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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
  • G16B20/30—Detection of binding sites or motifs
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
  • G16B30/10—Sequence alignment; Homology search
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
  • G16B50/30—Data warehousing; Computing architectures
• • C—CHEMISTRY; METALLURGY
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

• the technical field of the invention is the identification of a biological species of interest by metagenomic analysis.
• PCR Polymerase Chain Reaction
• PCR allows an analysis specific to a biological species, which makes it a selective, sensitive, and possibly quantitative method. However, it assumes an a priori on the target biological species. If several biological species are sought, so-called multiplex PCRs must be carried out, which makes the process more complex.
• RNA gene present in different target biological species.
• this is for example the 16S RNA gene.
• PCR analysis is then said to be broad spectrum.
• broad-spectrum PCR is more difficult to implement, and supposes having an a priori on the target biological species to be identified.
• the targeting of a gene is described in EP2985350 or in the publication Stâmmler F. “Adjusting microbiome profiles for differences in microbial load by spike-in bacteria”, Microbiome (2016) 4:28.
• metagenomics makes it possible to sequence the genomes of several individuals of different biological species in a given medium. We can then determine the species actually present in the sample, as well as their relative abundances. Metagenomics sequences the genomes of several individuals of different species in a given medium, and this without a priori on the biological species in the sample, whether bacterial, viral or human. We then have an analysis of the different genomes of the species biologicals of a sample. We can then determine which species are present, as well as their relative abundances.
• High Throughput sequencing also referred to by the acronym HTS (High Throughput Sequencing).
• HTS High Throughput Sequencing
• bioinformatics allowing rapid computer processing of biological information resulting from sequencing, has improved.
• High throughput sequencing now makes it possible to generate enough sequences to obtain a representative inventory of the different species present in the sample. This is a commercially available analytical method, the use of which is becoming relatively common.
• Document WO2018 / 069430 describes an application of metagenomic analysis for the identification of pathogens as well as markers of resistance to antibiotics.
• the inventor proposes a method for detecting, and optionally quantifying, a biological species of interest, or even different biological species of interest, in a sample, by implementing a metagenomic analysis of the sample.
• the method makes it possible to establish an indicator relating to the correct progress of the biological or bioinformatic steps of the metagenomic process.
• An object of the invention is a method for detecting a biological species of interest potentially present in an analysis sample, the biological species of interest exhibiting a known or partially known genome, the analysis sample comprising a mixture of different biological species, the process comprising the following steps:
• step b) assignment of the sequences resulting from step b) from a base of reference sequences; (ii) determining a quantity of sequences assigned to the biological species of interest; the method being characterized in that it comprises, prior to step b), the addition of a calibrator, the calibrator being a biological species added according to a known concentration, in the analysis sample, the calibrator having a known genome, and in that step c) comprises
• the quantities of sequences respectively assigned to the biological species of interest and to the control biological species are normalized by a reference quantity.
• the reference amount can for example be a total amount of sequences produced during sequencing.
• the method may include taking into account a decision threshold, with which the concentration of the species of interest is intended to be compared.
• the decision threshold is preferably expressed in a unit corresponding to a number of sequences per unit of volume (or of mass), for example in Genome Equivalent per mL.
• the decision threshold may depend on the biological species considered.
• the calibrator has one of the characteristics described below, taken in isolation or according to the technically feasible combinations:
• the calibrator is such that the size of its genome is between 0.1 times to 10 times the size of the genome of the biological species of interest;
• the calibrator has a different genome from that of endogenous organisms
• the concentration of the calibrator is between 0.001 times and 1000 times, and preferably between 0.01 and 100 times the decision threshold taken into account;
• the biological species of interest is a bacterium, the calibrator exhibiting an intact membrane or cell wall;
• the biological species of interest is a virus, the calibrator having a protein envelope; the genome of the calibrator has a number of GC (Guanine-Cytosine) type bases of between 75% and 125% of the number of GC (Guanine-Cytosine) type bases of the genome of the biological species of interest.
• GC Guanine-Cytosine
• Step d) can include:
• the estimation of the concentration of biological species of interest can then include a calculation of a product of the first ratio by the second ratio and by the concentration of the calibrator added to the analysis sample.
• Step d) can include:
• the method may comprise, following step d), a step e) of taking into account the decision threshold and of comparing the concentration resulting from step d) with the decision threshold.
• FIG. 1 schematizes the main steps of a method according to the invention.
• FIG. 2A shows a comparison of quantifications of a biological species of interest, in this case S. aureus, respectively by implementing the steps described below (y-axis) and a reference method (x-axis ), by cultivation.
• FIG. 2B shows a comparison of quantifications of a biological species of interest, in this case S. aureus, respectively by implementing the steps described below (y-axis) and a reference method (x-axis ), by quantitative PCR.
• FIG. 3 shows a statistical distribution of the normalized quantity of sequences, corresponding respectively to different biological species of interest, measured on test samples considered as not comprising said biological species of interest.
• FIG. 4 is a figure showing a comparison between concentrations of biological species of interest respectively estimated by culture (x-axis) and by metagenomic analysis (y-axis).
• the objective of the method is to be able to detect the presence of a biological species of SOI interest in a sample.
• SOI stands for "Species of Interest”.
• the method can allow absolute quantification of the species of interest SOI, so as to allow a comparison with a decision threshold SD.
• biological species it is understood a microorganism, for example a bacterium, or a virus, a fungus, an archaeobacterium, an amoeba, a protist, a microalgae.
• a biological species can also be a cell or any other material or entity comprising a sequenceable nucleic acid.
• the biological species of interest When the sample is obtained from a human or animal organism, the biological species of interest may be a pathogenic species. When the sample is taken from an industrial process or from the environment, the biological species of interest may be a species considered to be a contaminant, or a species of interest having an importance in an industrial process or in the environment, and whose presence or concentration is to be controlled.
• the species of interest has a known or partially known genome.
• the genome, or its known portion is made up of sequences, called sequences of interest.
• the method can simultaneously address several species of interest. Also, the term a species of interest is to be interpreted as meaning at least one species of interest.
• the decision threshold SD is a threshold making it possible to characterize a load of the biological species of interest, for example a microorganism, as a function of the targeted application. It is for example established on the basis of a regulatory, health or industrial limit.
• the decision threshold can be a concentration below which the presence of the bacterium corresponds to colonization, that is to say a non-pathological development, and beyond which the presence of the bacteria is considered to be pathological, corresponding for example to an infection.
• the detection threshold corresponds to a compliance value, such that beyond the detection threshold, the sample is considered as non-compliant, and that below the detection threshold, the sample is considered to be compliant.
• the concentration of the biological species of interest is greater than or equal to the decision threshold, it is defined as being critical. In certain applications, for example in the manufacture of products undergoing fermentation, a concentration of biological species of interest may be considered critical if it is below a decision threshold, the latter corresponding to a minimum admissible concentration of the biological species.
• the sample is generally a sample taken from the environment or from an organism, dead or alive, or even from an agri-food or manufactured product.
• the sample may also have been taken at an industrial facility for process control purposes.
• the sample contains different biological species, not having the same genome.
• the sample results from a sample from an organism, for example a human or animal organism, the sample contains a significant amount, or even a majority, of cells originating from the organism taken.
• the genomes of human or animal organisms are 1000 to 100,000 times larger in size than the genomes of prokaryotic organisms.
• the sample generally comprises biological species naturally present in the sample, and not likely to cause pathology or critical contamination.
• the sample when the sample is a bronchoalveolar sample, it contains a bacterial flora naturally present in the lungs. When the sample is a stool sample, it contains bacterial flora naturally present in the digestive tract.
• the biological species of interest when the biological species of interest is a bacterium or a virus, the nucleic acids originating from the biological species of interest may be in the minority in the sample.
• the sample comprises so-called "matrix" species, endogenous in the sample, and capable of masking the metagenomic information relating to the biological species of interest.
• matrix species representative of these media.
• the matrix contains the cells making up the organism.
• the sample undergoes extraction of nucleic acids (DNA and / or RNA), followed by a sequencing process, according to the principles of metagenomic analysis.
• the sequencing process can be preceded by an amplification process.
• the sequencing can be a complete sequencing of the genome, usually designated by the term “whole genome sequencing” (WGS), in particular a complete sequencing of the shotgun type.
• WGS whole genome sequencing
• An inventory of gene sequences of the different species constituting the sample is thus obtained.
• All, or almost all, of the nucleic acid of the different species constituting the sample is sequenced, by a high throughput sequencing method.
• Bioinformatic means then make it possible to identify sequences of interest associated with the biological species of interest, and to determine a quantity thereof, generally a standardized quantity, as described below.
• the computer resources are based on a database of reference sequences, for example complete reference genomes within the framework of a WGS-type process mentioned above.
• the database comprises at least the genomes, total or partial, of the biological species of interest potentially present in the sample. It also comprises the genome, total or partial, of a so-called control biological species, the latter being described below.
• a genomic description of the different species constituting the sample is obtained by sequencing.
• the sequences corresponding to the biological species of interest and those corresponding to the control species are then identified from among the inventoried genomic sequences.
• the method comprises the steps described below, in connection with FIG. 1.
• Step 10 sample collection.
• the sample is taken from a living human organism, for diagnostic aid.
• the invention is not limited to an application in the field of living organisms.
• the sample can be taken in an industrial or hospital environment, so as to verify compliance with a decision threshold.
• Step 20 adding a kind of control.
• One of the objectives of the invention is to evaluate to what extent a metagenomic analysis is exploitable. In particular, this involves evaluating the compliance of all the steps from sample preparation, excluding sampling, to the bioinformatic analysis of the sequencing data.
• SPC a species of control
• One function of the control species is to allow control of the correct progress of the nucleic acid extraction and sequencing steps, described below.
• the SPC control species may be a known biological species, the genome of which is also known, preferably in its entirety.
• the SPC control species can be a naturally occurring biological species. It can also be an artificial species, for example an RNA (ribonucleic acid) packaged.
• the SPC control species is not initially present in the collected sample, or in a negligible amount.
• the content of SPC control species initially present in the sample that is to say present before the addition, and preferably at least 10 times lower, or preferably at least 100 or 1000 times lower than the added concentration C S PC of the SPC control species in the sample.
• the SPC control species can for example be a bacterium. It is important that the concentration of the added control species is controlled.
• control species can be chosen taking into account the aspects listed below:
• control species must preferably be distinguished from the organisms naturally present in the sample, or endogenous organisms, as well as from the species of interest sought: thus, the bioinformatic tool can accurately identify the sequences resulting from the SPC sequencing.
• control species is preferably detectable by high throughput sequencing, while not being predominant in the sample.
• control species be such that :
• the size of its genome is preferably similar, or at least comparable, to the size of the genome of the biological species of interest. More particularly, the size of the genome of the control species is between 0.1 times to 10 times the size of the genome of the biological species of interest.
• the CSPC concentration of the control species can be determined based on the decision threshold.
• the CSPC concentration of the added SPC control species may for example be between 0.001 times and 1000 times, and preferably between 0.01 and 100 times the decision threshold.
• the nucleic acids of the control species SPC undergo a treatment similar to the nucleic acids of the species of interest during the steps of sample preparation, extraction and sequencing, and preferably:
• the percentage of GC bases is preferably close to the percentage of GC base of the biological species of interest; By close to is meant between 75% and 125%, and preferably between 80% and 120%.
• control biological species preferably comprises, when the biological species of interest is a bacterium, a membrane or an integral cell wall or, when the biological species of interest is a virus, an envelope protein. This condition also makes it possible to monitor the lysis steps or the extraction of nucleic acids from the biological species of interest.
• the nucleotide sequences of the control species do not preferably contain genomic markers, such as for example markers of resistance to antibiotics, markers of virulence, so as not to distort the results of a possible antibiotic sensitivity test by the presence of such markers in the genome of the biological species of interest.
• the nucleotide sequences of the control species do not contain any other gene of clinical or industrial interest and the presence of which can be controlled.
• control species is preferably easy to handle, in particular:
• control species must not form spores, or only marginally.
• control species must exhibit a sensitivity to lysis close to that of the biological species of interest.
• control species is present in the form of beads, each bead comprising a calibrated concentration of control biological species in lyophilized form.
• control species can be used, or that several species of control, of different types, can be used.
• Different biological control species can be used for the same biological species of interest.
• the control species forms a calibrator.
• a calibrator different from the control species, is added to the sample. The calibrator allows an estimate of the concentration of the species of interest.
• the added CSPC concentration of the SPC control species is preferably precisely known. Indeed, it can make it possible, subject to certain conditions being met, to quantify the concentration of biological species of interest in the sample, the control species then forming a calibrator.
• the term added concentration refers to the concentration of the control species in the sample due to the addition of the control species.
• control species fulfills the control function quality of the steps of the metagenomic analysis, as well as the function of calibrator, allowing a quantification of the concentration of the biological species of interest.
• step 20 there is an added concentration CSPC of the control species in the sample.
• the added concentration C S PC can be expressed in GEq / mL (equivalent genome per mL).
• Step 30 lysis and extraction of nucleic acids.
• the cells of the sample and in particular the cells of the biological species of interest and of the control species, undergo lysis, to allow extraction of their DNA.
• Different strategies can be considered:
• lysis can be configured to preferentially target the biological species of interest; the control species must exhibit the same sensitivity to lysis as the biological species of interest, or a sensitivity to lysis considered to be equivalent.
• the lysis can include a first lysis, intended to essentially lyse cells other than the species of interest.
• a first lysis can for example be envisaged when the biological species of interest is very much in the minority compared to the cells of a matrix making up the sample.
• the released nucleic acids are evacuated, then a second lysis is carried out, targeting the biological species of interest.
• the control species is preferably resistant to the first lysis, and not resistant to the second lysis.
• the DNA is extracted from the sample, for example according to the extraction method described in WO2014 / 114896.
• the DNA extracted from the sample can consist essentially of the DNA of the matrix, that is to say of the environment from which the sample was taken.
• the sample can undergo selective capture and / or amplification, mainly targeting sequences and / or specific physicochemical modifications of the genomes of the biological species of interest.
• the control species comprises the sequences and / the physicochemical modifications targeted by the selective capture or amplification.
• the sample may undergo depletion primarily targeting the DNA of the template. In this case, the control species does not contain sequences or physicochemical modifications which may be targeted by the depletion.
• Step 40 Amplification and sequencing.
• the DNA fragments optionally undergo amplification which may be of the targeted type, for example by PCR (Polymerase Chain Reaction), or non-targeted, for example by WGA (Whole Genome Amplification).
• WGS Whole Genome Sequencing
• SBS sequencing by synthesis
• nanopore nanopore
• the aim of sequencing is to provide digital sequences of nucleic acids, called reads.
• Sequencing comprises a preparation of libraries of sequences (or library preparation), optionally followed by an amplification step, then by a sequencing step proper.
• the amplification and the sequencing can be implemented by the MiSeq platform, marketed by the company Illumina.
• the DNA can be chopped up at random, so as to obtain nucleic acid sequences of a targeted average length, generally an average length between 50 bases and 300 bases.
• a targeted average length generally an average length between 50 bases and 300 bases.
• WGS Whole Genome Sequencing
• sequencer reads the bases of the sequenced DNA fragments, so as to obtain so-called “reads” sequences, each “read” corresponding to a sequence decoded by the sequencer.
• sequences resulting from the sequencing are then aligned with respect to genomes stored in a database, including in particular the genome of the biological species of interest sought and the genome of the control species. Sequencing is an operation known to those skilled in the art. Details relating to the sequencing operations are given for example in the documents cited in connection with the prior art, in particular WO2018 / 069430 or in the publication Rupfug E cited above.
• the sequencer transmits files corresponding to the measurements carried out including the "reads" to a data processing unit.
• the latter comprises a memory, in which are stored instructions allowing the implementation of sequencing algorithms.
• the sequencing algorithms make it possible to identify, for each sequence, the genome comprising the sequence, from among a plurality of genomes stored in a database. They also make it possible to establish the position of each sequence on the genome to which it belongs, and to carry out assemblies between the different sequences belonging to the same genome.
• sequencing data relating to the different biological species of the sample is available. It is in particular an identification of each species and a quantity of sequences assigned to each identified species. In particular, there is a number of R S oi sequences assigned to the biological species of interest and a number of R S P C sequences assigned to the control species.
• Step 45 Identification of the species to which the reads belong.
• this step implemented by the data processing unit, the origin of each of the reads is identified, in terms of bacterial species.
• This step generally known as “binning”, “taxonomic binning”, or “assignment”, comprises the comparison of each of the reads with digital sequences of nucleic acids from a reference database.
• binning software are for example Kraken, (Wood and Salzberg, “Kraken: ultrafast metagenomic sequence classification using exact alignments", Genome Biology, 2014), or “Wowpal Wabbit” (Vervier et al., “Large-scale machine learning for metagenomics sequence classification ", Bioinformatics, 2015), or” BWA-MEM “(Li,” Aligning sequence reads, clone sequences and assembly contigs with BWA-M EM “, Genomics, 2013).
• a read is assigned to a species of interest if it is entirely included in a genome representative of the species of interest stored in the database.
• the amounts of sequencing data resulting from step 45 do not have the same size for each of the samples.
• the number of sequences generated by the sequencing depends on the quality and the quantity of DNA of the different biological species composing the sample. It is therefore preferable, or even necessary, to standardize the quantity of sequences associated with a species relative to a reference quantity. Normalization depends on the type of sample analyzed and the metagenomic analysis applied.
• the reference quantity may for example be a total number of sequences produced for the sample analyzed.
• the standardized amount of sequences associated with each species, divided by the reference amount is usually multiplied by 1 E 6 so as to obtain a standardized amount corresponding to an amount per million of sequence (or RPM, acronym for Read Per Million).
• the reference quantity may be, without being exhaustive:
• a total number of sequences associated with all identified microorganisms a total number of sequences associated with an organism from which the sample is extracted: for example, when the organism is a human body, a total number of sequences associated with the human genome can be determined;
• reference species it is meant an endogenous or exogenous species which is considered to be present constantly in different samples taken.
• the reference species can be the control species.
• Step 50 is carried out for the biological species of interest (or for each biological species of interest), as well as for the control species (or for each SPC control species or for each calibrator).
• the biological species of interest SOI or for each biological species of interest
• the control species SPC or for each control species or for each calibrator
• N denotes the fact that the quantity is standardized.
• the term quantity can denote a standardized quantity.
• Step 60 Interpretation.
• This step is an important step of the invention. It is a question of knowing to what extent the results of the sequencing are interpretable.
• the method comprises a determination of a confidence level that can be attributed to the preceding steps, and in particular the steps 30 to 50 described above.
• the level of confidence is assigned thanks to the type of control, and in particular thanks to the fact that the species of control has been introduced beforehand at step 30.
• This step uses detection thresholds DTsoi and DTSPC, respectively associated with the biological species of interest SOI and with the control species SPC.
• the detection thresholds can be established on the basis of statistical detection thresholds respectively determined for the biological species of interest and the control species.
• the statistical detection thresholds are established beforehand, during a step 100 described later.
• a statistical detection threshold corresponds to the most low value of an analyte concentration measured by a detection method, which is statistically different from that measured, under the same conditions, when the analyte is absent from the sample.
• Each detection threshold can be equal to the statistical detection threshold, or be determined from the statistical detection threshold, in particular being k times equal to the statistical detection threshold, k being a non-zero real.
• the interpretation aims to compare the standardized quantities of RN S oi and RN S P C sequences, respectively assigned to the biological species of interest SOI and to the control species SPC, with their respective detection thresholds.
• the biological species of interest can be considered to be detected with an acceptable level of confidence when the standardized quantity of sequences assigned to the biological species of interest is greater than or equal to the detection threshold associated with it.
• the kind of control Based on the comparison, four situations can be distinguished:
• RNsoi 3 DTsoi and the level of confidence are considered sufficient, the respective detections of the biological species of interest and of the control species are confirmed.
• the SOI species of interest is considered to be present in the sample, with a sufficient level of confidence. Its Csoi concentration can be estimated, from:
• step 45 the number of sequences (or the number of normalized sequences), assigned to the biological species of interest, resulting from step 45;
• Lsoi are respectively the genome lengths of the control species and of the biological species of interest.
• a is a correction factor determined empirically, on the basis of training samples for which the concentration of the biological species of interest is known.
• the concentration of the biological species of interest is also expressed in the same unit.
• the sequencing comprises an assembly of the sequences respectively associated with the control species and with the biological species of interest, as well as a determination of a coverage rate Cov of the assemblies for each of the species.
• concentration C S oi of the biological species of interest can then be calculated according to the following equation:
• Covsoi are respectively the coverage rates determined for the control species and the biological species of interest.
• the coverage rate is usually designated by the Anglo-Saxon term “Coverage” and expresses an average number of times a base is sequenced at a given position of the genome, as described in the publication Lacoste C et al. “High throughput DNA sequencing in clinical practice”, Archives de Pediatrie 2017; 24: 373- 383.
• a 1 is a correction factor determined empirically, on the basis of training samples for which the concentration of the biological species of interest is known.
• step 61 can be carried out with a biological species, different from the control species, and forming a calibrator.
• a control species is used during step 60, to confirm the detection of the biological species of interest
• step 61 that is to say the quantification
• the characteristics of the calibrator are similar to those of the control species, and correspond to the characteristics described in connection with step 20.
• the quantification, using the calibrator can be carried out using the expression ( 1) or the expression (). Expression (1) becomes:
• Rc AL is the number of sequences, preferably normalized, assigned to the calibrator
• L CAi _ is the length of the calibrator genome
• C CA L is the concentration of calibrator added to the sample
• a is a correction factor as described in connection with (1).
• no control species is used.
• a calibrator is used, and the concentration of the biological species of interest is implemented from the number of sequences, preferably normalized,
• This step involves a comparison of the added concentration the type of control and the decision threshold SD, so that:
• Step 63 the concentration of biological species of interest cannot be estimated, but it can be considered to be above the decision threshold. Without being able to quantify the concentration of the biological species of interest, it is possible to conclude that the decision threshold has been crossed. Step 63
• the step comprises an estimate of a minimum detectable concentration of the biological species of interest.
• the minimum detectable concentration Cmin S oi of the biological species of interest corresponds to the lowest concentration that can be distinguished from the background noise, It is assimilated to the concentration, in genome equivalent, corresponding to the detection threshold DTsoi of the biological species of interest.
• the minimum detectable concentration can be determined from:
• Lsoi are respectively the genome lengths of the control species SPC and of the biological species of interest SOL
• a is the correction factor described in connection with equation (1).
• Step 63 comprises a comparison of the decision threshold SD with the minimum detectable concentration Cminsoi such that:
• the detection of the biological species of interest can be considered negative: the concentration of biological species of interest in the sample is less than or equal to the decision threshold.
• the sample comprises at least one species at a high concentration, and generating a high number of sequences, which produces a masking effect of the other sequences of interest.
• the confirmation of the presence of the biological species of interest, at a concentration above the decision threshold, and its possible quantification, are used as a diagnostic aid.
• the SPC control species provides both a function of checking the conformity of the metagenomic analysis as well as a function of calibrator, allowing quantification of the biological species of interest in sample.
• an SPC control species and a calibrator, different from the control species are added to the sample. These are for example two different bacterial species.
• the SPC control species performs a function of checking the conformity of the metagenomic analysis.
• the calibrator allows quantification of the biological species of interest in the sample, according to equations (1) or () or (2).
• the calibrator preferably has the same characteristics as the control species, the latter being described in connection with step 20.
• the SPC control species is added to a first concentration. It is assigned a detection threshold and step 60 is implemented by comparing a quantity of normalized sequences assigned to the control species, resulting from step 50, with the detection threshold associated with the control species.
• the calibrator is also added to the sample, at a second concentration.
• a detection threshold is assigned to it.
• the quantification can be carried out by taking into account a standardized quantity of sequences associated with the calibrator, as well as the detection threshold associated with it.
• the calibrator can be added prior to lysis or following lysis and prior to sequencing.
• several calibrators are added to the sample, each calibrator being chosen for one or more species of interest.
• groups of bacterial species may react in a significantly different way to the processes of nucleic acid extraction, for example Gram + bacteria and Gram- bacteria.
• a calibrator consisting of a Gram + bacteria is added. when one or more species of interest are Gram + and a calibrator consisting of a Gram bacterium - when one or more species of interest are Grams -
• the species of interest may consist of bacteria and viruses.
• a first calibrator is bacterial and a second calibrator is viral helper is viral.
• Step 100 Establishment of detection thresholds.
• control species and the biological species of interest are respectively associated with detection thresholds.
• the detection threshold is established prior to the interpretation of the results, using training samples, not including said species. These are negative samples relative to the species considered. These samples are representative of the sample analyzed. By representative, it is meant that these training samples comprise a population of biological species comparable to that of the sample analyzed, both from a qualitative and quantitative point of view. The absence of a biological species of interest and / or of the control species in each test sample can be verified by a standard method of culture and / or PCR type.
• sequencing is carried out, preferably under the same conditions as described in connection with steps 30 to 45.
• a quantity of sequences assigned to the species considered is determined. This amount is preferably normalized, as described in connection with step 50.
• the detection thresholds respectively associated with the biological species of interest and with the control species by using respectively first training samples, not comprising the biological species of interest, and second training samples, not including the control species.
• the first training samples can be confused with the second training samples, in which case the associated detection thresholds the biological species of interest and the control species are determined with the same training samples.
• Sequencing is preferably performed on a statistically representative number of training samples. We thus obtain a statistical distribution of the normalized quantity of sequences. We then estimate a mean m of the distribution, as well as an indicator of dispersion, for example the standard deviation s or the variance s 2 .
• the detection threshold is estimated by adding, to the average m, n times the dispersion indicator, n being a real n is typically between 2 and 4.
• the detection thresholds respectively associated with the biological species of interest and with the control species are intended to be compared with the quantities of standardized sequences of the biological species of interest and of the control species, it is important that the normalization carried out during step 100 is similar to the normalization carried out during step 50.
• the steps described above can be carried out by simultaneously targeting several biological species of interest. This is also a notable advantage of metagenomic analysis, which allows different biological species to be addressed simultaneously. Another advantage of metagenomic analysis is the possibility of using several control species simultaneously. Thus, one control species can be used to target one or more biological species, while another control species can be used to target other biological species of interest. This is another advantage of metagenomic analysis.
• steps 61 to 64 can be implemented by using, for the same biological species of interest, different control species. This makes it possible to limit the risks of failure of the process, following a malfunction in the sequencing of a control species.
• an estimate of the presence of the biological species of interest in relation to the decision threshold is available.
• several quantifications can be obtained, according to equations (1), () in which case we can consider the mean or the median of the quantifications obtained, or the quantification considered as the most penalizing, that is to say that resulting in the highest concentration of biological species of interest or, more generally, closest to the decision threshold.
• Steps 61 to 64 are implemented by a computing unit, for example of the microprocessor type, from the sequencing data coming from steps 40, 45 and 50 and supplied by the processing unit.
• the sequencing data which correspond to data measured from the analysis sample, are thus transmitted, by wire or wireless connection, from the computing unit so as to execute one of steps 61 to 64.
• the microprocessor is linked to a memory comprising instructions for implementing steps 61 to 64.
• Bacillus subtilis was a good candidate to be used as a control species for the metagenomic sequencing of samples resulting from bronchoalveolar lavage (BAL) performed on human patients. It is known that this type of sample is likely to contain a large amount of human DNA originating from the patient.
• BAL bronchoalveolar lavage
• the metagenomic sequencing of such samples can help in the diagnosis of pneumonia acquired in a hospital environment, for diagnostic aid.
• the clinical decision threshold is established at 1.0 E4 CFU / mL, the acronym CFU standing for Colony Forming Unit.
• the analysis protocol includes removal of the DNA from the patient during a prior lysis.
• the sample was treated with a lysing agent specifically targeting the cells of the patient.
• a lysing agent is for example described in WO2014 / 114896.
• the released DNA was then removed by enzymatic action and washing.
• the sample was then subjected to a second mechanical and chemical lysis so as to extract the bacterial DNA.
• the protocol Prior to the lysis steps, the protocol provides for the addition of a species of control in the sample.
• the biological species forming the control species must be resistant to lysis of human cells, while being sensitive to lysis of bacterial cells.
• certain bacteria in particular Gram-positive bacteria, are difficult to lyse.
• the metagenomic sequencing carried out aims to detect and possibly quantify about 20 biological species of interest, each species of interest being a bacterium included in the following list: Acinetobacter baumannii, Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Haemophilus influenzae, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumophila, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia stuartii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcus pneumoniae.
• the SPC control species must also be able to be sequenced with an efficiency comparable to the species of interest listed above.
• the sequencing efficiency depends essentially on the size of the genome as well as the GC content. (Guanine - Cytosine).
• the control species had to have a genome size of between 1.9 and 6.6 megabases, as well as a GC content of between 33% and 66%.
• the concentration of the control species, added to the sample was set at 1.0 E4 CFU / mL, ie a concentration comparable to the decision threshold mentioned above.
• Bacillus subtilis exhibited the characteristics required to be used as a control species.
• the Bacillus subtilis genome size is 4.12 Mb (megabases) and has a GC content of 43.6%.
• Bacillus subtilis is commercially available in the form of "BioBalls" type beads (registered trademark) - Biomérieux manufacturer. These are water-soluble beads containing a calibrated concentration of Bacillus subtilis, which makes it possible to adjust the concentration of the added control species.
• Bacillus subtilis is a relevant biological species to form a control species, in a sample obtained by BAL, and with the analysis protocol described at the beginning of the example.
• This example describes the detection and quantification of Staphylococcus aureus in a sample taken by bronchoalveolar lavage (BAL) by applying the double lysis protocol described in Example 1 and steps 10 to 50 described above.
• Example 2 A cohort of 13 samples from BAL was used. Following the conclusions of Example 1, the control species used was Bacillus subtilis, added to each sample at a concentration close to the decision threshold (1.0 E4 CFU / mL). In this example, the control species was obtained by rehydration of a Bioball Multishot 10 E 8 - Bacillus subtilis ATCC 19659 (Biomérieux), in 1.1 mL of PBS buffer (Saline Phosphate Buffer). The control species was diluted to 1.0 E6 CFU / mL in PBS and 10 ⁇ L are added to 600 ⁇ L of sample. An added concentration of the control species of 1.7 E4 CFU / mL is thus obtained.
• the control species used was Bacillus subtilis, added to each sample at a concentration close to the decision threshold (1.0 E4 CFU / mL).
• the control species was obtained by rehydration of a Bioball Multishot 10 E 8 - Bacillus subtilis ATCC 19659 (Biomérieux),
• each sample was processed within a maximum of 48 hours after collection. As previously indicated, each sample underwent a first lysis specific to human cells. Unlyysed cells were pelletized and treated with DNAse I. Before extraction of human DNA, DNAse was deactivated by heating and adding EDTA (Ethylenediaminetetraacetic). Each sample was then subjected to a second lysis, by being added to a lysis tube containing a mixture of glass beads of 1 mm diameter and of Zr / Si beads of 0.1 mm diameter. Lysis is obtained by stirring for 20 minutes. DNA was extracted from the lysate using the easyMAG (trademark) Biomérieux platform. Elution was carried out in a volume of 25 ⁇ L. The extracts were stored at -20 ° C.
• the sequencing libraries were prepared in 2x250 paired-end with the Nextera (registered trademark) XT DNA Library preparation kit (manufacturer Illumina).
• the samples were sequenced using the MiSeq platform (registered trademark) with the "MiSeq reagent kit V3" kit (Illumina).
• the sequences were processed with a processing unit using the KRAKEN VO 10.5b software using an internal sequence database.
• This database comprises, in particular, the sequences of the human genome as well as the sequences of 20 biological species of interest described in Example 1.
• the number of sequences produced on each sample varied between 331,000 and 17,000,000. numbers of sequences associated with the control biological species (Bacillus subtilis) and with the biological species of interest (S. Aureus) were normalized to reads per million (RPM).
• Table 1 shows the sequencing results for 13 culture positive samples. Columns 1 to 7 correspond respectively:
• the SPC control species acts as a calibrator, in the sense that it is used during the quantification step.
• SOI NA and SPC NA correspond respectively to the fact that the number of sequences associated with the biological species of interest SOI and with the control species SPC are not sufficient to allow assembly.
• NA stands for Non-Assembled.
• Samples 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12 and 13 correspond to the configuration described in connection with step 61, in which a quantification of the species of interest is possible, for example according to expression (1) and expression ().
• Sample 8 corresponds to the configuration described in connection with step 64: the results cannot be interpreted. Additional investigations have shown, for this sample, a failure of the sequence demultiplexing step. This scenario is interesting because it shows that taking into account the type of control makes it possible to avoid the production of a "false negative" result.
• concentration Csoi was estimated by equation ().
• the sequences associated with the control species SPC or with the biological species of interest SOI are sometimes not assemblable; in this case, the biological species of interest is not quantifiable according to this protocol, whereas it is by using equation (1).
• FIG. 2A shows a comparison of the quantification of S. aureus by culture (x-axis) and by sequencing (y-axis).
• FIG. 2B shows a correlation between the results of quantification by metasequencing (equation (1) - y-axis) and by quantitative PCR (x-axis).
• the detection of the 20 pathogenic bacterial species of interest was tested on samples obtained from bronchoalveolar washings (BAL) or mini bronchoalveolar washings (mini BAL). .
• the SPC control species (B. subtilis) is obtained identically to Example 2, the concentration added to each sample being 1.7 E4 CFU / mL.
• the decision threshold is 1.0 E4 CFU / mL for BAL samples, and 1.0 E3 CFU / mL for mini BAL samples.
• Two sample cohorts were collected: a training cohort, comprising 46 samples (23 BAL and 23 mini-BAL) and an analysis cohort, comprising 40 samples (33 BAL and 7 mini-BAL).
• Example 2 The sample underwent a double lysis, as described in connection with Example 2. The sequencing was carried out as described in Example 2.
• the quantity of sequences was normalized in reads per million reads associated with bacterial species (RPMb), cf. step 50.
• the detection threshold DTsoi was determined by considering only the training samples for which the biological species of interest is considered as not detected. The species of interest is considered as not detected in a sample, when the result of microbiological culture of the sample is negative for the detection of the SOI in question and negative for the detection of MetaPhlAn marker sequences specific for the SOI in question.
• Figure 3 shows the statistical distributions of the amount of sequence, normalized, on negative training samples relative to the species of interest.
• the x-axis corresponds to each species of interest, while the y-axis corresponds to the normalized quantity of sequences associated with the species of interest.
• the median value line included in the rectangle
• the 25% and 75% fractiles limits of the rectangle
• the ends of each vertical line correspond to the 1% and 99% fractiles. It is observed that the distributions are very variable from one another, which justifies that a detection threshold DTsoi be established for each biological species of interest.
• a detection threshold DTsoi was determined, according to step 100 previously described. If psoi denotes the average of the normalized number of sequences assigned to the species of interest, and s £ oi is their standard deviation, the detection threshold DTsoi is obtained "at 3 sigmas", according to the expression:
• the detection threshold DT S P C DT S.
• Subt m s associated with B. subtilis has been defined. We took into account 7 training samples without addition of B. subtilis. The mean p s was determined . subt m s of the normalized number of sequences assigned to B. subtilis, as well as their standard deviation s subt s The detection threshold 0 ⁇ B. subtms is such that:
• SD decision threshold
• the “Infection” population corresponds to the 20 occurrences detected by culture at concentrations equal to or greater than the clinical thresholds, namely 1.0 E3 CFU / mL for the miniBAL samples and 1.0 E4 CFU / mL for the BAL samples.
• the “Colonization” population corresponds to the 900 occurrences not detected by culture or detected by culture at concentrations below the clinical thresholds, namely 1.0 E3 CFU / mL for the miniBAL samples and 1.0 E4 CFU / mL for the BAL samples
• the 920 occurrences correspond to the analyzes, by microculture, of the 46 training samples, considering respectively the 20 biological species of interest.
• FIG. 4 represents, for different samples, quantifications of biological species carried out by culture (abscissa axis) and by metagenomic analysis (ordinate axis).
• the black circles correspond to a species chosen from Acinetobacter baumannii, Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Escherichia coli, Haemophilus influenzae, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumoniaii, Legionella pneumoniaii, Legionella pneumoniaii, Morganeus mirabilisophila, Proteus vulgaris, Providencia stuartii, Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia and Streptococcus pneumoniae. Clear triangles correspond to Staphylococcus aureus.
• Figure 4 shows that for a species of interest, or for a group of species of interest, the “Colonization” and “Infection” populations can also be differentiated from the results of quantification by genome equivalent sequencing (GEq).
• the metagenomic threshold (SD) is defined by taking into account the first half percentile of the concentrations measured on the “Infection” population, the value thus obtained is 5.5 E 3 GEq / mL.
• a metagenomic threshold forming a decision threshold SD, making it possible to separate respectively the samples whose concentration in the biological species of interest is located below or beyond a critical value.
• the critical value can in particular correspond to the decision threshold SD described above.
• the concentration of a species of interest, determined by sequencing, is then compared to the decision threshold associated with it.
• the decision threshold generally depends on the biological species considered. It is then possible to establish a decision threshold for a biological species considered or for a group of biological species. Two different biological species can be associated with two different decision thresholds.
• Tables 2A to 2C collate the results obtained, each table respectively collating the results of samples 1 to 13, 14 to 27 and 28 to 40.
• the first line of each table contains the references of each sample.
• the second line represents the detection (+) or non-detection (-) of the control species SPC compared to the detection threshold which is associated with it: cf. step 60.
• TP True Positive - Vrai Positif
• FN False Negative
• the metagenomic analysis allowed the detection of 19 additional occurrences, compared to the microbiological culture. These occurrences are designated by FP (False Positive - False Positif) or FP + in Tables 2A to 2C.
• the 5 FP + occurrences correspond to detections for which MetaPhlAn markers and BLAST alignments (acronym for Basic Local Alignment Search Tool) make it possible to confirm the presence of the species of interest in the sample, despite its non-detection by culture.
• MetaPhlAn markers and BLAST alignments ascronym for Basic Local Alignment Search Tool
• the FP occurrences correspond to false positives for which the number of reads associated with the species of interest are too low for confirmation by the search for MetaPhlAn markers and BLAST alignments. These additional occurrences are also probably due to a better sensitivity of the metagenomic test compared to detection by microbiological culture, however the absence of confirmation does not allow to exclude a defect of specificity of the metagenomic test.
• the control of the metagenomic test makes it possible to limit the risk of false negatives, this situation is clearly illustrated by the non detection of E. cloacae in sample 27.
• a particular application of the invention to so-called “shotgun” sequences has been described.
• the invention also applies to targeted sequences, for example to so-called 16S sequences.
• a step of amplifying the targeted genes is implemented in order to multiply their copies in the sample.
• the reads used by the invention are then the reads corresponding only to the targeted genes.
• Bacillus subtilis as a control species has been described during a metagenomic analysis of BAL or mini-BAL type samples.
• another control species can be used, provided that it satisfies all or part of the criteria described in connection with step 20. It can for example be a species chosen from: Bacillus stearothermophilus , Synechocystis sp. PCC6803, Pelagibacter ubique, Methanocaldococcus jannaschii, Aeropyrum pernix, Kocuria rhizophila, Azospirillum lipoferum, Lactococcus lactis, Synechococcus sp. WH 7805, Schizosaccharomyces pombe, Pantoea stewartii, Phage T4, Pichia pastoris, Armored DNA Quant TM.
• control species have been described in the form of elements comprising nucleic acids included in or encapsulated in membranes (bacterial membrane, capsid, etc.). This characteristic is used for the conformance control function of the metagenomic analysis, in particular to know if the nucleic acid extraction process has worked as expected.
• the calibrator can consist of acids free nucleic acids added to the sample or to the DNA extract in known quantity.
• control and calibration species all at once, namely before the step of extracting the nucleic sequences.
• the calibrators can be added at a later stage, preferably after the sample lysis step in the case of naked nucleic acids in order to avoid the destruction of the latter.
• the method according to the invention makes it possible in particular to assay the biological species of interest in a sample.
• the method according to the invention is supplemented by a step of determining an antibiotic therapy according to the species identified and assayed in the sample, and of administration of the determined antibiotics. to the patient.
• the method makes it possible to aid in the diagnosis of contamination of a sample by a species of interest, the latter possibly being a bacterium or a fungus.
• a species of interest possibly being a bacterium or a fungus.
• This allows a definition of an appropriate treatment (antibiotic in the case of a bacterium, antifungal in the case of a yeast or a fungus), on the basis of the identity of the species of interest, but also on the basis of any marks of antimicrobial resistance detected in the genome.
• the concentration of the biological species when the concentration of the biological species is greater than the decision threshold, this can be considered to signify the occurrence of an anomaly.
• Appropriate remedial treatment is decided, aimed at remedying the anomaly.
• the species of interest in the agri-food sector, can be a bacterium.
• the remedial treatment may be a removal or destruction of food products intended for sale, and / or cleaning of a production facility.
• the application relates to health control, for example health control of an installation, for example part of a hospital, so as to prevent nosocomial infections.
• the proven presence of an undesirable biological species leads to a remedial treatment such as cleaning or decontamination.
• the invention can be implemented in the field of health, as a diagnostic aid, or, more generally, in the field of the analysis of samples taken from the environment, or in industrial processes, for example the food industry, industry, pharmaceutical or cosmetics industry. It can also be implemented in health control.

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