High-throughput methods for identifying and recovering gene clusters of interest

Abstract

The present invention relates to methods for identifying and recovering a gene cluster of interest.

Description

Technical Field

[0001] This invention relates to methods for identifying / locating target gene clusters. In particular, this invention relates to the discovery of novel biosynthetic gene clusters (BGCs) in prokaryotic and eukaryotic cells.

[0002] Therefore, this invention is applicable to screening clone libraries.

[0003] Accordingly, the present invention relates to methods compatible with high-throughput research, and these methods are compatible with the economical and efficient detection of multiple gene clusters. Background Technology

[0004] There is a general need to identify new products, especially secondary metabolites, from host organisms.

[0005] Such metabolites may include clinically valuable molecules, such as antimicrobial and antifungal products, especially in the context of antimicrobial resistance, as reported by Lewis (“The Science of Antibiotic Discovery”; Cell181, 2020).

[0006] However, characterizing and isolating such metabolites is also challenging.

[0007] Such metabolites are typically produced by the enzymatic biosynthesis of the products of more than one gene, which are usually clustered into gene clusters.

[0008] Furthermore, such gene clusters may not be easily expressed under laboratory conditions. Alternatively, when cloned into microorganisms belonging to different species or even different genera, such gene clusters may be expressed differentially.

[0009] When the corresponding host is cultured under different conditions, such gene clusters may also be expressed differentially.

[0010] In addition, such gene clusters include multiple genes, resulting in high molecular weight nucleic acids that may be difficult to clone, expensive to synthesize, or expensive to sequence in an unbiased manner.

[0011] Therefore, there is a need for new methods that can identify, clone, and recover such gene clusters in an efficient and cost-effective manner, especially in microorganisms.

[0012] One strategy involves cloning the entire genome, which may contain gene clusters, as a random genomic fragment library, and then screening the clones to locate and recover the target fragment.

[0013] Screening cloning libraries to locate a small number of target clones remains a time-consuming and resource-intensive step. This is typically accomplished, for example, by using PCR strategies and primers targeting conserved regions of such clusters. The amplicon can then be sequenced to provide additional insights into the properties of the amplified material, which can be used for deduplication.

[0014] On the other hand, performing PCR on each individual clone would be impractical. Therefore, it has been recommended to reduce the number of PCR steps required to locate clones in a given clone library.

[0015] Owen et al. (“Mapping gene clusters within arrayed metagenomic libraries to expand the structural diversity of biomedically relevant natural products”; PNAS, vol. 110, no. 29, 2013) taught multiplex sequencing of barcoded PCR amplicon from clonal libraries.

[0016] Lam et al. (“Evaluation of a Pooled Strategy for High-Throughput Sequencing of Cosmid Clones from Metagenomic Libraries”; 2014 PLoS ONE 9(6): e98968.doi:10.1371 / journal.pone.0098968) taught a pooled sequencing strategy that involves combining clones into a single sample for sequencing and assembly, and then using previously obtained Sanger end tags to recover specific clone sequences.

[0017] Libis et al. (“Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences”; Nat Commun., 10(1):3848, 2019) and WO2021041397A2 taught the use of degenerate PCR primers targeting conserved regions to amplify clonal pools, combined with a prediction algorithm that requires the identification of domain pairs that exhibit non-random occurrence.

[0018] Libis et al. (“Multiplexed mobilization and expression of biosynthetic geneclusters”; 13:5256, 2022) taught a variant of the aforementioned method that also relies on degenerate primer amplification, but also includes the step of directly cloning large and complete gene clusters into the final shuttle vector.

[0019] Crits-Christoph et al. (“Novel soil bacteria possess diverse genes for secondary metabolite biosynthesis”; Nature, 558, 440-444, 2018) elucidated a case in which a near-complete genome of microorganisms from a soil ecosystem was reconstructed de novo using metagenomic methods derived from genome analysis. They also evaluated the performance of degenerate primers for detecting target gene clusters, concluding that such primers may not be able to amplify genetically divergent sequences.

[0020] Therefore, although such methods remain effective in identifying gene clusters encoding clinically valuable metabolites, they are still highly dependent on primer selection and are thus often limited to predetermined clusters; in particular certain biosynthetic gene cluster (BGC) classes, and other conserved sequences to which primers can bind.

[0021] Furthermore, PCR screening aimed at physically recovering clones corresponding to BGCs from the pool is time-consuming and often produces truncated BGCs containing primer binding sites but not the entire target sequence.

[0022] Therefore, new methods are still needed to identify, recover, and isolate target gene clusters in cells and other microorganisms in an unbiased, efficient, and complete manner.

[0023] It is still necessary to compensate for the bias caused by the need for degenerate primers in amplicon-based sequencing.

[0024] The purpose of this invention is to meet the above-mentioned needs. Summary of the Invention

[0025] Therefore, the present invention relates to a method for identifying at least one target gene cluster, the method comprising the following steps:

[0026] a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster;

[0027] b) Divide the cloned library into at least a first plurality of independent partitions and a second plurality of independent partitions:

[0028] - The first plurality of independent partitions contain a plurality of clones, and the plurality of independent partitions thereby form a plurality of clone pools;

[0029] - The second plurality of independent partitions contain multiple clones from the clone pool, thereby forming multiple clone heaps;

[0030] Among these steps, high molecular weight nucleic acids from the cloning pool and the cloning stack are fragmented;

[0031] c) Determine all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the cloning pool and the cloning stack to provide multiple sequence reads, with the sequencing depth of the multiple sequence reads being less than 30×, for example less than 20×; thereby identifying the at least one target gene cluster.

[0032] Therefore, this method is applicable to identifying target gene clusters in clone libraries and in cells from which said clone libraries were previously obtained.

[0033] Therefore, this method is applicable to locating target gene clusters in clone libraries and in cells from which said clone libraries were previously obtained.

[0034] Therefore, this method is applicable to the recovery of target gene clusters from clone libraries and from cells from which said clone libraries were previously obtained. Detailed Implementation

[0035] In this invention, the following terms have the following meanings:

[0036] The terms “comprising,” “substantially consisting of,” and “composed of” may be replaced by any of the other two terms. The terms and expressions used are descriptive rather than restrictive, and the use of such terms and expressions does not exclude any equivalents of the features shown and described or portions thereof, and various modifications may be made within the scope of the claimed technology.

[0037] Nouns without quantifiers may refer to one or more elements they modify (e.g., "reagent" may mean one or more reagents) unless the context clearly indicates that one or more elements are being described. As used herein, the expressions "one" or "at least one" encompass "one" or "more than one"; they encompass "multiple" or "more than two," and may include three, four, five, six, or even more than six.

[0038] The term “about” as used in this article refers to a value within 10% of the base parameter (i.e., plus or minus 10%), and the term “about” is used at the beginning of a series of values ​​to modify each value (i.e., “about 1, 2 and 3” means about 1, about 2 and about 3).

[0039] The term "nucleic acid fractionation" refers to the process of randomly breaking high-molecular-weight nucleic acids (e.g., typically large target DNA) into smaller fragments. Nucleic acid fractionation can be performed before cloning nucleic acid fragments into vectors or before sequencing the fragments.

[0040] The term "shotgun sequencing" refers to sequencing individual fragments of nucleic acids (such as DNA or RNA). This term is not intended to limit it to a specific type of shotgun sequencing, although the fragmentation step is typically performed using enzymes or mechanically. Therefore, the process allows high-molecular-weight nucleic acids (e.g., often large target DNA) to be randomly broken into smaller fragments, and the ends of these smaller fragments are sequenced.

[0041] The term "sequencing depth" or "read depth" refers to the number of times a given base or nucleotide in a nucleic acid is read during a given sequencing process. Therefore, the term can correspond to the number of unique sequence reads aligned to a reference sequence, a reference genome, or a region in a de novo assembly. In a non-restrictive manner, sequencing depth can be equal to or greater than 0.01×, 0.1×, 0.5×, 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, or higher. In a non-restrictive manner, sequencing depth can also be equal to or less than 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×. The sequencing depth value that can be considered standard will depend on the type of sequencing technology. For example, a sequencing depth considered "standard" or "high" may therefore correspond to 20× or higher; for example, 30× or higher. For example, a sequencing depth considered "low" may therefore correspond to less than 20×; for example, less than 10×, less than 5×, or even less than 1×.

[0042] The term "completeness" or "genome coverage" refers to the percentage of base pairs covered by sequencing that correspond to a reference sequence (e.g., a gene or gene cluster). Therefore, sequencing completeness can be expressed as the percentage of the entire length of the reference sequence or a portion of the reference sequence covered by nucleic acid sequence reads. Sequencing completeness can also be quantified for a given length of nucleic acid sequence portion; for example, as the percentage of the reference sequence covered by a 1-kb nucleic acid sequence portion.

[0043] The term "gene" refers to a biological unit of heredity—a gene that is capable of self-replication and located at a specific location (locus) on a particular chromosome. In one embodiment, the particular chromosome is a bacterial chromosome. The term bacterial chromosome is used interchangeably with the term bacterial genome herein. As used herein, the term "gene cluster" refers to a group of closely adjacent genes located on the same chromosome whose products work synergistically in specific aspects of primary or secondary cellular metabolism.

[0044] The term "gene cluster" refers to a set of genes that share a common function or functional product. Genes typically reside within the genomic DNA in physically adjacent locations (e.g., within one centimole (cM)). Gene clusters can exist in prokaryotic or eukaryotic cells.

[0045] The term "biosynthetic gene cluster" or "BGC" or "metabolite gene cluster" refers to a cluster (i.e., two or more) of physically adjacent genes encoding a metabolite (e.g., a secondary metabolite), and thus contains a polynucleotide sequence encoding the function required for the synthesis and activity of such a metabolite. In a non-limiting manner, this term may therefore encompass BGCs containing polynucleotide sequences encoding one or more metabolites. Specifically, BGCs considered herein include those standardized by the "Minimal Information about a Biosynthetic Gene Cluster" (MIBiG) and / or antiSMASH as defined by the Genome Standards Consortium. Therefore, unless otherwise stated, the term may, in a non-limiting manner, include BGCs used for the synthesis of metabolites selected from: alkaloids, polyketides, sugars, non-ribosomal peptides (NRPs), post-translational modified peptides (RiPPs), and terpenes.

[0046] The terms "secondary metabolites" or "specific metabolites" refer to compounds that do not participate in primary metabolism and are therefore distinct from the more general macromolecules that constitute the fundamental mechanisms of life, such as proteins and nucleic acids. Many metabolites have important biotechnological applications in biomedical and drug discovery research, as well as in agriculture, aquaculture, and the chemical industry. Some examples include: antibacterial products such as penicillin and daptomycin; antifungal products such as amphotericin B; cholesterol-lowering products such as lovastatin; anticancer products such as bleomycin; and immunomodulatory products, including rapamycin and cyclosporine.

[0047] As used herein, the term "library," such as "clonal library," "genomic fragment library," "multi-genomic fragment library," or "high molecular weight DNA insert library," refers to any collection of nucleic acids, particularly any collection of DNA, that can be stored and propagated in a microbiome (or clone) through a molecular cloning process; thus, it can include cDNA libraries, genomic or multi-genomic libraries, and / or any synthetic mutant library, which may, for example, include variants of nucleic acid sequences, such as any mutant nucleic acid sequence derived from an endogenous sequence. Therefore, unless otherwise stated, the term "clonal library" can be applied indiscriminately to a cloning vector containing one or more nucleic acids, or to a host cell containing said cloning vector. For example, the term "clonal library" can refer to a cloning vector that replicates autonomously in a host cell, such as a prokaryotic cell, for example, Gram-positive or Gram-negative bacteria.

[0048] As used herein, the term "cloning vector" refers to a nucleic acid molecule capable of autonomous replication in a host cell, such as colloids, Fors plasmids, bacteriophages, bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes (PACs), yeast artificial chromosomes (YACs), and fungal artificial chromosomes (FACs). Cloning vectors typically contain: one or a few restriction endonuclease recognition sites to allow nucleic acid molecules to be inserted in a deterministic manner without losing the vector's essential biological function; and a nucleotide sequence encoding a marker gene suitable for identifying and selecting cells transformed with the cloning vector. Marker genes typically include genes that provide chloramphenicol resistance or apopramine resistance.

[0049] As used in this article, the term "host cell" refers to any cell capable of replicating and / or transcribing and / or translating a foreign gene. Therefore, "host cell" refers to any prokaryotic cell (including but not limited to Escherichia coli) or eukaryotic cell (including but not limited to yeast cells, fungal cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells).

[0050] As used herein, the terms “DNA library,” “genomic DNA library,” “multi-genome library,” “cDNA library,” and “environmental DNA library” refer to a library containing multiple genetic constructs, each containing an inserted polynucleotide sequence in the form of DNA. Each of these terms has its common meaning as known and used in the art.

[0051] As used herein, the terms "polynucleotide" or "nucleic acid" mean any single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, and, as non-limiting examples, include coding and non-coding sequences of genes, sense and antisense sequences, exons, introns, genomic DNA, cDNA, precursor mRNA, inRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, genetic constructs, vectors, and modified polynucleotides. References to nucleic acids, nucleic acid molecules, nucleotide sequences, and polynucleotide sequences should be understood similarly. Detailed Implementation

[0052] The inventors believe that screening the entire clone library to locate a few target clones is a time-consuming and resource-intensive process. The inventors initially hypothesized that the existence of gene clusters (especially biosynthetic gene clusters) could be assessed with minimal effort through nucleic acid fragmentation followed by nucleic acid sequencing (especially shotgun sequencing).

[0053] Therefore, the inventors infer that the PCR and amplicon sequencing strategies previously used to assess the presence of target genes can be easily and completely replaced by fragmentation and sequencing steps, even for sequencing of cloned libraries such as BAC, while maintaining efficiency in identifying new and diverse target gene clusters.

[0054] Therefore, the inventors now propose combining primer-free techniques that rely on nucleic acid fragmentation with pooling-based methods; particularly a multiplexed divide-and-conquer pooling strategy. This strategy exhibits several key advantages compared to alternative PCR screening described in the prior art.

[0055] As a primer-free technique, it is not limited to predetermined biosynthetic gene cluster (BGC) categories, nor does it require the targeted category to have conserved sequences that primers can bind to. In particular, all potential BGC categories or any other arbitrary DNA sequence can be detected by shotgun sequencing, provided that a relevant reference sequence is available for alignment.

[0056] Advantageously, the method of the present invention can therefore be applied to large clonal libraries containing high molecular weight nucleic acids, which may contain such gene clusters.

[0057] In contrast to PCR screening, which typically produces truncated gene clusters containing primer binding sites but not the entire target sequence, the integrity of the target region captured on the insert can be assessed using shotgun sequencing without first physically recovering the corresponding clones from the pool. This step saves a significant amount of effort wasted on recovering truncated sequences, which, for large gene clusters, often account for more than half of the clones recovered through PCR screening.

[0058] A complete reference gene cluster sequence allows for the selection and prioritization of target gene clusters (e.g., BGCs) because it can be used to assess the putative novelty and gene composition of the captured BGCs. Furthermore, more refined analysis of the read data can reveal novel gene insertions present within the captured BGCs that are not present in the reference sequence.

[0059] According to a first main embodiment, the present invention therefore relates to a method for identifying at least one target gene cluster, the method comprising the following steps:

[0060] a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster;

[0061] b) Divide the cloned library into at least a first plurality of independent partitions and a second plurality of independent partitions:

[0062] - The first plurality of independent partitions contain a plurality of clones, and the plurality of independent partitions thereby form a plurality of clone pools;

[0063] - The second plurality of independent partitions contain clones from the clone pool, thereby forming multiple clone heaps;

[0064] Among these steps, high molecular weight nucleic acids from the cloning pool and the cloning stack are fragmented;

[0065] c) Determine all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the cloning pool and the cloning stack; thereby identifying the at least one target gene cluster.

[0066] According to a particular embodiment, the present invention relates to a method for identifying at least one target gene cluster, the method comprising the following steps:

[0067] a) Provide a clone library containing multiple clones of high molecular weight nucleic acids, which may encode or contain a target biosynthetic gene cluster;

[0068] b) Divide the cloned library into at least a first plurality of independent partitions and a second plurality of independent partitions:

[0069] - The first plurality of independent partitions contain a plurality of clones, and the plurality of independent partitions thereby form a plurality of clone pools;

[0070] - The second plurality of independent partitions contain multiple clones from the clone pool, thereby forming multiple clone heaps;

[0071] Among these steps, high molecular weight nucleic acids from the cloning pool and the cloning stack are fragmented;

[0072] c) Determine all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the cloning pool and the cloning stack to provide multiple sequence reads, with the sequencing depth of the multiple sequence reads being less than 30×, for example less than 20×; thereby identifying the at least one target gene cluster.

[0073] Advantageously, the method of the present invention is therefore applicable to locating the target gene cluster in a clone library, and / or for locating the target gene cluster in cells or cells from which the clone library was previously obtained.

[0074] Advantageously, the method of the present invention is therefore applicable to the recovery of the target gene cluster in a clone library, and / or the recovery of the target gene cluster in cells or cells from which the clone library was previously obtained.

[0075] According to a particular embodiment, the present invention therefore relates to a method for identifying at least one target gene cluster, particularly multiple target gene clusters, in a cell or a clonal library thereof, preferably multiple BGCs in a eukaryotic or prokaryotic cell or a clonal library thereof, the method comprising the following steps:

[0076] a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster;

[0077] b1) Divide the clone library into a first plurality of independent partitions, the first plurality of independent partitions containing a plurality of clones, thereby forming a plurality of clone pools;

[0078] b2) Fragment the high molecular weight nucleic acids from the cloning pool and determine all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the cloning pool, thereby identifying multiple cloning pools that contain at least one occurrence of the at least one target gene cluster.

[0079] c1) Divide the clone library into a second plurality of independent partitions, the second plurality of independent partitions containing multiple clones from the clone pool, thereby forming multiple clone heaps;

[0080] c2) Fragment the high molecular weight nucleic acid from the cloned mass and determine all or part of the nucleic acid sequence of the fragmented high molecular weight nucleic acid in the cloned mass;

[0081] d) Based on the nucleic acid sequence from the clone cluster, identify the clone containing the target gene cluster in the library, thereby identifying the at least one target gene cluster.

[0082] According to the described embodiment, determining all or part of the nucleic acid sequence of the fragmented high molecular weight nucleic acid (e.g., in a cloning pool and / or cloning stack) may include providing multiple sequence reads, with the sequencing depth corresponding to the multiple sequence reads being less than 30×, for example less than 20×.

[0083] The second plurality of independent partitions may contain multiple clones from the cloning pool, thus forming multiple cloning clusters. Advantageously, the second plurality of independent partitions (i.e., the second partition) do not necessarily contain all clones in the first plurality of partitions (i.e., the first partition) whose nucleic acid sequences have been determined. Ideally, it may contain only clones in the first cloning pool that statistically contain the target gene cluster (e.g., BGC). Thus, one advantage is that sequencing is limited to portions of the cloning library in other partitions (e.g., the second partition, and optionally other additional partitions).

[0084] In view of the above, based on the nucleic acid sequences determined in the first plurality of regions, the characteristic of forming a second plurality of independent partitions may be that they contain all or only a portion of the clones of the first plurality of regions. For example, those independent partitions may contain all, substantially all, or only a portion of the clones of the first plurality of regions.

[0085] According to a particular implementation, the clone library is divided into at least a second or more independent partitions, each of which contains clones from each clone pool, thereby forming multiple clone heaps.

[0086] When the method requires further partitioning steps, such as third and fourth partitions (thus forming a third plurality and a fourth plurality), they may therefore optionally be characterized in the same manner; that is, the independent partition from each further partitioning step may contain all or only a partial clone of the previous partitions (e.g., the first plurality, or alternatively the second plurality, and / or any other partitions in the preceding order).

[0087] According to a particular embodiment, the present invention relates to a method for identifying at least one target gene cluster in a cell, particularly multiple target gene clusters in a cell or a clone library thereof, preferably multiple BGCs in a eukaryotic cell or a clone library thereof, comprising the following steps:

[0088] a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster;

[0089] b1) Divide the clone library into a first plurality of independent partitions, each of which contains a plurality of clones, thereby forming a plurality of clone pools;

[0090] b2) Fragmenting the high molecular weight nucleic acids from the cloning pool;

[0091] b3) Determine all or part of the nucleic acid sequence of the fragmented high molecular weight nucleic acid in the cloning pool to identify multiple cloning pools that contain at least one occurrence of the at least one target gene cluster;

[0092] c1) Divide the clone library into a second set of independent partitions, each containing clones from each clone pool, thus forming multiple clone heaps;

[0093] c2) Fragmenting the high molecular weight nucleic acids from the clone stack;

[0094] c3) Determine all or part of the nucleic acid sequence of the fragmented high molecular weight nucleic acid in the cloned mass;

[0095] d) Based on the nucleic acid sequence from the clone cluster, identify the clone containing the target gene cluster in the library, thereby identifying the at least one target gene cluster.

[0096] According to the implementation scheme, steps b3) and / or c3) of determining the nucleic acid sequence may include providing multiple sequence reads, with the sequencing depth corresponding to the multiple sequence reads being less than 30×, for example less than 20×.

[0097] According to one particular embodiment, the method of the present invention is used to identify multiple gene clusters in eukaryotic cells or their clone libraries; said eukaryotic cells are particularly fungi, preferably filamentous fungi, such as fungi belonging to the genus Aspergillus.

[0098] According to a particular embodiment, the method of the present invention is used to identify multiple gene clusters in a clone library; the clone library is preferably derived from eukaryotic cells, particularly fungi, preferably filamentous fungi, such as fungi belonging to the genus Aspergillus.

[0099] According to one particular embodiment, the method of the present invention is used to locate multiple gene clusters in a cloning library; the cloning library is preferably obtained from eukaryotic cells, particularly fungi, preferably filamentous fungi, such as fungi belonging to the genus Aspergillus.

[0100] According to some embodiments, the gene cluster is a biosynthetic gene cluster (BGC). According to some other embodiments, the gene cluster is not a biosynthetic gene cluster (BGC).

[0101] According to one particular embodiment, the method of the present invention is used to identify multiple biosynthetic gene clusters (BGCs).

[0102] According to one particular embodiment, the method of the present invention is used to identify multiple biosynthetic gene clusters (BGCs) in eukaryotic cells.

[0103] According to a particular embodiment, the plurality of biosynthetic gene clusters (BGCs) are used for the synthesis of: terpenes, NRPS, RiPP-like compounds, T1 PKS, NRPS-like compounds, melanin, butyrolactone, T3 PKS, and T2 PKS. PKS, ectoine, NAPAA, β-lactone, Class III lanethionide, PKS-like, lassopeptide, LPA, CDPS, RRE-containing proteins, redox cofactors, thiopeptides, Class I lanethionide, phenazine, hgIE-KS, Class II lanethionide, indole, ladderane, linaridin, aryl polyene, Class IV lanethionide, β-lactam, nucleoside, oligosaccharide, Class V lanethionide, transAT-PKS, guanidinotide, siderophores, aminoglycosides / aminocyclools ("amglyccycl"), thioamide-NRP, styracin, furan, cyanobactin, phosphate glycolipids, phosphonates / salts, transAT-PKS-like, botromycin, thioamides.

[0104] According to a particular embodiment, the plurality of biosynthetic gene clusters (BGCs) are used to synthesize nonribosomal peptides (NRPS), polyketides (PKS), terpenes, aminoglycosides, bacteriocins, lasso peptides, lanothioneins, or combinations thereof.

[0105] According to a particular embodiment, the plurality of biosynthetic gene clusters (BGCs) are used to synthesize nonribosomal peptides (NRPS), polyketides (PKS), terpenes, aminoglycosides, bacteriocins, lasso peptides, lanothiopeptides, nucleosides, phosphonates, or combinations thereof.

[0106] According to a particular implementation, the at least one target gene cluster is a BGC for the synthesis of NRPS or PKS.

[0107] According to a particular implementation, the at least one target gene cluster is a BGC used to synthesize a combination of NRPS and PKS.

[0108] According to one embodiment, the size of the at least one target gene cluster (e.g., a biosynthetic gene cluster) is less than 300 kbp; for example, less than 200 kbp, less than 100 kbp, less than 20 kbp, or even less than 10 kbp.

[0109] According to a particular embodiment, the size of the at least one target gene cluster (e.g., a biosynthetic gene cluster) is at least 10 kbp; for example, at least 20 kbp, for example, at least 100 kbp.

[0110] According to a particular embodiment, the size of the at least one target gene cluster is at least 20 kbp, for example, ranging from 20 kbp to 300 kbp, for example, ranging from 20 kbp to 200 kbp.

[0111] According to a particular embodiment, the size of the at least one target gene cluster is at least 20 kbp; therefore, it can be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, or 300 kbp.

[0112] According to a particular embodiment, the clone library is characterized in that it consists of or contains biosynthetic gene clusters.

[0113] According to one specific embodiment, the cloning library is characterized in that the size of the high molecular weight nucleic acid is at least 10 kbp; for example, at least 20 kbp, for example, at least 100 kbp. According to one specific embodiment, the cloning library is characterized in that the size of the high molecular weight nucleic acid is at least 20 kbp; therefore, it may include 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 kbp.

[0114] According to a particular embodiment, the clone library is characterized in that the size of the high molecular weight nucleic acid is at least 10 kbp; the clone library is further characterized in that the clone library is composed of or contains biosynthetic gene clusters.

[0115] According to a particular embodiment, the clone library is characterized in that the size of the high molecular weight nucleic acid is at least 20 kbp, for example, in the range of 20 kbp to 300 kbp, for example, in the range of 20 kbp to 200 kbp.

[0116] According to a particular embodiment, the clone library is characterized in that the size of the high molecular weight nucleic acid is at least 20 kbp; therefore, it may include 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, and 300 kbp.

[0117] According to a particular embodiment, the cloning library is characterized in that it consists of a cloning vector library selected from the following: sclerotia, Fors plasmids, bacteriophages, bacterial artificial chromosomes (BAC), P1-derived artificial chromosomes (PAC), yeast artificial chromosomes (YAC), and fungal artificial chromosomes (FAC).

[0118] According to a particular embodiment, the clone library contains or consists of shuttle vectors, particularly shuttle vectors suitable for replication in multiple cells belonging to different genera or kingdoms, more particularly one cell belonging to the genus Streptomyces and another cell belonging to the genus Escherichia; preferably shuttle vectors suitable for replication in species of Escherichia coli and Streptomyces.

[0119] According to one particular implementation, the shuttle vector is adapted to replicate and integrate the genome in multiple prokaryotic cells belonging to different genera, such as in Streptomyces species.

[0120] According to a particular embodiment, the clone library is provided in the form of prokaryotic cells; the prokaryotic cells are, for example, Gram-positive or Gram-negative bacteria, such as those belonging to the genus Escherichia, preferably Escherichia coli.

[0121] According to a particular embodiment, the method is used to identify multiple target gene clusters in a cell or its library; wherein the method further includes the step of recovering high molecular weight nucleic acids from the library in which the target gene clusters are detected.

[0122] It is worth noting that if the target gene cluster is assumed to exist statistically only on one clone in the pool, then determining the integrity of the target gene cluster (e.g., BGC) on a given clone will be greatly simplified after nucleic acid fragmentation and nucleic acid sequencing (e.g., by shotgun sequencing).

[0123] In fact, if the target gene cluster (e.g., BGC) exists in more than one clone in the pool, then even if no single clone contains the complete sequence, it is possible to observe the alignment of reads from overlapping clones with the full-length sequence of the gene cluster.

[0124] Therefore, during the first partitioning of the library, it is highly advantageous to construct a pool containing a number greater than the expected number of clones containing partial or complete target gene clusters. This number can be statistically estimated by considering the ratio between the number of nucleotides captured in the clone library and the number of nucleotides in the target genome from which the library was created.

[0125] Furthermore, while locating clones via triangulation necessarily involves allocating resources and effort to analyze multiple dimensions and partitions (e.g., multiple pool partitions), the proposed sequential pooling strategy minimizes these resources and effort by locating only selected clones containing the complete target gene cluster.

[0126] In fact, dividing the clone library into first-order clone pools and sequencing (e.g., shotgun sequencing) of these first-order pools has three additional advantages: obtaining definitive information about the nature and integrity of the clusters, and obtaining partial information about their location in the clone library.

[0127] Starting from this step, based on the level of each gene cluster (which can be assigned a priority score) and the economies of scale associated with simultaneously detecting other target gene clusters in the same pool, it is possible to carefully decide to allocate additional resources to obtain the precise cloning location of the target gene cluster.

[0128] Typically, deciding to locate too few gene clusters will result in higher costs per cluster because it is almost impossible to take advantage of economies of scale, and therefore requires sequencing of a large number of pools or pools with a large number of clones.

[0129] Conversely, attempting to locate too many or all of the target gene clusters in a clone library may result in diminishing returns and also lead to high costs per cluster.

[0130] The possibility of allocating or not allocating resources to locate a given cluster after the initial sequencing step provides options for a variety of optimal objectives, such as minimizing the cost per cluster or the total number of pooling rounds during the procedure.

[0131] The obtained alignments are typically analyzed for read coverage (or “sequencing depth”) to reveal which reference sequences can be assigned enough reads that are regularly distributed across the complete reference sequence.

[0132] Advantageously, the method according to the invention is therefore applicable to locating gene clusters even at low or very low sequencing depths, while maintaining a large selection of gene clusters suitable for large numbers of clone libraries; particularly a large selection of BGCs.

[0133] Advantageously, the method described herein is further characterized by the step of determining all or part of the nucleic acid sequence of fragmented high molecular weight nucleic acids in a cloning pool and / or cloning stack, thereby providing multiple sequence reads, which can be performed at different levels of sequencing depth; for example, at low sequencing depth, while maintaining its ability to effectively identify or recover the target gene cluster.

[0134] According to a specific embodiment, the method is characterized in that the sequencing depth corresponding to the plurality of sequence reads is less than 20×, for example less than 19×, 18×, 17×, 16×, 15×, 14×, 13×, 12×, 11×, 10×, 9×, 8×, 7×, 6×, 5×, 4×, 3×, 2×, or less than 1×. According to a specific embodiment, the method is characterized in that the sequencing depth corresponding to the plurality of sequence reads is less than 20×, for example less than 19×, 18×, 17×, 16×, 15×, 14×, 13×, 12×, 11×, 10×, 9×, 8×, 7×, 6×, 5×, 4×, 3×, 2×, or less than 1×; and the method is used to identify at least one gene cluster of at least 10 kbp; for example, at least 20 kbp or larger. According to a particular embodiment, the method is characterized in that the sequencing depth corresponding to the plurality of sequence reads is less than 20×, for example less than 19×, 18×, 17×, 16×, 15×, 14×, 13×, 12×, 11×, 10×, 9×, 8×, 7×, 6×, 5×, 4×, 3×, 2× or less than 1×; and the method is used to identify at least one biosynthetic gene cluster (BGC).

[0135] According to some exemplary embodiments, when applied to the identification of gene clusters (e.g., BGCs), the method shows that it can recover said gene clusters by identifying multiple sequence reads at low to very low sequencing depths; particularly sequencing depths of 10× or less; for example, 2× or less, such as 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×.

[0136] According to some implementation schemes, the sequencing depth is equal to or greater than 0.01×, 0.02×, 0.03×, 0.04×, 0.05×, 0.06×, 0.07×, 0.08×, 0.09×, 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, or 2.0×.

[0137] According to some implementation schemes, the sequencing depth ranges from 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9× or 2.0× to 10×, 20× or 30×. According to some implementation schemes, the sequencing depth ranges from 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9× or 2.0× to 10× or 20×. According to some implementation schemes, the sequencing depth ranges from 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9× or 2.0× to 10×.

[0138] According to a particular implementation, the method is characterized in that the integrity of the sequencing (such as the percentage of a reference sequence (e.g., a gene cluster or a portion thereof) covered by nucleic acid sequence reads) is higher than a predetermined threshold; for example, higher than at least 10%.

[0139] According to a particular implementation, the method is characterized in that the integrity of the sequencing (such as the percentage of a reference sequence (e.g., a gene cluster or a portion thereof) partially covered by a 1 kb nucleic acid sequence) is higher than a predetermined threshold; for example, higher than at least 10%.

[0140] According to a particular implementation, the cloning pool forming the cloning cluster statistically contains at least one occurrence of the target gene cluster.

[0141] According to a particular implementation, each of the plurality of cloning pools statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence.

[0142] According to a particular implementation, each of the multiple clone clusters statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence.

[0143] The above embodiments can be advantageously combined. Thus, according to a particular embodiment, the cloning pools forming the cloning clusters statistically contain at least one occurrence of the target gene cluster; and / or each cloning pool from the plurality of cloning pools statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence; and / or each cloning cluster from the plurality of cloning pools statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence.

[0144] According to a particular implementation, the cloning pools forming the cloning clusters statistically contain at least one occurrence of the target gene cluster; and each cloning pool from the plurality of cloning pools statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence; and each cloning cluster from the plurality of cloning pools statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence.

[0145] Advantageously, the step of fragmenting high molecular weight nucleic acids from the cloning pool can thus provide insights into how to divide a second set of independent parts.

[0146] In view of the above, it is foreseeable that the step of dividing the clone library into a second plurality of independent partitions, the second plurality of independent partitions containing clones from each clone pool, is guided by the nucleic acid sequence determined from all or part of the fragmented high molecular weight nucleic acids in the previous clone pool.

[0147] In particular, the cloning pools that form the cloning clusters can be rearranged so that, based on the first determination of the nucleic acid sequence, each cloning pool from the cloning clusters statistically contains at least one occurrence of the target gene cluster.

[0148] Therefore, according to a particular embodiment, the present invention relates to a method for identifying at least one target gene cluster in a cell or its clone library, particularly multiple target gene clusters in a cell, preferably multiple BGCs in a eukaryotic cell or its clone library, comprising the following steps:

[0149] a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster;

[0150] b1) Divide the clone library into a first plurality of independent partitions, each of which contains a plurality of clones, thereby forming a plurality of clone pools;

[0151] b2) Fragment the high molecular weight nucleic acid from the cloning pool and determine all or part of the nucleic acid sequence of the fragmented high molecular weight nucleic acid in the cloning pool, thereby identifying multiple cloning pools that contain at least one occurrence of the at least one target gene cluster.

[0152] c1) Divide the clone library into a second set of independent partitions, each containing clones from each clone pool, thus forming multiple clone heaps;

[0153] c2) Fragment the high molecular weight nucleic acids from the cloned mass and determine all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the cloned mass;

[0154] d) Based on the nucleic acid sequences from the clone cluster, identify clones containing the target gene cluster in the library, thereby identifying the at least one target gene cluster;

[0155] Optionally, the cloning pool forming the cloning cluster statistically contains at least one occurrence of the target gene cluster;

[0156] Optionally, each of the multiple cloning pools statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence;

[0157] Optionally, each of the multiple clone clusters statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence.

[0158] According to a preferred embodiment, the method includes the following steps:

[0159] a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster;

[0160] b) Divide the clone library into a first plurality of independent partitions, the first plurality of independent partitions containing a plurality of clones, thereby forming a plurality of clone pools, fragmenting the high molecular weight nucleic acids from the clone pools, and determining all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the clone pools, thereby identifying a plurality of clone pools containing at least one occurrence of the at least one target gene cluster.

[0161] c) Divide the clone library into a second set of independent partitions, each set of independent partitions containing clones from each clone pool, thereby forming multiple clone stacks, fragmenting the high molecular weight nucleic acids from the clone stacks, and determining all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the clone stacks.

[0162] d) Based on the nucleic acid sequence from the clone cluster, identify the clone containing the target gene cluster in the library, thereby identifying the at least one target gene cluster;

[0163] Among them, the cloning pool that forms the cloning cluster statistically contains the presence of at least one target gene cluster;

[0164] Optionally, each of the multiple cloning pools statistically contains less than two occurrences, particularly at most one occurrence, of the at least one target gene cluster;

[0165] Optionally, each of the multiple clone clusters statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence.

[0166] According to one particular implementation, the method includes multiple steps of partitioning the clone library, thereby forming more than two partitions, such as more than two clone pools and more than two clone heaps.

[0167] According to a particular implementation scheme, the step of providing a cloned library may include the following sub-steps:

[0168] (i) Provide one or more cells that may encode or contain the target gene cluster (e.g., BGC and / or a gene cluster of at least 10 kbp in size);

[0169] (ii) Isolating nucleic acids from said cells or multiple cells;

[0170] (iii) Fragmenting the nucleic acid into high-molecular-weight nucleic acids that may encode or contain the target gene cluster; and

[0171] (iv) Prepare multiple clones using the high molecular weight nucleic acid to prepare the clone library.

[0172] According to some embodiments, the method according to the invention includes the step of comparing a sequence read with all or part of a reference sequence.

[0173] According to a specific implementation scheme, the step of determining the sequence of high molecular weight nucleic acids in cloning pools and cloning stacks may include the following sub-steps:

[0174] (i) Determine all or part of the nucleic acid sequence of the corresponding fragmented high molecular weight nucleic acid, thereby providing multiple sequence reads;

[0175] (ii) The sequence read is compared with all or part of a reference sequence to determine the sequence of the high molecular weight nucleic acid.

[0176] According to one specific embodiment, the sequencing depth corresponding to the plurality of sequence reads is less than 30×. According to one specific embodiment, the sequencing depth corresponding to the plurality of sequence reads is less than 20×; for example, less than 1×.

[0177] According to a particular embodiment, the present invention relates to a method for identifying at least one target gene cluster (e.g., BGC), preferably multiple gene clusters, comprising the following steps:

[0178] a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster;

[0179] b) Divide the cloned library into at least a first set of independent partitions and a second set of independent partitions:

[0180] - The first plurality of independent partitions contain a plurality of clones, and the plurality of independent partitions thereby form a plurality of clone pools;

[0181] - The second plurality of independent partitions contain multiple clones from the clone pool, thereby forming multiple clone heaps;

[0182] Among these steps, high molecular weight nucleic acids from the cloning pool and the cloning stack are fragmented;

[0183] c) Determine all or part of the nucleic acid sequence of the corresponding fragmented high molecular weight nucleic acid to provide multiple sequence reads, with a sequencing depth of less than 30×, for example less than 20×, corresponding to the multiple sequence reads;

[0184] d) The sequence read is compared with all or part of a reference sequence to determine the sequence of the high molecular weight nucleic acid, thereby identifying the at least one target gene cluster.

[0185] The procedure for determining all or part of the nucleic acid sequence of fragmented high molecular weight nucleic acids in cloning pools and cloning stacks, providing multiple sequence reads; including:

[0186] - Multiple sequence reads of fragmented high-molecular-weight nucleic acids from the first partition (e.g., the cloning pool); and

[0187] - Multiple sequence reads of fragmented high-molecular-weight nucleic acids from a second partition (e.g., a clone heap).

[0188] According to some implementation schemes, both sets of sequence reads (i.e., from the first partition and the second partition) are provided at a sequencing depth of less than 30×, for example, less than 20×; the sequencing depth of each plurality of sequence reads can be the same or different. For example, the sequencing depth corresponding to the first partition can be higher than the sequencing depth corresponding to the second partition. Alternatively, the sequencing depth corresponding to the first partition can be lower than the sequencing depth corresponding to the second partition.

[0189] According to some implementation schemes, the sequencing depth corresponding to multiple reads in the first partition can be less than 30×, and the sequencing depth corresponding to multiple reads in the second partition can be less than 30×. According to some implementation schemes, the sequencing depth corresponding to multiple reads in the first partition can be less than 20×, and the sequencing depth corresponding to multiple reads in the second partition can be less than 20×. According to some implementation schemes, the sequencing depth corresponding to multiple reads in the first partition can be less than 30×, and the sequencing depth corresponding to multiple reads in the second partition can be less than 20×. According to some implementation schemes, the sequencing depth corresponding to multiple reads in the first partition can be less than 20×, and the sequencing depth corresponding to multiple reads in the second partition can be less than 30×. According to some implementation schemes, the sequencing depth corresponding to multiple reads in the first partition can be less than 10×, and the sequencing depth corresponding to multiple reads in the second partition can be less than 10×.

[0190] According to some implementation schemes, more than one set of sequence reads can be obtained from a given (e.g., a first or second) partition. This can be particularly advantageous when a set of sequence reads is below a reference integrity threshold. The reference integrity threshold can be adjusted based on the number of sequence read sets for a given partition and / or a given partition group. For example, the reference threshold can be at or below a predetermined percentage of X-kb nucleic acid sequence reads covering the full length of the partition / reference sequence; where X can be 20-kb; 15-kb, 10-kb, 9-kb, 8-kb, 7-kb, 6-kb, 5-kb, 4-kb, 3-kb, 2-kb, 1-kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 b, or less than 500 bp (e.g., less than a predetermined percentage of 1-kb nucleic acid sequence reads covering the full length of the partition / reference sequence).

[0191] According to some implementation schemes, the step of determining the sequence of high molecular weight nucleic acids in cloning pools and cloning stacks may include the following sub-steps:

[0192] (i) Determine the nucleic acid sequence of the corresponding fragmented high molecular weight nucleic acid portion to provide the first multiple sequence reads;

[0193] (ii) Compare the first plurality of sequence reads with all or part of the reference sequence; and

[0194] (iii) Optionally, when the first plurality of sequence reads are at or below a reference integrity threshold, the nucleic acid sequence of the second part of the corresponding fragmented high molecular weight nucleic acid is determined, thereby providing a second plurality of sequence reads.

[0195] According to the aforementioned implementation scheme, the multiple sequence read segments may include:

[0196] - The first or more sequence reads of fragmented high-molecular-weight nucleic acids from the first partition (e.g., the cloning pool); and

[0197] - At least a second or more sequence reads of fragmented high molecular weight nucleic acids from the first partition (e.g., a cloned heap).

[0198] According to the aforementioned implementation scheme, the multiple sequence read segments may include:

[0199] - The first or more sequence reads of fragmented high-molecular-weight nucleic acids from a second partition (e.g., a cloning pool); and

[0200] - At least a second or more sequence reads of fragmented high molecular weight nucleic acids from a second partition (e.g., a cloned heap).

[0201] According to some implementation schemes, the number of sequence reads is sufficient to align at least one read with a reference sequence; for example, aligning at least about 10 reads with a reference sequence; aligning at least about 100 reads with a reference sequence; aligning at least about 1000 reads with a reference sequence.

[0202] According to some embodiments, the method according to the invention includes the step of assembling all or part of previously aligned sequence reads. According to other embodiments, the method according to the invention does not include the step of assembling previously aligned sequence reads.

[0203] According to some implementation schemes, the sequencing depth is less than 1000×; for example, less than 500×; for example, less than 100×; for example, less than 20×; for example, less than 10×; for example, less than 1×.

[0204] According to some implementation schemes, for at least one step of determining the nucleic acid sequence of fragmented high molecular weight nucleic acid, the sequencing depth is less than 1×; for example, for determining all or part of the nucleic acid sequence of fragmented high molecular weight nucleic acid in the first partition, the sequencing depth is less than 1×.

[0205] Therefore, according to some implementation schemes, the steps of determining all or part of the nucleic acid sequence of the fragmented high molecular weight nucleic acid in the first and second partitions provide multiple sequence reads; including:

[0206] - Multiple sequence reads of fragmented high-molecular-weight nucleic acids from the first partition (e.g., cloning pool), wherein the sequencing depth is less than 1000×; for example, less than 100×; for example, less than 10×; for example, less than 1×; and

[0207] - Multiple sequence reads of fragmented high molecular weight nucleic acids from a second partition (e.g., a cloning heap), where the sequencing depth is less than 1000×; for example, less than 100×; for example, less than 10×; for example, less than 1×.

[0208] According to some implementation schemes, each clone from multiple clones is assigned a coordinate in the corresponding clone pool.

[0209] According to some implementation schemes, each clone from multiple clones is assigned coordinates in a corresponding clone pool and clone heap.

[0210] According to some implementations, the method includes the step of determining the coordinates of a clone containing the at least one target gene cluster in the library.

[0211] According to some implementations, the method includes the step of locating the coordinates of a clone containing the at least one target gene cluster in the library.

[0212] According to one specific implementation, the method is a computer-implemented method. According to the implementation, one or more steps are computer-implemented; for example, the step of determining clones containing the target gene cluster based on nucleic acid sequences from a cloning pool and / or cloning stack.

[0213] According to one specific implementation, the method includes the step of recovering from a library, preferably isolating clones containing at least one target gene cluster.

[0214] According to one specific implementation, the method includes the steps of replicating the recovered clone and / or culturing cells containing the recovered clone in a suitable culture medium. Attached Figure Description

[0215] Figure 1 This is a schematic diagram illustrating the general concept of locating the target BGC from a cloned library through several rounds of partitioning followed by shotgun sequencing.

[0216] Figure 2 This diagram illustrates a multiplexed divide-and-conquer strategy for efficiently triangulating multiple target BGCs in a clone library. After sequencing the pool constituting the first partition, only the portion of the library predicted to contain selected clones with complete target BGCs is retained to form new partitions (the second partition, and optionally one or more additional partitions). The selection of clones to form subsequent partitions can be computationally optimized, thereby enabling efficient parallel triangulation of multiple target BGCs in a cost- and / or time-efficient manner.

[0217] Figure 3The coverage of the reference BGC from Example 1 using sequencing reads from a single sample is shown. Different lines represent different sequencing depths achieved through random downsampling in the mapping step. A coverage of 2× is sufficient to determine that the variant of the target reference BGC in that sample is complete. On the X-axis, positions across the target reference BGC are expressed in bases, from 0 to 100,000. On the Y-axis, coverage is expressed as the average sequencing depth over a 1-kb bin.

[0218] Figure 4 shows a multipanel histogram illustrating the observed coverage (percentage of 1-kb bins covered) of the targets from Example 1. Each plot represents a different sequencing depth achieved through random downsampling during the mapping step, where 4A corresponds to 20× sequencing depth (left subplot) and 10× sequencing depth (right subplot), and 4B corresponds to 2× sequencing depth (left subplot) and 0.2× sequencing depth (right subplot). The X-axis represents the number of BGCs. The Y-axis provides the observed coverage, expressed as the percentage of 1-kb bins with at least one read in the BGC. Targets predicted as complete using the complete dataset are highlighted in light gray. Notably, even at 2× coverage, a threshold can be established to distinguish between complete and incomplete targets.

[0219] Figure 5 This is a graph illustrating the number and diversity of NRPSs located in the two sequencing steps from Example 1. Each node is a reference BGC predicted to be a complete NRPS type in at least one plate of the inventor's clone library according to the first sequencing step. Edges connect structurally similar BGCs according to Big-SCAPE (default parameter). Dark-filled nodes are reference BGCs located at the well level after the second sequencing step. Nodes with dark borders are those considered of interest due to their novelty. All complete NRPSs are shown, including those considered less interesting (grey borders). The sequential nature of this workflow allows targeting specific BGCs and thus targeting the most interesting and diverse BGC groups while mapping is in progress. Importantly, interesting BGCs that are not currently fully mapped (shown in white with dark borders) can be mapped at the expense of additional sequencing steps.

[0220] Example

[0221] The present invention is further illustrated by the following embodiments.

[0222] Example 1:

[0223] Materials and Methods

[0224] Streptomyces strain collection

[0225] In accordance with local regulations relating to the Nagoya Protocol, approximately 1 g of topsoil was collected from 96 sites in the Île-de-France region of France. Microbial colonies with morphological characteristics associated with the genus Streptomyces were isolated from each sample using standard isolation procedures. Barcoded universal 16S primers (27F: 1492R: Each strain underwent PCR, and the amplicon was sequenced on a MinION sequencer (Oxford Nanopore Technologies) with an R10.4.1 flow cell. Reads associated with each strain were compared with a database of genomes present on RefSeq using Blester, and strains with higher than 98% 16S nucleotide identity to genomes annotated as *Streptomyces* were selected for long-term storage in glycerol stock solution.

[0226] High molecular weight DNA extraction and BAC library construction

[0227] Spore stocks of 140 *Streptomyces* species were cultured individually in TSB medium. Mycelia were harvested immediately after reaching the stationary phase. The mycelia were centrifuged, and 20 μL of each precipitate was pooled together. The pooled mycelia were washed and centrifuged in 2 volumes of buffer (200 mM NaCl; 10 mM TrisHCl pH 8; 100 mM EDTA). The pooled mycelia were incorporated into agarose inserts according to the instructions of the CHEF Bacterial Genomic DNA Plug Kit and treated with lysozyme and proteinase K. BAC library construction was performed by the service provider (CNRGV-INRAE, Toulouse) according to standard methods. Briefly, the high molecular weight DNA present in the inserts was partially digested with BamHI and ligated into the pAGIBAC vector, which was modified to contain *Streptomyces* shuttle elements (OriT, apopramine-resistant, ...). The pOJ436 fragment of the integrase gene was used. The ligation product was transformed into E. coli DH10B, and the resulting transformants were picked into 384-well plates containing LB and chloramphenicol.

[0228] Initial partitioning and shotgun sequencing

[0229] Each 384-well plate containing the BAC library was replicated in a new 384-well plate containing fresh culture medium to ensure sufficient biomass for each clone. After 24 hours at 37°C, all wells were merged into a single pool for each plate. All 384 BACs present in each pool were extracted together using a standard BAC extraction protocol. The concentration of the resulting purified DNA was normalized before sequencing by the service provider. Each extracted pool was sequenced on a Novaseq sequencer (Illumina) using 2... 150 bp paired-end reads were sequenced to produce approximately 1 gigabyte per pool. FastP was used for read quality control to remove low-quality reads and for trimming. Reads were aligned to the *E. coli* DH10B genome and BAC vector sequences using bbmap (BBtools can be found at the main website: BBMap – Bushnell B. – sourceforge.net / projects / bbmap / ). Only unaligned reads were further analyzed.

[0230] Existence and completeness prediction

[0231] Using conservative parameters, reads were aligned to a database of reference BGCs using bbmap to generate per-nucleotide coverage and 1 kb bin coverage (covbinsize = 1000, maxsites2 = 5000, delcoverage = f). An arbitrary 50% per-nucleotide coverage was used to create a whitelist of reference BGCs that might be partially present in the library. The bin coverage of the whitelisted references was analyzed in more detail to predict which BGCs might be complete (or truncated, or nonexistent) and in which pools. Specifically, the inventors considered that 98% or more of the references with an average bin coverage greater than 50% likely received reads from closely related BGCs and were predicted as complete. The last bin was ignored in the analysis because it might be shorter than 1 kb, and its coverage could introduce statistical bias. Similarly, the inventors considered that if a reference had 66% to 98% of its bins covered (i.e., an average coverage greater than 50% per bin), the reference was incomplete and might be difficult to distinguish from complete references. Smaller truncated regions are not considered to pose a problem for positioning.

[0232] Referencing BGC's database

[0233] A database of reference BGCs was constructed by analyzing all Actinobacteria genomes present in RefSeq using the antiSMASH software (Blin et al., Nucleic Acids Research, 51, W1, 2023). Regions detected by antiSMASH were used as proxies defining BGCs and their associated boundaries. BGCs were deduplicated using dededupe.sh (from the BBTools suite) with conserved parameters (minidentity = 99, e = 1000, ac = f). The resulting FASTA contained more than 300,000 loosely deduplicated antiSMASH regions, which were considered "reference BGC sequences" for the purposes of this experiment. The bbmap index was constructed using this file.

[0234] BGC interest assessment and prioritization

[0235] The antiSMASH outputs of reference BGCs predicted as complete were manually reviewed to evaluate the relative importance of the cluster. Each BGC was assigned an integer score from 0 to 5. References that might produce known natural products received a score of 0 and were removed from the analysis. All references with a score of 1 or higher were considered valid targets. Higher scores were intended to prioritize the localization of some BGCs over others if resources allocated to localization were limited.

[0236] Pooling design for subsequent shotgun sequencing rounds

[0237] A linear programming algorithm is used to find board combinations that have the most high-scoring references, where each high-scoring reference has exactly one prediction of a complete occurrence without any incomplete references. Each reference with a score of n is arbitrarily considered equivalent to three references with a score of n-1. The algorithm is run iteratively, removing references found in previous iterations, to find board combinations that allow the location of all references.

[0238] Subsequent shotgun sequencing rounds and localization

[0239] Aside from the pool configuration, this step is identical to the first shotgun sequencing step. In short, once culture is complete, cells are transferred well-to-well from the selected plate to a 384-well receiving plate (all A1 to A1, etc.). Sequencing samples are then formed by merging all wells in each row (correspondingly, column), resulting in 16 (correspondingly, 24) pools. The sequencing results are analyzed using the same workflow described above to identify which pools contain which references at which coverage values. For each target BGC, rows and columns are predicted based on the highest per-nucleotide coverage in each corresponding pool.

[0240] Determination of BGCs through long-read sequencing and localization

[0241] The localization was further determined by randomly selecting 10 clones predicted to contain one of the non-fuzzy localizations of the BGC. Sequencing of the corresponding BGC was performed on a MinION sequencer (Oxford Nanopore Technologies), and the presence of sequences closely related to the expected reference sequence was determined in all cases.

[0242] result

[0243] To demonstrate the concepts described herein, multiple biosynthetic gene clusters (BGCs) derived from *Streptomyces* bacteria were successfully isolated into cloning vectors maintained in *Escherichia coli*. *Streptomyces* is a favorable candidate because it contains many BGCs encoding previously unknown natural products. Soil samples were collected and *Streptomyces* strains were isolated using standard isolation techniques. The 16S locus of these strains was amplified by PCR and sequenced to confirm their *Streptomyces* genus membership. 140 *Streptomyces* strains with different 16S sequences were retained for demonstration purposes.

[0244] Selected *Streptomyces* strains were cultured and harvested to construct bacterial artificial chromosome (BAC) libraries using standard techniques. The first step was to culture each strain individually to accumulate sufficient DNA. Genomic DNA from all strains was then extracted and merged to form high molecular weight (HMW) libraries. These HMW DNA fragments were then electroporated into *E. coli* and maintained as bacterial artificial chromosomes (BACs). Finally, *E. coli* transformants were plate-inoculated and individually picked into 384-well plates to form BAC libraries of *Streptomyces* inserts, with each clone stored in a separate well. Thus, each well contained a clone expected to contain a BAC, which consisted of a random DNA insert from one of the selected *Streptomyces* strains. This step resulted in the creation of a BAC library consisting of 18,432 clones. Based on analysis of a subset of 10 randomly selected clones, the average size of these inserts was estimated to be approximately 100 kb.

[0245] It is expected that only a small fraction of clones will be the target clones: those containing a subset of complete BGCs that have never been reported to be associated with known secondary metabolites and are not present in the MIBiG database (Terlouw et al., Nucleic Acids Research, Vol. 51, No. D1, 2023).

[0246] The goal is to locate the target clone without wasting resources on other clones. A sequential, multi-round pooling strategy is provided to analyze intermediate partitions and guide the design of subsequent partitions to locate as many target BGCs as possible while minimizing the required time and resources.

[0247] For each 384-well plate of the library, all clones from each plate were merged and shallow sequenced. Reads were aligned against a database of deduplicated known BGC sequences (“Reference”). Since the expected captured BGCs have sequences closely related to but distinct from the Reference, alignment parameters were set to allow for error between the original reads and the Reference sequences.

[0248] The resulting alignments are analyzed for read coverage to predict which references in which pools are likely complete. Since the inventors are only interested in the completeness of the BGC, they only need to check that the reads are roughly distributed across the full length of the reference sequence. For each sequencing epoch, reads from each pool are mapped to the reference BGC sequence. If their completeness passes a predetermined threshold, detailed coverage is checked to determine if the core BGC is indeed complete. Advantageously, when the target is considered interesting and complete, one or more sequencing steps can be introduced / provided to seek more locational information. It is also advantageous to design different pools for each sequencing step to obtain additional information about multiple target nucleic acid sequences / targets.

[0249] Using shotgun sequencing as a screening tool requires only a small sequencing depth. Figure 3 and 4A (B), which in turn allows for the merging of more samples. Advantageously, assembly steps that are known to be difficult for sequences with repetitive structural domains (such as BGC) can be bypassed.

[0250] A complete reference BGC list is expected to be used in the first sequencing step to prioritize BGC selection. A large number of BGCs were detected across all BGC families, including those encoding genes for the synthesis of one or more of the following compounds and / or peptides: terpenes, NRPS, RiPP-like, T1 PKS, NRPS-like, melanin, butyrolactone, and T3. PKS, T2PKS, ectoine, NAPAA, β-lactone, Class III lanethionide, PKS-like peptides, lasso peptides, LPA, CDPS, proteins containing RRE, redox cofactors, thiopeptides, Class I lanethionide, phenazine, hgIE-KS, Class II lanethionide, indole, ladderane, linaridin, aryl polyene, Class IV lanethionide, β-lactam, nucleoside, oligosaccharide, Class V lanethionide, trans-AT-PKS, guanidinonucleotides, siderophores, aminoglycosides / aminocyclools, thioamide-NRP, styracin, furan, cyanobacterial peptides, phosphoglycolipids, phosphonates / salts, trans-AT-PKS-like peptides, borzomycin, thioamidide. Some BGCs belong to multiple classes. The characteristics of BGC reference sequences are used for specific targeting of BGCs due to their novelty. Figure 5 161 BGCs were deemed valuable, new, and therefore worthy of recycling. Some of these BGCs appeared only once throughout the entire library, while others had complete or incomplete copies on multiple boards. At this point in the analysis, the board location of each of these BGCs was known, but their hole locations were unknown.

[0251] First, PCR and amplicon sequencing strategies were completely replaced by shotgun sequencing. Samples were shotgun sequenced with shallow coverage, and reads were aligned against a database of deduplicated known BGC sequences (“reference”). Since the expected captured BGCs have sequences closely related to but distinct from the references, alignment parameters were set to allow for error between the original reads and the reference sequences. Read coverage analysis was performed on the resulting alignments to reveal which references could be assigned sufficient, regularly distributed reads across the entire reference sequence to determine if the reference (or its closely related variants) could be captured in one or more clones.

[0252] Secondly, the sequential multi-round pooling strategy allows for the analysis of intermediate partitions and guides the design of subsequent partitions to locate as many target BGCs as possible while minimizing the required time or resources. One implementation of this concept can be seen in this embodiment. For each 384-well plate of the library, all clones of the plate are merged and sequenced as a single sample. Analysis of this first dimension using read coverage data reveals which reference BGC variants are expected to be present in each pool and therefore in the corresponding plate.

[0253] In particular, the analysis detected a large number of BGCs, of which 161 were considered novel and of interest. Some of these BGCs appeared only once throughout the entire library, while others had complete or incomplete copies on multiple plates.

[0254] A new pool set was designed to maximize the number of localized BGCs with the fewest sequencing steps. To this end, subsets of different number of plates were identified to maximize the number of complete target BGCs with exactly one copy in each subset.

[0255] As a demonstration, after predicting that it contains 63 complete target BGCs (each fully present on exactly one BAC in the entire subset), a subset of 24 boards was selected. While subsets of several boards could be formed for subsequent analysis to reclaim more BGCs, this embodiment focuses only on this first subset of 24 boards. To obtain the precise location of each target BGC, pools were formed by clustering clones belonging to the same row in the 24 boards (16 rows in total, thus producing a total of 16 pools), and by clustering clones belonging to the same column in the 24 boards (24 columns in total, thus producing a total of 24 pools).

[0256] Since the intended BGC is expected to exist in exactly one clone in exactly one of the 24 plates, it is expected to exist in exactly one of the 16 pools corresponding to the row and one of the 24 pools corresponding to the column, thus allowing for simultaneous triangulation of the precise location of the clone containing each intended BGC.

[0257] Analysis of read coverage data from plate, row, and column dimensions revealed the precise localization of 55 complete-purpose BGCs. These BGCs were then individually transferred to heterologous hosts to assess the production of their associated natural products using standard analytical chemistry methods.

[0258] This experimental demonstration showcases the key features of the method: it enables parallel evaluation of the presence and location of a large number of BGCs that may belong to a wide variety of different categories, significantly reducing the cost per BGC in the procedure. Furthermore, this example demonstrates that shotgun sequencing can be used even at shallow sequencing depths, requiring far fewer resources than assembling the exact sequences present in the library into contigs.

[0259] Example 2:

[0260] This embodiment focuses on a reference BGC (GCF_000716685.1.contig0007.region001), which is a T2PKS, and its related BGC is located according to a procedure similar to that detailed in Embodiment 1.

[0261] Compared to the procedure described in Example 1, the inventors here used three sequencing steps (instead of two), and worked at a lower sequencing depth.

[0262] For the first sequencing step (PL), each sample is equivalent to 8 wells in a pooled 384-well plate. Assuming the BAC has 100 kb insert, 10 kb backbone, and no contamination (including contamination from the host strain), this gives a theoretical sequencing depth of 6× for 2 Gb of data. ).

[0263] For the second sequencing step (MS), each sample is equivalent to up to 21 pooled 384-well plates. A list of plates is selected for each well so that the combination of PL and MS data provides plate localization.

[0264] Finally, the rows and columns of the last partition (S) of the plate were sequenced in different samples as previously described by the inventors to resolve well localization.

[0265] Relevant metrics were provided, including bin coverage for each sequencing step. Specifically, for partition PL, the BGC was considered complete in the three bins (internal reference numbers: PL-GEN-12-8 and PL-GEN-12-7) of the first sequencing step. The BGC was then considered complete in bin MPST-240823-S0M6 of the second sequencing step. Knowing which bins comprised these bins, it could be inferred that only bin 55 of library 12 could explain the observed signal (data not shown). This bin was included in the final sequencing step to complete well localization of the BGC (and others). Using merged rows and columns (partition S), complete localization of the BGC in well K17 of bin 55 of library 12 was obtained. Note that all three sequencing steps used very low sequencing depths, which are unsuitable for de novo assembly. In this specific instance, the average read coverage was less than 3×. Nevertheless, target localization was still resolvable.

[0266] The localization of the BGC was subsequently validated in long-read sequencing quality control experiments. Read mapping showed successful recovery of the variant GCF_000716685.1.contig0007.region001, a sequence close to but distinct from the reference, with a complete core of 35 to 40 kb. Specifically, the percentage of shared sequence identity of the recovered BGC was 92%.

Claims

1. A method for identifying at least one target gene cluster, the method comprising the following steps: a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster; b) Divide the cloned library into at least a first plurality of independent partitions and a second plurality of independent partitions: - The first plurality of independent partitions contain a plurality of clones, and the plurality of independent partitions thereby form a plurality of clone pools; - The second plurality of independent partitions contain multiple clones from the clone pool, thereby forming multiple clone heaps; Among these steps, high molecular weight nucleic acids from the cloning pool and the cloning stack are fragmented; c) Determine all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the cloning pool and the cloning stack to provide multiple sequence reads, with a sequencing depth of less than 30× corresponding to the multiple sequence reads; thereby identifying the at least one target gene cluster.

2. The method according to claim 1, used to identify multiple gene clusters in a eukaryotic cell or its clone library; wherein the eukaryotic cell is particularly a fungus, preferably a filamentous fungus, such as a fungus belonging to the genus Aspergillus.

3. The method according to claim 1 or 2, used for identifying multiple biosynthetic gene clusters (BGCs) in fungi or their clone libraries.

4. The method according to claim 3, wherein the plurality of biosynthetic gene clusters (BGCs) are used to synthesize nonribosomal peptides (NRPS), polyketides (PKS), terpenes, aminoglycosides, bacteriocins, lasso peptides, lanothiopeptides, nucleosides, phosphonates / esters, or combinations thereof.

5. The method according to any one of claims 1 to 4, wherein at least one target gene cluster is a BGC for synthesizing NRPS or PKS.

6. The method according to any one of claims 1 to 5, wherein the size of the at least one target gene cluster is at least 20 kbp, for example, ranging from 20 kbp to 200 kbp.

7. The method according to any one of claims 1 to 6, wherein the cloned library is characterized in that the size of the high molecular weight nucleic acid is at least 20 kbp, for example, in the range of 20 kbp to 200 kbp.

8. The method according to any one of claims 1 to 7, wherein the clone library is characterized in that it comprises a clone vector library selected from the following: Glutamates, Fors plasmids, bacteriophages, bacterial artificial chromosomes (BAC), P1-derived artificial chromosomes (PAC), yeast artificial chromosomes (YAC), and fungal artificial chromosomes (FAC).

9. The method according to any one of claims 1 to 8, wherein the clone library comprises or is composed of a shuttle vector, the shuttle vector being adapted to replicate in multiple cells belonging to different genera or kingdoms, more particularly one belonging to the genus Streptomyces and another to the genus Escherichia; preferably the shuttle vector is adapted to replicate in Escherichia coli and transfer to Streptomyces spp. species.

10. The method according to any one of claims 1 to 9, the method comprising the following steps: a) Provide a clone library containing multiple clones with high molecular weight nucleic acids that may encode or contain a target gene cluster; b) Divide the clone library into a first plurality of independent partitions, the first plurality of independent partitions containing a plurality of clones, thereby forming a plurality of clone pools, fragmenting the high molecular weight nucleic acids from the clone pools, and determining all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the clone pools, thereby identifying a plurality of clone pools containing at least one occurrence of the at least one target gene cluster. c) Divide the clone library into a second plurality of independent partitions, the second plurality of independent partitions containing clones from each clone pool, thereby forming a plurality of clone pools, fragmenting the high molecular weight nucleic acids from the clone pools, and determining all or part of the nucleic acid sequences of the fragmented high molecular weight nucleic acids in the clone pools; d) Based on the nucleic acid sequences from the clone cluster, identify clones in the library that contain the target gene cluster, thereby identifying the at least one target gene cluster; The cloning pool that forms the cloning cluster statistically contains at least one occurrence of the target gene cluster.

11. The method according to any one of claims 1 to 10, comprising a plurality of steps of partitioning the clone library; thereby forming more than two partitions.

12. The method according to any one of claims 1 to 11; The steps for determining the sequences of high molecular weight nucleic acids in the cloning pool and the cloning stack include: (i) Determine all or part of the nucleic acid sequence of the corresponding fragmented high molecular weight nucleic acid, thereby providing multiple sequence reads; (ii) The sequence read is compared with a reference sequence to determine all or part of the sequence of the high molecular weight nucleic acid.

13. The method of claim 12, wherein the sequencing depth corresponding to the plurality of sequence reads is less than 20×; for example, less than 1×.

14. The method according to any one of claims 1 to 13, wherein each of the plurality of cloning pools statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence; and / or each of the plurality of cloning heaps statistically contains less than two occurrences of the at least one target gene cluster, particularly at most one occurrence.

15. The method according to any one of claims 1 to 14, comprising the step of recovering from the library, preferably isolating clones containing the at least one target gene cluster.

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