of the species of interest in the sample. The use of a control species ensures that the method of extraction and possible amplification of nucleotide sequences is performed accurately. The amount of the control species introduced into the sample is known and can therefore be used to quantify the concentration or the minimum detectable concentration of the species of interest. Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors have adapted the above methods to use real-time sequencers that deliver digital nucleotide sequences (also called "reads" and corresponding to DNA or RNA sequences) as nucleotide fragments in a sample are read, because these fragments generate analytical results more quickly, for example, within hours or tens of minutes. [Means for solving the problem]

[0006] A first subject of the present invention is a method for detecting a target species possibly present in a sample, the target species having a known or partially known genome and the sample comprising a mixture of different species, the method comprising the following steps: - a) adding a control species to the sample, the control species having a known genome and the control species being added to the sample at a known concentration; -b) extracting nucleic acid from the sample; -c) sequencing a portion of the nucleic acid sequences extracted in step b) using a real-time sequencer for a predetermined period of time or until a predetermined number of sequencing runs are obtained; - d) following step c), assigning the sequences obtained from step c) to species of interest and to control species; - e) updating the amount of sequences associated with the target species and the control species, respectively, such that the amount of sequences associated with the control species and the target species is: · in the first iteration of step e), add the amount of sequences assigned to the control and target species in step d) to the amount of initial sequences; In each iteration of step e), the amount of sequences (n i SOI ,n i SPC ) to the amount of sequences associated with the control and target species obtained from the previous iteration; - f) comparing the amount of sequences associated with the target species updated in step e) with a sequence number threshold; and / or comparing the amount of sequences associated with the control species updated in step e) with a control threshold; - g) depending on at least one comparison made in step f), detecting the presence of the biological species of interest in the sample or proceeding to step h); -h) Repeat steps c) to g) until an iterative stopping criterion is reached.

[0007] According to one embodiment, step d) of each iteration is carried out for a predetermined period of time or until a predetermined number of sequences have been sequenced.

[0008] The method may be as follows: - if in step f) of the iteration the amount of sequences related to the species of interest is greater than a threshold number of sequences; - if in step e) of the iteration the amount of sequences associated with the control species is strictly greater than 0; Step g) includes estimating the concentration of the species of interest as a function of: - the amount of sequences related to the species of interest at the last update (i.e., in the last step e); - the amount of sequences related to the control species at the time of the last update; -Addition concentration of control species.

[0009] The method may be as follows: - if in step f) of the iteration the amount of sequences related to the species of interest is greater than a threshold number of sequences; - if in step e) of the iteration the amount of sequences associated with the control species is 0; During step g), the concentration of the species of interest is considered to be greater than the concentration of the control species.

[0010] The method can be as follows. - if in step f) of the iteration the amount of sequences related to the species of interest is less than a threshold number of sequences; - if in step f) of the iteration the amount of sequences associated with the control species is greater than the control threshold; In step g), if the amount of sequence associated with the species of interest is zero, the concentration of the species of interest is considered to be zero.

[0011] The method may be as follows: - if in step f) of the iteration the amount of sequences related to the species of interest is below a threshold number of sequences; - if in step f) of the iteration the amount of sequences associated with the control species is greater than the control threshold; If during step g) the amount of sequences associated with the species of interest is not zero, step g) comprises an estimation of the concentration of the species of interest which depends on: - the amount of sequences related to the species of interest; - the amount of sequences related to the control species; -Depends on the concentration of the control species added. The concentration of the target species can be estimated from the ratio:

number

[0012] According to one possibility, the method comprises taking into account a decision threshold, the method aiming at comparing the concentration of the species of interest with the decision threshold, the method being such that in step a) the concentration of the control species is between 0.01 and 100 times the decision threshold.

[0013] According to one possibility, in step h), an iteration stopping criterion is reached when: - the amount of sequences related to the species of interest exceeds a sequence count threshold; - and / or the amount of sequences associated with the control species exceeds the control threshold; - and / or the cumulative duration of step c) reaches a predetermined maximum duration; - and / or the cumulative number of sequencing determinations in step c) reaches a predetermined maximum number; - and / or the number of iterations reaches a predefined maximum number of iterations.

[0014] According to one embodiment, the method comprises, following detection of the presence of a species of interest in a sample, the following steps: - i) sequencing a portion of the nucleotide sequence extracted in step b) using a real-time sequencer for a defined period of time or until a defined number of sequence determinations is obtained; - j) following step i), assigning the sequences obtained from step i) to the detected species of interest; - k) updating the amount of sequences associated with the species of interest, so that the amount of sequences associated with the species of interest is · in the first iteration of step k), the amount of sequences assigned to the species of interest detected in step j) is added to the amount of sequences obtained from the last iteration of steps c) to g); · in each iteration of step k), the amount of sequences assigned to the species of interest in step j) is added to the amount of sequences obtained from the previous iteration of step k); -l) Repeat steps i) to k) until an iterative stopping criterion is reached. Steps i) through k) may be repeated until a predetermined number of iterations is reached.

[0015] According to one possibility, - during each iteration of steps i) to k), following step k), the method comprises determining a sequencing depth for the biological species of interest, the sequencing depth corresponding to the ratio between the cumulative length of sequences associated with the species and the length of the genome of the species. - Steps i) to k) are repeated until a predetermined sequencing depth is reached.

[0016] According to one embodiment, following the cessation of the repetition of steps i) to k), a sequence representative of the genome of the species of interest is detected, the representative sequence comprising an antibiotic resistance marker or a virulence marker of the species of interest. The sequencing device is preferably configured to perform successive sequencing of different sequences of the nucleic acid one by one, the sequencing of each sequence being considered to be performed in real time.

[0017] A second subject of the invention is a device for the metagenomic analysis of a sample, comprising: - a fluid chamber intended to receive the sample; - a sequencer configured to perform real-time sequencing of the sample; - a processing unit comprising a bioinformatics module programmed to assign sequences obtained from the sequencer to species, and a control module programmed to carry out steps c) to h) of the method according to the first subject of the invention.

[0018] A third subject of the invention is a computer-readable or downloadable recording medium comprising instructions for carrying out steps c) to h) of the method according to the first subject of the invention.

[0019] The present invention will be better understood by reading the disclosure of exemplary embodiments presented in the remainder of the description with reference to the figures listed below. [Brief description of the drawings]

[0020] [Figure 1]Figures 1 and 2 show diagrammatically the main steps of the method according to the invention: Figure 1 relates to the phase for the detection of the species of interest. [Diagram 2] FIG. 2 relates to a phase for the characterization of each target species detected in the detection phase. [Diagram 3] FIG. 3 shows a schematic diagram of an apparatus configured to implement the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The aim of the method is to be able to detect the presence of a target species SOI in a sample. The acronym SOI stands for "target species". In the case of detection, the process can allow absolute quantification of the target species SOI, so as to allow a comparison with a decision threshold SD.

[0022] By biological species is meant a microorganism, such as a bacterium, a virus, a fungus, an archaea, an amoeba, a protist, or a microalgae. A biological species may also be a cell or any other object or entity that contains sequenceable nucleic acid.

[0023] If the sample is obtained from a human or animal organism, the target species may be a pathogenic species or a species identified as affecting the health or function of the human or animal body, such as an enterobacteria species, or a bacterium that affects the efficacy of an anti-cancer treatment. If the sample is obtained by sampling from an industrial process or the environment, the target species may be a species that is considered a contaminant, or a species of interest that has significance in the industrial process or environment and whose presence or concentration it is desired to monitor.

[0024] A species of interest represents a known or partially known genome, or a known portion thereof, that is comprised of sequences that are referred to as sequences of interest.

[0025] The method can address multiple target species simultaneously, and thus the term "target species" should be interpreted as meaning at least one target species.

[0026] The decision threshold SD is a threshold that allows characterizing the load of the target biological species, for example, microbial, depending on the target application. It is set, for example, in the light of regulatory or sanitary or industrial restrictions. For example, if the application is used to aid clinical diagnosis and the target biological species is bacteria, the decision threshold can be the concentration below which the presence of bacteria corresponds to a normal presence, i.e. non-pathological development, and above which the presence of bacteria is considered pathological, for example corresponding to an infectious disease. If the invention is applied to industrial processes, the decision threshold corresponds to a pass value, above which the sample is considered not to pass, and below the detection threshold the sample is considered to pass. Whatever the application, if the concentration of the target biological species is equal to or greater than the decision threshold, it is defined as important. In certain applications, for example in the production of fermentation products, if the concentration of the target biological species is lower than the decision threshold, it can be considered as critical. The decision threshold corresponds to the minimum acceptable concentration of the biological species.

[0027] The sample is generally a sample taken from the environment, from a dead or living organism, or from an agricultural food or manufactured product. The sample may also be taken from an industrial facility for process control purposes. Thus, the sample includes various species that do not have identical genomes. In particular, when the sample is obtained from a sampling of an organism, such as a human or animal organism, the sample includes a significant amount or an overwhelming amount of cells from the organism from which the sample was taken. The genome of a human or animal organism has a size that is 1000 to 100000 times larger than the genome of a prokaryotic organism. Furthermore, the sample generally includes species that naturally occur in the sample and are less likely to cause pathology or significant contamination. For example, if the sample is a bronchoalveolar sample, the sample includes the bacterial flora naturally occurring in the lungs. If the sample is a stool sample, the sample includes the bacterial flora naturally occurring in the digestive tract. Thus, if the species of interest is a bacterium or a virus, the nucleic acid of the species of interest may be a minority of the nucleic acid in the sample.

[0028] The sample contains what are called "matrix" species, which are endogenous to the sample and tend to obscure metagenomic information about the biological species of interest. For example, if the sample is taken from yogurt, a piece of meat, or a vaccine, it will contain matrix species representative of these media. If sampling from an organism, the matrix will contain the organism's constituent cells.

[0029] An important aspect of the present invention is that the presence of a species of interest in a sample is detected using a real-time sequencer of the prior art. This type of sequencer makes it possible to obtain usable data related to the sequences present in the sample more quickly than conventional sequencers. The real-time sequencer is configured to continuously collect signals representing the successive detection of bases in a single sequence. Thus, the real-time sequencer is configured to generate sequences in real time as the sequences are decoded. The sequencing of the different sequences present in the sample is performed continuously, with each sequence being sequenced one after the other. This does not exclude the possibility of certain sequences being subjected to simultaneous sequencing in parallel. For example, if the sequencer is a nanopore sequencer, each nanopore "reads" many different sequences in parallel. Each nanopore can read many different sequences sequentially.

[0030] Another advantage of such a sequencer is the possibility to perform a real-time analysis of sequences, the amount of sequences read is related to the duration of the sequencing. Depending on the available sequencing results, the sequencing may continue or may be stopped, providing a time gain and high flexibility of use. The sequencer may be of the nanopore type as described in the prior art, and more generally, the invention relates to the use of a sequencer configured to generate sequences in real time. These sequences are identified after sequencing. The sequencer is therefore configured to generate sequentially a list of identified sequences, these identified sequences being denoted by the term "reads". The list of reads is generated at regular intervals, for example every minute, for example 10. It is also possible to parameterize the amount of sequences read in each list. The amount of sequences (or reads) in each list may be, for example, 4000.

[0031] Similar to the situation in patent application WO2021 / 013900, one of the objectives of the present invention is to evaluate the extent to which metagenomic analysis can be used. In particular, it is necessary to evaluate the suitability of the collective steps from sample preparation (excluding sampling) to the bioinformatics analysis of the sequencing data. For this purpose, a control species, called SPC, an acronym for Sample Processing Control, is added to the sample. One function of the control species is to allow monitoring of the effective progress of the nucleic acid extraction and sequencing steps described below. The control species SPC may be a known biological species, the genome of which is also known, preferably in its entirety. The control species SPC may be a natural biological species. It may also be an artificial species, for example an encapsulated RNA (ribonucleic acid). Preferably, the control species SPC is not initially present in the sample taken or, if it is present, is present in negligible amounts. Preferably, the content of the control species SPC initially present in the sample, i.e. before addition, is a function of the control species SPC concentration C of the control species SPC added to the sample. SPC The control species SPC may be, for example, a bacterium. The concentration of the control species SPC added, C SPC It is important that the amount of oxygen consumed is controlled.

[0032] The control species may be selected taking into consideration the following aspects: - The control species should preferably be different from the naturally occurring or endogenous organisms in the sample and from the search species of interest, so that bioinformatics tools can accurately identify sequences from the sequencing of SPCs. - The amount of sequence assigned to the control species during sequencing must be sufficient to allow accurate detection of the corresponding sequence of the biological species of interest without obscuring useful information. In other words, the control species is preferably not predominant in the sample but is detectable by high-throughput sequencing. In particular, if one wishes to determine a positive (concentration of the species above the decision threshold) or negative (concentration of the species below the decision threshold), the control species preferably exhibits at least one of the following characteristics: The size of its genome is preferably similar or at least comparable to the size of the genome of the species of interest. More specifically, the size of the genome of the control species is between 0.1 and 10 times the size of the genome of the species of interest. Control species concentration C SPC can be set according to the decision threshold. The concentration C of the added control species SPC SPC is, for example, between 0.001 and 1000 times the judgment threshold, and preferably between 0.01 and 100 times. The nucleic acid of the control species SPC undergoes the same treatment as the nucleic acid of the target species in the sample preparation, extraction and sequencing steps. The percentage of GC (guanine, cytosine) bases is preferably close to the percentage of GC bases of the target species, where close means between 75% and 125%, preferably between 80% and 120%. The control species preferably contains an intact cell wall or membrane if the target species is a bacterium, and preferably contains a protein shell if the target species is a virus. This condition also makes it possible to monitor the process of lysis or extraction of the nucleic acid of the target species. - preferably, the nucleotide sequence of the control species does not contain genomic markers, such as markers of resistance to antibiotics, or virulence markers, so that the results of potential tests for susceptibility to antibiotics are not compromised by the presence of such markers in the genome of the biological species of interest. Preferably, the nucleotide sequence of the control species does not contain other genes of clinical or industrial interest, the presence of which is likely to be checked. The control species is preferably easy to manipulate, in particular · Non-harmful to humans or the environment; and / or be resistant to thermal treatments such as freeze-drying or freezing, thereby allowing for easy storage. - The control species should not produce spores or should produce only a few. - The control species must have a lysis sensitivity close to that of the biological species of interest. The control species may take the form of balls, each ball containing a calibrated concentration of the control biological species in lyophilized form.

[0033] It should be noted that a single control species SPC may be used, or multiple control species of various types may be used.

[0034] The method first involves detecting the species of interest, with the steps described below in conjunction with FIG.

[0035] Step 10 : Collect a sample. In this example, the sample is taken from a living human organism for the purpose of aiding diagnosis. However, the invention is not limited to application in the field of living organisms. Samples can be taken from natural, industrial or hospital environments to verify suitability with respect to the decision threshold.

[0036] Step 20 : Add control species. The added concentration of control SPC, C SPC is preferably known precisely, e.g., with as much precision as desired for the quantification of one or more target species. In particular, it allows the concentration of the target species in the sample to be quantified if certain conditions are met, and the control species then forms a calibrator. The term "added concentration" denotes the concentration of the control species in the sample by addition of the control species.

[0037] The description of steps 30 to 60 is advantageously based on the addition of a single type of control species to the sample, which then performs the functions of quality control and calibrator in the metagenomic analysis steps and allows the quantification of the concentration of the species of interest.

[0038] At the end of step 20, the sample is spiked with a control species concentration C SPC There is a control species added at a concentration of C SPC is preferably expressed in GEq / mL (genomic equivalents per mL).

[0039] In addition, the clinical detection threshold in bacteriology (e.g., the threshold above which an infection is diagnosed) is generally expressed in CFU / mL (colony forming units), but the proposed quantification method allows the quantification of nucleic acids without information on genome equivalence. Therefore, for SPC, it is recommended to obtain a correlation between the number of colony forming units by culture in a gel medium or the like and the equivalence of genome numbers by quantitative PCR or the like. For example, it has been confirmed that one colony forming unit corresponds to one genome in BioBall® Bacillus subtilis strain ATCC 19659 BioBall MultiShot 10E8 (bioMerieux, catalog #416721).

[0040] The spike concentration can be defined as a function of the decision threshold SD. The spike concentration C of the control species SPC is preferably equal to or close to the decision threshold SD, for example within about ±50%. Therefore, 0.1 SD≦C SPC ≦10SD. Control species added concentration C SPC The effects are described below.

[0041] Step 30 :Dissolution and extraction of nucleic acids. In this step, the cells of the sample, in particular the cells of the biological species of interest and the control species, are lysed to allow their DNA to be extracted. Various strategies are possible. Dissolution can be parameterized to preferentially target the biological species of interest. In such a scenario, the control species should have the same or comparable dissolution susceptibility as the biological species of interest. Lysis can include a first lysis intended essentially to lyse cells other than the species of interest. Such a first lysis can be envisaged, for example, when the biological species of interest is very few in number with respect to the cells of the matrix constituting the sample. Following the first lysis, the released nucleic acids are removed and then a second lysis is carried out targeting the biological species of interest. In such a scenario, the control species is preferably resistant to the first lysis and not to the second lysis.

[0042] Following lysis, DNA is extracted from the sample, for example according to the extraction methods described in WO2014 / 114896.

[0043] Step 40: Sequencing The DNA extracted from the sample is sequenced by a sequencer, for example a nanopore sequencer, which allows real-time access to the read sequence. It may be, for example, a MinION® sequencer from Oxford Nanopore Technologies. The sequencer is connected to a dedicated fluid chamber that contains a nanopore. The sample is introduced into the fluid chamber. The sequencer is connected to a processing unit for analyzing the identified sequence. The processing unit includes a microprocessor configured to execute instructions from a bioinformatics software to identify the analyzed sequence. The processing unit is, for example, a MinIT computer that includes a computing environment dedicated to sequencing.

[0044] The invention applies in particular to so-called "real-time" sequencers having the following characteristics: - the ability to read all the sequences present in the sample. In contrast to sequencers based on fixing the sequences on a support in order to read them, these sequencers therefore sequence a given maximum number of sequences, which corresponds to the number of elements for fixing the nucleotide sequences. This means, in particular, that the number of sequences generated is proportional to the duration of the sequencing. -Ability to output raw data (e.g., readings or raw signals such as potentials) at the same rate as they are collected, or ability to output raw data in batches at a defined frequency for batches of arrays of a given size, or ability to output raw data periodically at a fixed frequency (e.g., a batch of raw data every minute or every 10 minutes).

[0045] A particular feature of real-time sequencing is that the RNA or DNA sequence reads are available in real-time or quasi-real-time as they are output from the sequencer. Quasi-real-time means that the reads (or measured raw signals, such as potentials) are output from the sequencer in batches at the end of a period that is much shorter than the total period required for sequencing the acids present in the sample, or at the end of a period that is much shorter than the total consumption period of the reagents required for sequencing. Thus, each batch is generated in a few minutes or tens of minutes. Real-time sequencers have the ability to generate sequences (reads) in real-time. However, it is generally assumed that a certain number of sequences are decoded in files generated at regular intervals. As an example, the Oxford Nanpore MinION Sequencer generates ".fast5" files in batches of 4000 sequences of potential differences in quasi-real-time by default. This can be converted by a computer to a .fastq file in batches of 4000 reads in quasi-real-time. The fast5 extension stands for raw data file. The fastq extension stands for file that stores sequences and associated quality scores. This allows the method to be implemented interactively, with the sequence being able to be stopped or continued depending on the results obtained.

[0046] The sequencing process can in particular be carried out iteratively, with each iteration corresponding to a given number (or batch) of sequences or to a sequencing process carried out over a given period of time. During each iteration, following the sequencing process, the sequences are potentially assigned to control species SPCs or to the biological species of interest SOI, or to the biological species of interest or other species present in the sample.

[0047] Each iteration has a corresponding rank i, where i is an integer between 1 and I, and I corresponds to the maximum number of iterations described in connection with step 70 or the maximum number of sequences (reads) obtained. During each iteration, sequencing is performed on the target species SOI and the control species SPC with a sequence number n i SOI and n i SPC Assign.

[0048] The following steps are described in relation to a target species SOI, but they can be performed in a number of different target species.

[0049] Step 50: Update the sequence number. During this step, the number of sequences N assigned to the target and control species in step 40, respectively, i-1 SOI ,N i-1 SPC are the number of sequences associated with the target species SOI and the control species SPCs, respectively, in the previous iteration. This step corresponds to updating the number of sequences associated with the target species SOI and the control species SPCs, respectively, according to the following formula:

number

number

[0050] where n i SOI and ni SPC are the number of sequences assigned to the target and control species, respectively, in step 40 of iteration i. i corresponds to the number of sequences assigned to a species at iteration i, while this notation N i corresponds to the total number of sequences associated with the species in all iterations up to iteration

[0051] In the first iteration (i=1), a given initial value N 0 SOI and N 0 SPC These initial values ​​are generally 0.

number

[0052] According to one possibility, the sequence N i SOI or N i SPC The number of can be normalized by the reference amount, which is, for example, the total amount N of sequences generated during sequencing up to the current iteration. i i may be also possible.

[0053] Step 60: Compare with threshold During this step, the number of sequences associated with each target species SOI, N i SOI is the sequence number S, which corresponds to the minimum number of reads associated with a species of interest SOI required to stop sequencing and begin a quantitative interpretation related to the presence of the species of interest in a sample. SOI The sequence number threshold corresponds to a sequence number threshold or read number threshold associated with the species of interest.

[0054] Number of sequences related to control species SPCs, N i SPC is the control threshold S SPC is compared with the control threshold S SPC is the number of reads associated with each SOI in the array S SOIThis corresponds to the minimum number of SPC-associated reads to stop sequencing if a threshold number of reads is not reached. -N i SOI SOI and N i SPC SPC If so, see step 61; -N i SOI ≧S SOI and N i SPC If >0, see step 62; -N i SOI ≧S SOI and N i SPC If =0, see step 63; -N i SOI SOI and N i SPC ≧S SPC If so, see step 64; The determination of the threshold for the number of sequences and the control threshold will be described later.

[0055] Step 61 During this step, no conclusions can be drawn regarding the presence of the target SOI in the sample. Re-iteration of steps 40 through 60 is necessary unless an iteration stopping criterion is reached, as described in connection with step 70.

[0056] Step 62 : During this step it is possible to conclude the presence of the target species SOI in the sample and to quantify its concentration, for example by the following formula:

number

[0057] Where: -L SPC and L SOI are the genome lengths of the control and target species, respectively. ​​​-α is a correction factor empirically determined based on training samples with known concentrations of the target species. The correction factor α accounts for differences in the efficiency of the sequencing procedure for the target and control species. As a default, α=1 can be considered. This unitary value produces an absolute amount sufficient to determine a sample as positive or negative relative to the decision threshold. When spike concentrations are expressed in GEq / mL, the concentration of the target species is also expressed in the same units.

[0058] Step 63 During this step, the species of interest will likely be present in the sample at a concentration higher than that of the control species, but the absolute concentration of the SOI cannot be determined.

number

[0059] Step 64 During this step, N i SOI = 0, it can be concluded that the target species SOI is not present in the sample. If not, its concentration can be quantified using the formula set forth in step 62.

[0060] Step 70 : Steps 40 to 60 are repeated until an iteration stopping criterion is reached. Preferentially, each iteration corresponds to a predefined sequencing duration, preferably identical for each iteration i, or a predefined number of sequences read (number of reads). The iteration stopping criterion is considered to be reached when the condition leading to step 62 or 63 or 64 is reached.

[0061] The iteration stopping criterion may be a number of iterations equal to the maximum number of iterations I, said number being predetermined. For example, the iteration stopping criterion may be reached when the sequencing period is greater than or equal to a predetermined period, such as 12 hours, or when the total number of sequences obtained from the sequencer is greater than a predetermined maximum number.

[0062] Step 80 caveat. Step 80 is N I SOI SOI and N I SPC SPC i.e., if following the last iteration of rank I, the number of sequences associated with the species of interest and the control, respectively, is less than the sequence number threshold and the control threshold, respectively, in which case a warning signal is generated, indicating that the presence or absence of the species of interest in the sample cannot be confirmed.

[0063] Step 90 Stop Following step 70 or possibly step 80, the method can stop. According to one possibility, several different species of interest are analyzed simultaneously. If one of the conditions indicated in steps 62, 63 and 64 is encountered, the sequencing can be stopped and each species of interest is interpreted according to these criteria.

[0064] Optionally, following steps 62, 63 or 64, if the presence of a species of interest in the sample is reported, one may have complementary genomic information regarding the identified species of interest. The information of interest may in particular be the presence of a particular marker in the genome, for example an antibiotic resistance marker or a toxicity marker. Antibiotic resistance or toxicity markers are known.

[0065] ​​The iterative steps 100 to 130 described below in connection with FIG. 2 can be carried out to confirm the presence of specific markers in the genome of the identified target species. If i’ represents the rank of the iteration that has reached the iteration stop criterion of step 70, steps 100 to 130 are carried out such that i’ < i ≦ I corresponds to the rank of the last iteration.

[0066] Step 100 This step is similar to step 40. Only the sequences corresponding to the various types of the target SOI detected after the iteration from step 40 to 80 are numbered.

[0067] Step 110 This step is similar to step 50, except that the number of sequences of each type of the target is updated according to the following formula.

Number

[0068] Step 120 : Steps 100 to 110 are repeated unless the iteration stop criterion is reached. The iteration stop criterion may be the reaching of the maximum number of iterations I. Note that the latter may correspond to the maximum sequencing period or the maximum number of sequences identified.

[0069] According to one possibility, during each iteration, the depth of sequencing of the species SOI of interest is determined. The depth of sequencing corresponds to the ratio between the cumulative length of the sequences related to the species of interest and the length of the genome of the species of interest. When the depth of sequencing reaches the threshold depth, the iteration stop criterion is reached. The iteration is stopped.

[0070] Step 130 : Characterization In this step, the sequences corresponding to the desired markers are identified and numbered. Number of arrays S SOI and the control threshold S SPC Determination of threshold value The implementation of the above described methods is subject to prior decisions. - At each step, 60, the number of sequences of the desired species, N i SOI The number of sequences to compare is S SOI threshold; - and the number of control sequences N at each step 60 i SPC A control threshold S to compare SPC .

[0071] Different concentrations of the species of interest SOI in the sample, C SOI Taking into account the above, we estimated the sequencing time required to generate the number of reads corresponding to 1, 10, 100, 1000 and 10000 species SOIs of interest, respectively.

[0072] The sequencing time TTR (time to result) is estimated according to the following formula:

number

[0073] Table 1 shows the sequencing times (in hh:mm). This is the number of sequences S SOI and the concentration C of the target species SOI expressed as a function of the decision threshold SD SOI The control species is at concentration C SPC = SD, and the sum of human DNA and commensal flora is considered to be equal to 100 SD. In this case, the total amount of DNA is C ADNtot =101SD+C SOI The sample is equal to the other three samples (N sample = 4), each sample is sequenced simultaneously at approximately T fastq = R at 3 minute frequency fastq The same amount of DNA is sequenced using a sequencer that generates a read file containing =4000 reads.

[0074] [Table 1]

[0075] In Table 1, the concentration C SOI If is equal to the decision threshold SD, then the number of sequences S in 30 min of sequencing SOI If we want to reach the threshold of,the number of sequences,S, SOI It is observed that the threshold value of should not exceed 100. In particular, the number of sequences S SOI The higher the threshold, the longer the analysis time. SOI The higher the threshold, the longer it takes to reach it. SOI = 1 is quickly reached even when the concentration of the target species is low. However, too low a value of S SOI The adoption of ,increased risk of false positives and negatively impacted the,accuracy of quantification.

[0076] Moreover, too low a value limits the possibility of detecting other target species that may be present at lower concentrations. For example, a sequence number S of 100SOI Using a threshold of 0.1 allows for the detection of another target species SOI' present at 100-fold lower concentration based on a single read. Therefore, if the majority is present at more than 100-fold concentration, the number of sequences S SOI The high threshold of can be particularly important: it allows the detection of a few other SOIs, termed SOI', at concentrations 100-fold lower than the SOI but higher than the decision threshold SD.

[0077] Based on the results shown in Table 1, it can be concluded that the threshold value of the number of sequences SSOI is preferably between 5 and 500, or between 50 and 500. If a short analysis time is a priority, the number of sequences S SOI A relatively low threshold for the number of sequences, close to 50 or even lower, is chosen, recognizing the fact that a threshold for S that is too low can lead to false positives. If the dynamics of the measurement and the possibility of simultaneous detection of different target species present at different concentrations take precedence, a threshold for the number of sequences, S, close to 500 or even higher, can be chosen at the expense of analysis time. SOI A high threshold is selected.

[0078] The control threshold S for the implementation of this method SPC We investigated the effect of the genomic DNA concentration on the SOI relative to the SD. In the absence of the target species, a significant amount of DNA may be present in the sample. In clinical samples, the origin of DNA is diverse, e.g., DNA from the patient's cells or from the commensal flora. DNA may also result from atypical infections. Thus, the concentration of genomic DNA from microorganisms other than the SOI may reach amounts far exceeding the decision threshold SD. Table 2 shows the DNA concentration C relative to the decision threshold SD. ADN For the difference between the number of sequences corresponding to the control species, the control threshold S SPC The sequencing time is shown to be higher than the concentration of the species of interest, C SOI We assumed that SD corresponds to the decision threshold. The aim is to determine the control threshold S for the sequencing time of samples where SOI is not detected. SPC The aim of the study is to determine the impact of changes in

[0079] [Table 2]

[0080] DNA concentration is SD (C ADN = SD), control threshold S between 5 and 3125 for sequencing in less than 30 minutes SPC Thus, for these samples, deletion of the SOI can be concluded in less than 30 minutes of sequencing.

[0081] Since sequencing times are significantly increased when the DNA concentration exceeds the SD, it is necessary to select a control threshold that is not too high to obtain a negative result for the detection of the SOI of the species of interest. For example, for a clinical sample with an atypical infection with a pathogen concentration equal to 1000 SD, a control threshold of S SPC = 125, no negative results are obtained for the detection of the SOI of the species of interest prior to at least 6 hours of sequencing. Therefore, the number of sequences selected, S SOI It is recommended that the threshold for is not too low to allow detection of an SOI, distinct from the species of interest, but allowing the sequencer to generate enough reads to identify another species of interest whose presence should be reported if its concentration is higher than the SD, as may occur in the context of an atypical infection.

[0082] Addition concentration of control species C SPC Decision In the previous paragraph, the control species SPC is the concentration C equal to the decision threshold SD. SPC The inventors found that the control species was present at a concentration of C SPC The effect of changing

[0083] Table 3 shows the results of the various concentrations (C SPC = 0.01 SD;C SPC = SD and C SPC = 100SD), the time required to return the results (hh:mm), and various concentrations of SOI between 0.0001SD and 10000SD C SOI The detection results for

[0084] The "Results" column states: - The presence of concentrations expressed relative to the decision threshold SD in the "Result" column indicates that SOI(N i SOI >0) and SPC(N i SPC >0) was detected, indicating that this concentration could be calculated. i SOI >0 corresponds to step 62 or step 64. - Entry "NEG" is the SOI(N i SOI = 0), but the number of reads associated with SPCs exceeded the control threshold set at 25 (N i SPC >25). This is because i SOI =0 corresponds to step 64. - The entry ">SPC" is the SPC(N i SPC No reads were associated with the SOI (N i SOI >100) was set to 100. SOI Under these conditions, the concentration of the target species, C SOI It is not possible to calculate the concentration of the control species, C SPC This corresponds to step 63.

[0085] First, a low concentration C equal to 0.01 SD SPC Considering the above. C SOI For >0.1 SD, the duration obtained is essentially the same as in Table 1. Thus, the amount of SPC added is proportional to the number of sequences S SOI However, since there is no SPC reading, it is not possible to calculate the concentration of the SOI, and the entry "SPC" is not sufficient for the comparison of this concentration to the SD. SOIIn the case of <0.001SD, result C SOI To return <SD, more than 12 hours of array determination is required. In this context, if the total time of array determination is limited to 12 hours, negative results cannot be returned for these samples.

[0086]

Table 3

[0087] Concentration C equal to the decision threshold SPC is the concentration C SPC increases the detection limit for <0.01SD. However, this allows the concentration of the species of interest to be quantified according to a larger dynamic range (between 0.01SD and 1000SD).

[0088] Next, a high concentration C of 100 times the decision threshold <SD was considered. A large amount of SPC can return results very quickly. This is because within the range of the SOI of the evaluated concentration, results are returned within just 3 minutes after the start of array determination. However, compared to the test performed with <SD = C, at least a 10-fold increase in the detection limit is observed. A possible explanation for this is that a high concentration of SPC reduces the number of readings related to the SOI accordingly. Therefore, the sensitivity decreases. SPC SPC

[0089] Also, adding a large amount of SPC can return quantitative results over a wider range than in the case of the lowest concentration.

[0090] Therefore, various analysis parameters as follows can be selected. - The concentration C of the control species added to the sample SPC ; - The threshold of the number of arrays S SOI ; - The control threshold of S SPC

[0091] ​​​Parameterization allows the performance level of the above-mentioned methods to be adapted in terms of sequencing rapidity, quantitative dynamics, and required sensitivity.

[0092] For example, in the context of clinical diagnostics requiring rapid detection with high detection sensitivity and precision in quantification of SOI detected near the clinical decision threshold, C SPC ≒SD (e.g., C SPC between 0.1SD and 10SD, or between 0.01SD and 100SD), and the number of sequences S SOI The threshold value is between 5 and 500, and the control threshold S SPC It is advantageous to add between 5 and 125 SPC.

[0093] Example 1 In a first example, Bacillus subtilis was used as a control species for metagenomic sequencing of samples obtained from bronchoalveolar lavage (BAL) performed on human patients. It is known that this type of sample tends to contain a significant amount of human DNA derived from the patient. 38 samples were sequenced. The maximum sequencing time was set to 12 hours. Each replicate contained 4000 reads.

[0094] The volume of each sample was 600 μL. The control species was added at a concentration of 1.7E4 CFU / mL, where CFU stands for colony forming units, a threshold considered to be just above the clinical threshold that distinguishes between the normal presence of bacteria and the presence of bacteria representative of an infection.

[0095] To remove DNA from the patient, the analysis protocol involves removing DNA from the patient in the course of a first lysis. In the first lysis, the sample was treated with a lysis agent that specifically targets the patient's cells. This type of lysis agent is described, for example, in WO2014 / 114896. The released DNA was then removed by enzymatic action and washing. The sample then underwent a second mechanical and chemical lysis to extract the bacterial DNA. Mechanical lysis was performed using glass beads with a diameter of 1 mm and Zr / Si microbeads with a diameter of 0.1 mm, by stirring lasting for 20 minutes. DNA was extracted from the lysate using the Easymag (Biomerieux) platform. The volume resulting from the elution was 25 μL. The DNA extract was stored at -20 °C.

[0096] Preparation of the sequence library and barcoding of DNA fragments was performed using the Rapid PCR Barcoding kit (Oxford Nanopore Technologies). Four samples were processed in parallel. Sequencing was performed using a MinION sequencer (Oxford Nanopore Technologies) connected to a MinIT computer (Oxford Nanopore Technologies). Sequencing data were processed by the WIMP software of the EPI2ME suite (What is in my Pot?). This software allows to separate the sequences of each sample using barcodes (barcodes with molecular markers that assign an identifiable label to each sample), to identify the bacterial, viral or fungal species that corresponds to each sequence read, and to count the number of reads associated with each identified species.

[0097] Steps 40 to 70 were repeated for a series of 4000 reads. The detection thresholds associated with the target and control species, respectively, were 100 reads and 25 reads. 20 different target species were taken into account: Escherichia coli, Klebsiella oxytoca, Klebsiellapneumoniae, Klebsiella aerogenes, Enterobacter cloacae, Serratia marcescens, Proteus mirabilis, Proteus vulgaris, Hafnia alvei, Citrobacter freundii, Citrobacter koseri, Morganella morganii, Providencia stuartii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter baumannii, Legionella lapneumophila, Haemophilus influenzae, Staphylococcus aureus and Streptococcus pneumoniae.

[0098] When the concentration of each target species was quantified, the concentration was compared to the metagenomic threshold of 5.53 GEq / mL, which is the decision threshold at which it can be concluded that the target species is pathologically present in the sample obtained by BAL.

[0099] Thirty-eight samples were analyzed. When at least one species of interest SOI was detected, the median sequencing time was 28.5 min per sample. This is in line with the condition N obtained for at least one species of interest. i SOI ≧100 corresponds to the required sequencing time.

[0100] If at least one species of interest was not detected (N i SOI <100 and N i SPC ≥ 25), the median sequencing time was 2 hours and 13 minutes. i SPC>S SPC allows one to ensure that sequencing has been performed properly, thereby ensuring that negative samples are in fact true negatives.

[0101] The results of the sequencing by the above method were compared with the same kind of sequencing carried out over a 12-h fixation period. Table 4 summarizes the main results obtained. The performance level of each technique was compared by analyzing each sample by the reference technique, microbial culture.

[0102] [Table 4]

[0103] Sensitivity, specificity, positive predictive value (usually denoted PPV), and negative predictive value (usually denoted NPV) are determined by the number of true positives, false positives, true negatives, and false negatives.

[0104] The method was observed to have a performance level equal to or better than that obtained with 12-hour sequencing. The method was able to detect all infections with a median time extension of more than 11 hours and 30 minutes. Furthermore, in the absence of infections with all species of interest, detection of SPCs confirmed that the method worked properly and gave negative detection results in a median time of 2 hours and 13 minutes.

[0105] Four false negatives were observed, but these detection failures were not attributable to the method described. Two of the four false negatives were not detected by PCR, and were therefore considered to be false culture positives or errors in species identification by the reference technique. The other two false negatives corresponded to S. marcescens, with fewer than 100 reads and quantified at concentrations below the clinical decision threshold. The discrepancy between the method and the reference technique in these two cases was confirmed by PCR. Extending the sequencing time to 12 h did not improve the detection of these two infections with S. marcescens, suggesting an error by the WIMP software used for sequencing.

[0106] It was found that this method can improve the performance level of the test while decreasing the number of false positives and improving the specificity and positive predictive value. This is because when sufficient relevant information is available, i.e., N i SOI ≧S SOI When or N i SPC >S SPC , in order to appropriately stop the sequencing when one of these thresholds is exceeded. As soon as one of these thresholds is exceeded, the iterations are stopped and the concentration (or minimum concentration) of the species of interest can be quantified. Continuing the iterations up to the initially set maximum number of iterations, which corresponds to a total duration of 12 hours, proved to be not useful, or even desirable, since obtaining too large a number of sequences of the species of interest could result in false positives.

[0107] Example 2 In the second example, the same protocol was used as described in relation to the first example. Eleven BAL samples prepared as described above were analyzed. Eleven samples had previously been analyzed by bacterial culture and proved positive for at least one species of interest, with concentrations of 1.0 or more. E >4 CFU / mL. Eleven samples were analyzed to identify at least one of the 20 species of interest listed above. Repetitive steps 40 through 80 were performed. Six of the eleven samples were considered positive for at least one species of interest in less than 30 minutes of sequencing.

[0108] However, the amount of 100 sequences for each species of interest detected is too small to achieve a detailed characterization of their genome. Iterative steps 100 to 120 were performed on the samples to identify the presence of possible antibiotic resistance markers in the genome. Iterative steps 100 to 120 were performed until a total sequencing time of 12 hours was obtained.

[0109] In Table 5: - The first column (ref) corresponds to the reference for each sample. - the second column (SOI) corresponds to each species of object detected; - the third column (technique) corresponds to the detection technique: culture or implementation of the invention; -Fourth column (C SOI ) corresponds to the measured or estimated concentration of each detected bacterial species; - the fifth column (dep) corresponds to the sequencing depth as described above; - The sixth column (TTRID) corresponds to the duration in "hours:minutes" format of steps 40 to 80 to confirm the presence of the species of interest in the sample. The durations range from 3 minutes (sample C1-026) to 8 hours 37 (sample C1-060). Six samples were determined to be positive in less than 30 minutes.

[0110] - The following columns correspond to the presence of antibiotic resistance markers for 21 antibiotics. The letter R indicates resistance to an antibiotic observed by culture or predicted by the presence of a marker, and the letter S indicates susceptibility observed by culture. Based on the absence of a resistance marker for an antibiotic, no opinion can be made about susceptibility to that antibiotic. This is why there is no letter S in the row corresponding to the implementation of the invention. The letter R in grey indicates the presence of an antibiotic resistance marker revealed by sequencing but not confirmed by bacterial culture (letter S in grey). This is therefore a false positive by sequencing. The letter R in white on a black background indicates the presence of an antibiotic resistance marker revealed by bacterial culture but not detected by sequencing. This is a false negative by sequencing. Count 9 false negatives.

[0111] Of the nine false negatives (white letter R on a black background), eight come from samples with a low sequencing depth, i.e., less than 30. It would therefore be possible to reduce the proportion of false negatives by continuing sequencing until a sequencing depth threshold is reached. In this example, this threshold is equal to 30.

[0112] It will be appreciated that a notable advantage of the present invention lies in the possibility of obtaining increasingly accurate information about the composition of the sample as the iterations proceed. When the task at hand is to determine the presence of a species of interest, the present invention makes it possible to optimize the duration of the analysis by stopping the iterations as soon as the biological species of interest has been detected with sufficient reliability. The duration of the analysis is thus reduced without a loss of sensitivity.

[0113] [Table 5]

[0114] As shown in Example 2, following detection of species of interest, complementary iterations can be performed to obtain more precise information about the genome of each detected species of interest.

[0115] Although illustrated with sequencing performed by a MinION sequencer (Oxford Nanopore Technologies), the invention can be practiced with other types of sequencers that allow reading and real-time analysis of DNA fragments present in a sequencing library or directly in a sample.

[0116] A method is described in which the steps following the sequencing are carried out by a processing unit connected to the sequencer, in particular one or more processor or microprocessor-based units having a computer memory storing all steps 40 to 130 in the form of computer executable instructions, this memory being arranged to store the results and values, parameters and intermediate results of the method. This unit is advantageously connected to a screen for displaying the results of the method according to the invention for the attention of the user. This screen can constitute the screen of a mobile device (such as a smartphone) connected to the processing unit to receive and display the results. These steps can be carried out remotely, for example on a remote server connected to the sequencer, or directly or via a laboratory computing system. These steps can also be carried out on a mobile device (such as a smartphone, tablet, etc.) connected to the sequencer, for example a MinION, the assembly consisting of the MinION and the mobile device being easily mobile. These steps can also be carried out by different processing units. For example, one unit connected to the sequencer performs the bioinformatics steps (reading the signal, translating it into nucleotide bases, assembly, quality control, etc.), and another processing unit connected to the first unit performs the remainder of the method.

Claims

1. 1. A method for detecting a species of interest (SOI) that may be present in an analytical sample, wherein the species of interest has a known or partially known genome and the sample contains a mixture of different species, the method comprising the steps of: - a) A control species (SPC) is spiked into the sample, the control species having a known genome and the control species being at a known concentration (C SPC ) to the sample; - b) extracting nucleic acids from the sample; -c) sequencing a portion of the nucleic acid sequences extracted in step b) using a real-time sequencer for a predetermined period of time or until a predetermined number of sequences is obtained; -d) following step c), the sequence (n i SOI ,n i SPC ) to the species of interest and the control species; -e) the amount of sequences associated with the species of interest and the control species, respectively (N i SOI ,N i SPC ) so that the amount of sequences associated with the control species and the species of interest is: In the first iteration of step e), the amount of sequences assigned to the control species and the species of interest in step d) is calculated as the amount of initial sequences (N 0 SOI ,N 0 SPC ) to add; In each iteration of step e), the amount of sequences assigned to the control species and the target species in step d) (n i SOI ,n i SPC ) is the amount of sequences related to the control species and the species of interest obtained from the previous iteration (N i-1 SOI ,N i-1 SPC ) to add; -f) The amount of sequences related to the target species updated in step e) (N i SOI ) as the threshold for the number of sequences (S SOI ) and compare it to and / or The amount of sequences related to the control species updated in step e) (N i SPC ) is the control threshold (S SPC ) compared to; - g) depending on at least one comparison made in step f), detecting the presence of the biological species of interest in the sample or proceeding to step h); - h) Repeat steps c) to g) until an iteration stopping criterion is reached.

2. 2. The method of claim 1, wherein step d) of each iteration is performed for a predetermined period of time or until a predetermined number of sequences have been sequenced.

3. 3. The method of claim 1 or 2, - In step f) of the iteration, the species of interest (N SOI ) is determined by the sequence number threshold (S SOI ) if greater than; - In step e) of the iteration, the control species (N SPC ) if the amount of array associated with it is strictly greater than 0; Step g) Determine the concentration of the species of interest (C) depending on: SOI ) including estimates of - Species of interest in the last update (N i SOI ) the amount of sequence associated with; - Control species at the time of last update (N i SPC ) the amount of sequence associated with; -Addition concentration of control species (C SPC ).

4. 3. The method of claim 1 or 2, - In step f of the iteration, the species of interest (N i SOI ) is the number of sequences (S SOI ) is greater than the threshold; - In step e) of the iteration, the control species (N i SPC ) if the amount of the array associated with it is 0; During step g), the concentration of the desired species (C SOI ) is the concentration of the control species (C SPC ) is considered to be greater than the method.

5. 3. The method of claim 1 or 2, - in step f) of the iteration, the species of interest (N i SOI ) is determined by the sequence number threshold (S SOI ) if it is less than; - in step f) of said iteration, a control species (N i SPC ) is determined by the amount of sequence related to the control threshold (S SPC ) is greater than; In step g), the target species (N i SOI ) is 0, the concentration of the species of interest is considered to be 0.

6. 3. The method of claim 1 or 2, - in step f) of the iteration, the amount of sequences associated with the species of interest (N i SOI ) is the threshold for the number of sequences (S SOI ) if it is less than; - In step f) of the iteration, the amount of sequences associated with the control species (N i SPC ) is the control threshold (S SPC ) if greater than; In step g), the amount of sequences associated with the species of interest (N i SOI ) is not zero, step g) calculates the concentration of the species of interest (C) depending on: SOI ) including estimates of: - the amount of sequences related to the species of interest (N i SOI ); - the amount of sequences related to the reference species (N i SPC ); -Addition concentration of control species (C SPC ).

7. 4. The method of claim 3, wherein the concentration of the target species is estimated from the ratio of formula (1), [Equation 1] where: -L SPC and L SOI are the genome lengths of the control and target species, respectively; -N i SOI and N i SPC are the abundances of sequences associated with the target species and the control species resulting from the last update, respectively; -C SPC is the concentration of the control species added to the sample.

8. 3. The method of claim 1 or 2, wherein after an iteration stopping criterion is reached, - the amount of sequences related to the target species (N i SOI ) is the threshold for the number of sequences (S SOI ), - or the amount of sequences related to the control species (N i SPC ) is the control threshold (S SPC ), - The method generates information that does not confirm the presence or absence of the target species.

9. 3. The method according to claim 1 or 2, comprising taking into account a decision threshold (SD), the method being aimed at comparing the concentration of the target species with said decision threshold, the method being such that in step a) the concentration of the control species is between 0.01 and 100 times the decision threshold.

10. 3. The method according to claim 1 or 2, wherein in step h) an iteration stopping criterion is achieved when: - the amount of sequences related to the species of interest (N i SOI ) is the threshold for the number of sequences (S SOI ) exceed; - and / or the amount of sequences related to the control species (N i SPC ) is the control threshold (S SPC ) exceed; - and / or the cumulative duration of step c) reaches a predetermined maximum duration; - and / or the cumulative number of sequencing determinations in step c) reaches a predetermined maximum number; The number of iterations reaches a predetermined maximum number of iterations and / or the number of iterations reaches a predetermined maximum number of iterations.

11. 3. The method of claim 1 or 2, comprising, after detecting the presence of a target species in a sample, the following steps: - i) sequencing a portion of the nucleotide sequence extracted in step b) using a real-time sequencer for a predetermined period of time or until a predetermined number of sequences is obtained; -j) following step i), the sequence (N i SOI ) to the detected target species; -k) Target species (N i SOI ), wherein the amount of sequence associated with the target species is In the first iteration of step k), the amount of sequences assigned to the species of interest detected in step j) is multiplied by the amount of sequences obtained from the last iteration of steps c) to g) (N i’ SOI ) is added; In each iteration of step k), the desired species (n i SOI ) is added to the amount of sequence obtained from the previous iteration of step k); -l) Repeat steps i) to k) until an iteration stopping criterion is reached.

12. The method of claim 11, wherein steps i) through k) are repeated until a predetermined number of iterations is reached.

13. 13. The method of claim 12, wherein steps i) to k) are repeated until a predetermined number of iterations is reached, - during each iteration of steps i) to k), following step k), the method comprises determining a sequencing depth for the biological species of interest, said sequencing depth corresponding to the ratio between the cumulative length of the sequences associated with said species and the length of the genome of said species; - Steps i) to k) are repeated until a predetermined sequencing depth is reached.

14. 12. The method of claim 11, further comprising the step of detecting a sequence representative of the genome of said species of interest, said sequence comprising an antibiotic resistance marker or a virulence marker for said species of interest, following the termination of the repetition of steps i) to k).

15. 3. The method of claim 1 or 2, wherein the sequencer is configured to perform successive sequencing of different sequences of nucleic acid, one after the other, the sequencing of each sequence being considered to be performed in real time.

16. 1. An apparatus for metagenomic analysis of a sample, comprising: - a fluid chamber intended to receive the sample; - a sequencer configured to perform real-time sequencing of the sample; - a processing unit comprising a bioinformatics module programmed to assign sequences obtained from the sequencer to species and a control module programmed to carry out steps c) to h) of the method according to claim 1 or 2.

17. A computer readable or downloadable storage medium containing instructions for carrying out steps c) to h) of the method of claim 1 or 2.

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