WO2020073007A1 - Profilage du microbiome moléculaire pour la cicatrisation de plaies - Google Patents

Profilage du microbiome moléculaire pour la cicatrisation de plaies

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Publication number
WO2020073007A1
WO2020073007A1 PCT/US2019/054875 US2019054875W WO2020073007A1 WO 2020073007 A1 WO2020073007 A1 WO 2020073007A1 US 2019054875 W US2019054875 W US 2019054875W WO 2020073007 A1 WO2020073007 A1 WO 2020073007A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid sequence
reads
chimeric
sequence
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PCT/US2019/054875
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English (en)
Inventor
Christopher Szeto
Shahrooz Rabizadeh
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Nantomics, Llc
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Publication date
Application filed by Nantomics, Llc filed Critical Nantomics, Llc
Publication of WO2020073007A1 publication Critical patent/WO2020073007A1/fr

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    • 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
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations

Definitions

  • the field of the invention is omics analysis of microbiomes in a microenvironment of a wound, particularly using computational analysis to identify the wound microbiome and to improve wound healing
  • a microbiome is an ecological community of all monocellular and/or multicellular symbiotic or pathogenic microorganisms. Different species of animals, plants, or any other multicellular organisms are either surrounded or possess a microbiome, which affects their metabolism, immune system, endocrine system, and general health status. For example, the skin, periodontal pockets, and various portions of the intestinal tracks are host to numerous and distinct bacterial species, and in total bacterial cells outnumber the cells of a human host.
  • any substantive changes in the microbiome that breaks the balance in the microbiome may affect the status or condition of the host entity or any biological entity around the microbiome, necessitating a quick, yet thorough analysis of a microbiome covering a large variety of microorganisms.
  • US Pat. Pub No. 2017/0159108 to Budding discloses microbiome population analysis using taxonomic variations among DNA sequences of microbial 16S-23S rRNA internal transcribed region as primers to amplify various types of microorganisms and the length differences in the PCR products using such primers.
  • US Pat. Pub. No. 2017/0058430 to Watts discloses infection identification using whole metagenome sequence analysis of a sample. In Watt, human DNA is removed fro the human DNA- bacterial DNA mixture obtained from the wound sample, and the isolated bacterial DNA sequences are analyzed using k-mer based sequence analysis. Yet, all such methods require multiple steps of sample purification, amplification and/or sequencing, which may cause incomplete analysis of all microorgani sms present in the microbiome.
  • the inventive subject matter is directed to in silica omics analysis of a microbiome to identify the presence of one or more known microorganisms in a site of inj ury (e.g., traumatic wound, diabetic ulcer, burn, etc.) as well as any changes in the microorganisms that may alter the balance of the microbiome.
  • a treatment plan may be established or modified.
  • the inventors contemplate systems and methods of identifying the microbiome of a sample in silica.
  • a nucleic acid sequence which preferably comprises a plurality of short reads, from the sample (e.g., site of injury) is obtained.
  • the nucleic acid sequence is then aligned with a reference nucleic acid sequence to locate the reads relative to the reference nucleic acid sequence.
  • the located reads are then aligned with a chimeric nucleic acid sequence comprising at least two genomic nucleic acid sequences of at least two distinct microorganisms, for example, that are merged to form a single sequence file.
  • nucleic acid sequence can be identified from an alignment of the chimeric nucleic acid sequence and the nucleic acid sequence from the sample.
  • the nucleic acid sequence comprises at least one microorganism’s genomic nucleic acid sequence, which can be selected from bacteria, yeast, a fungus, a virus, and a mycoplasma.
  • the sample is a human tissue that includes the microorganisms from the site of injury, tissue damage, or infection
  • the nucleic acid sequence comprises a human genomic nucleic acid sequence and a non-human genomic nucleic acid sequence.
  • the chimeric reference nucleic acid sequence can further comprise a reference human genomic nucleic acid that is merged with the at least two microorganisms’ genomic nucleic acid sequences.
  • the method may also include steps of generating a patient chimeric nucleic acid sequence comprising the non- human genomic nucleic acid sequence merged at an end of the human genome nucleic acid sequence, and aligning the patient chimeric nucleic acid sequence and the chimeric reference nucleic acid sequence to identify the origin of the nucleic acid sequence.
  • the sample for determination of the microbiome will be obtained from the individual (typically a mammal such as a human, a pet or companion animal, or livestock), which may be taken in form of a swab, a rinse fluid, a biopsy, an exudate or aspirate, etc. Therefore, in at least some aspects, the human tissue is a diseased or injured tissue, but may also exclude human tissue and predominantly be a microorganism sample. Therefore, nucleic acid isolation from such samples may vary. However, it. is generally preferred that nucleic acid isolation will follow generally known procedures. Moreover, it should be appreciated that the nucleic acid will typically be DNA and/or RNA (which specifically includes mRNA as well as rRNA).
  • sequencing of the samples can be performed in numerous manners, including nextgen sequencing of the whole genome or exorae, as well as transcriptorae sequencing (e.g., RNAseq).
  • sequence information can be qualitative (i.e., base calls) as well as quantitative (e.g., copy number, numbers of transcripts, etc.)
  • the nucleic acid sequence from the sample and the chimeric reference nucleic acid sequence are in BAM, SAM, FASTQ, PASTA, or PASTA index format.
  • the nucleic acid sequence is aligned with a chimeric reference nucleic acid sequence using incremental synchronized alignment.
  • the step of identifying the origin of the nucleic acid sequence comprises determining a quantity of first reads of the nucleic acid sequence that are aligned with a first portion of the chimeric nucleic acid sequence.
  • a quantity of second reads of the nucleic acid sequence that are aligned with a second portion of the chimeric nucleic acid sequence can be determined and then a relative quantity of first and second microorganisms in the sample can be determined.
  • the first and second portions are nucleic acids sequences of the first and second microorganisms, respectively.
  • the method further comprises generating or updating a record of the sample according to the identification of the origin and/or providing or recommending a treatment that is specific to the origin. Also, where the nucleic acid sequence is obtained from the sample periodically, the method can further comprises generating or updating a record of the sample according to a change of the identification of the origin.
  • Another aspect of the inventive subject matter includes a method of determining a change in a microbiome of a biological entity in silica in this method, first and second samples from the biological entity are obtained at first and second time points.
  • the first time point can be before start of a therapeutic intervention (e.g., application of an antibiotic), and the second time point can be upon termination or conclusion of the intervention.
  • first and second nucleic acid sequences are obtained from the first and second samples, respectively.
  • each of the first and second nucleic acid sequence comprises a plurality of reads.
  • the first and second nucleic acid sequences can then be aligned with a reference nucleic acid sequence to locate the reads relative to the reference nucleic acid sequence.
  • the located reads then are aligned with a chimeric nucleic acid sequence comprising at least two microorganisms’ genomic nucleic acid sequences that are merged to form a single sequence file. Then, a change in the microbiome can be determined by comparing alignments of located reads derived from the first and second nucleic acid sequences with the chimeric nucleic acid sequence.
  • the first and second nucleic acid sequences comprise at least one microorganism’s genomic nucleic acid sequence, which can be bacteria, yeast, a fungus, a virus, and a mycoplasma.
  • the first and second samples are derived from a human tissue
  • each of the first and second nucleic acid sequences comprises a human genomic nucleic acid sequence and a non-human genomic nucleic acid sequence, respectively.
  • the chimeric reference nucleic acid sequence further comprises a reference human genomic nucleic acid that is merged with the at least two microorganisms’ genomic nucleic acid sequences.
  • the human tissue can be a diseased tissue.
  • the method can also include steps of generating first and second patient chimeric nucleic acid sequences, each comprising the non-human genomic nucleic acid sequence merged at an end of the human genome nucleic acid sequence and aligning the patient chimeric nucleic acid sequence and the chimeric reference nucleic acid sequence to identify the origin of the nucleic acid sequence.
  • the first and second nucleic acid sequences and the chimeric nucleic acid sequence are in BAM SAM, FASTQ, FASTA, or FASTA index format and/or the first and second nucleic acid sequence is aligned with a chimeric nucleic acid sequence using incremental synchronized alignment.
  • the change in the microbiome comprises at least one of a quantity change of at least one microorganism, a ratio change among a plurality of microorganisms and a mutation in at least one microorganism.
  • the quantity change is determined by measuring quantities of first and second reads of first and second nucleic acid sequences, wherein the first and second reads are aligned with a first portion of the chimeric nucleic acid sequence.
  • the ratio change is determined by measuring first and second reads of the first nucleic acid sequence and third and fourth reads of the second nucleic acid sequence, wherein the first and third reads, and second and fourth reads are aligned with a first portion or a second portion of the chimeric nucleic acid sequence, respectively.
  • the first and second portions are nucleic acids sequences of the first and second microorganisms, respectively or are nucleic acids sequences of different strains of same species of the microorganism.
  • the method further comprises generating or updating a record of the sample according to the change of the microbiome or providing or recommending a treatment according to the change of the microbiome.
  • FIG. 1 A is an illustrated example of a reference sequence comprising only human genome.
  • FIG. IB is an illustrated example of a chimeric nucleic acid sequence comprising a plurality of microorganisms’ genomic nucleic acid sequences.
  • FIG. 1C is an illustrated example of a chimeric nucleic acid sequence comprising a human genome sequence and a plurality of microorganisms’ genomic nucleic acid sequences
  • F IG. 2 shows three different scenarios of aligning the nucleic acid sequences with a reference sequence at an aligner and analyzing the aligned sequence via the aligner by comparing with a chimeric nucleic acid sequence.
  • FIG. 3 illustrates one embodiment of checkpoint inhibitors and response to immunotherapy during a wound healing process.
  • FIG. 4 illustrates one embodiment of three immune modulating compounds and control of cutaneous infection.
  • FIG , 5 illustrates one embodiment of nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) during the wound healing process
  • FIG, 6 illustrates one embodiment of compounds that synergize with a TLR2 ligand to induce IL-8 secretion.
  • FIG. 7 illustrates one embodiment of tissue polypeptide specific (TPS) antigen synergy with other germ line-encoded pattern recognition receptors (PRRs).
  • TPS tissue polypeptide specific
  • PRRs germ line-encoded pattern recognition receptors
  • FIG. 8 illustrates one embodiment of Bioluminescent S. aureus as a Tool to Identify Novel Therapeutics for MRSA
  • FIG. 9 illustrates one embodiment of time course of neutrophil recruitment following TPS treatment
  • FIG , 10 illustrates one embodiment of wound progression following infection with S. aureus.
  • FIG, 11 illustrates another embodiment of w ' ound progression following infection with S. aureus.
  • F IG. 12 illustrates one embodiment of bacterial enumeration - treatments significantly decrease wound size and bacterial burden.
  • FIG. 13 illustrates one embodiment of formulation development and formulation stability.
  • FIG. 14 illustrates one embodiment of TPS cream formulation. Results against surgical site infection by S. aureus NRS 384.
  • the inventors have discovered that the presence, quantities and/or ratio of one or more organisms in a microbiome in a site of injury such as a traumatic wound, a burn wound, and/or a chronic skin disease sample can be readily determined by comparing the sequence information of nucleic acid sequences obtained from the sample with a plurality of genomic sequences of microorganisms. Most typically, information of the microbiome may inform initial or subsequent treatment, and may also be used to further classify the type or severity of the injury. Moreover, omics analysis may also be used to determine the immune status in the site of injury or disease.
  • the inventors further discovered that the efficiency of the comparison of sequence information can be substantially increased when the plurality of genomic sequences of microorganisms are coupled to for a single hybrid or chimeric sequence. Consequently, in one especially preferred aspect of the inventive subject matter, the inventors contemplate a method of identifying microbiome of a sample in silico.
  • a nucleic acid sequence can be obtained from the sample, and the nucleic acid sequence is aligned with a chimeric nucleic acid sequence that includes a plurality of microorganisms' genomic nucleic acid sequences. Based on where and how the nucleic acid sequence aligns with the chimeric nucleic acid sequence, the source/origin of the nucleic acid sequence can be identified.
  • the term“patient” as used herein includes both individuals that are diagnosed with a condition (e.g., cancer) as well as individuals undergoing examination and/or testing for the purpose of detecting or identifying a condition.
  • the term “provide” or“providing” refers to and includes any acts of manufacturing, generating, placing, enabling to use, transferring, or making ready to use.
  • tissue samples can be obtained from the individual via a biopsy (including liquid biopsy, or obtained via tissue excision during a surgery or an independent biopsy procedure, etc.), which can be fresh or processed (e.g., frozen, etc.) until further process for obtaining nucleic acid information from the sample.
  • tissue or cells may be fresh or frozen.
  • the tissues or cells may be in a form of cell/tissue extracts.
  • the tissues or cells may be obtained from a single or multiple different tissues or anatomical regions.
  • samples may be obtained using biopsies, swabs. rinse fluids, from aspirates, etc. in preferred aspects, the sample will he obtained from a surface of an individual such as die skin, a mucous membrane, an open wound, ulcerated tissue, etc.
  • the sample will contain or is suspected to contain at least one microorganism (e.g., bacteria, yeast, virus, fungus, mycoplasma, etc.), and preferably its nucleic acid (e.g., DNA, RNA, etc.).
  • the sample may contain two or more different types of microorganisms (e.g., mixture of two distinct families of bacteria (e.g., streptococcus and E.coli, etc.) or two or more strains of the same species of microorganism.
  • the sample may contain a mixture of a host cell (e.g., human tissue, animal tissue, etc.) and one or more microorganisms.
  • a sample may be a biopsy sample fro the rectum, colon, skin, oral or gastric mucosa, trachea, lung, etc., each of which are known to contain microbial entities (e.g., virus, bacteria, yeast, etc.)
  • microbial entities e.g., virus, bacteria, yeast, etc.
  • nucleic acid information is whole genome information of all cells and/or microorganisms present in the sample.
  • nucleic acid information can be obtained by processing the sample to obtain DNA and/or RNA from the sample to further analyze relevant information.
  • the nucleic acid information can be obtained directly from a database that stores the nucleic acid information of the including DNA sequence analysis information and/or RNA sequence information (i.e., where the microorganism is an RNA virus, etc.) of a plurality of short reads at a length between 50-500 base pairs, preferably between 100-300 base pairs, that can be obtained by whole genome sequencing and/or exome sequencing (typically at a coverage depth of at least lOx, more typically at least 20x) and/or RNAseq using next generation sequencing.
  • DNA data may also be provided from an already established sequence record (e.g., SAM, BAM, FASTA, FASTQ, or VCF file) from a prior sequence determination.
  • data sets may include unprocessed or processed data sets, and exemplary data sets include those having BAM format, SAM format, FASTQ format, or FASTA format.
  • BAM format or as BAMBAM diff objects (e.g., US2012/0059670A1 and US2012/0066001A1).
  • computational analysis of the sequence data may be performed in numerous manners. In most preferred methods, however, analysis is performed in silico by location- guided synchronous alignment of a sample from a wound/injury and normal samples as, for example, disclosed in US 2012/0059670A1 and US 2012/0066001A1 using BAM files and BAM servers. Such analysis advantageously reduces false positive neoepitopes and significantly reduces demands on memory and computational resources.
  • Such obtained nucleic acid sequences can be aligned with a reference sequence to locate the short reads relative to the reference sequence and/or separate out some of the short reads as non-aligned sequences.
  • reference sequence may vary depending on the type of samples and/or type of microorganisms interested and/or type of analysis further required.
  • the reference sequence may include genomic sequence of one or more microorganisms.
  • the chimeric reference nucleic acid sequence includes at least 10, at least 20, at least 50, at least 100, or substantially all possible microorganisms that may be present in the sample.
  • the chimeric reference nucleic acid sequence may vary depending on the type of samples that are analyzed (e.g., human v.
  • the chimeric reference nucleic acid sequence may be constructed by types of microorganisms.
  • several different chimeric reference nucleic acid sequences can be constructed, each of which has merged genomic nucleic acid sequences of known microorganism of same species or same family (e.g., bacterial chimeric reference nucleic acid sequence, E.coli chimeric reference nucleic acid sequence having nucleic acid sequences of all known strains of E coli, etc.).
  • the chimeric reference nucleic acid sequence may also include a human genome sequence as shown in FIG. 1A or a human genome sequence merged with the two or more microorganism genome sequences as shown in FIG. 1C Typically, where the chimeric reference nucleic acid sequence is a human genome sequence merged with the two or more microorganism genome sequences, human genome sequence to form the chimeric reference nucleic acid sequence is derived from a whole genome nucleic acid sequences of a healthy tissue of a same individual where the sample to be analyzed are derived.
  • the human genome sequence to form the chimeric reference nucleic acid sequence is derived from genome sequences of a plurality of individuals, preferably stratified by gender, or an average or consensus sequence. Most typically, the reference genome will be or encompass the entire genome. However, smaller portions of the genome are also contemplated and include at least one chromosome, or two- five chromosomes, or five-ten chromosomes, or more than ten chromosomes. Alternatively, the reference genome may also be only representative of a portion (e.g., between 1-10%, between 10-30%, between 30-60%, or between 60-90%) of the entire exome or entire transcriptome.
  • the reference genome will typically include at least 10%, or at least 30% , or at least 50%, or at least 70% of the entire genome of the human (or other species).
  • the chimeric reference nucleic acid sequence can be constructed in a fusion chromosome format or structure (e.g., in a BAM format or file), in which genomic sequence of each human chromosome (e.g., chromosome 14, chromosome 15, etc.) is merged with the two or more microorganism genome sequences to form a plurality of chimeric reference nucleic acid sequence for each chromosome.
  • genomic sequence of each human chromosome e.g., chromosome 14, chromosome 15, etc.
  • the sequence database is then updated with the so produced chimeric nucleic acid sequence file.
  • the short reads can then be processed using incremental synchronized alignment with the reference sequence.
  • the genomic analysis is performed using a software tool in which a chimeric reference nucleic acid sequence is synchronized and incrementally compared against the nucleic acid sequence from the sample.
  • a software tool includes BAMBAM as previously described in W02013/074058A1, incorporated by- reference herein.
  • the form of such generated synchronously aligned sequences may vary depending on the type of reference sequence used in the alignment.
  • the reference sequence is a chimeric nucleic acid having a plurality of microorganisms’ genomic sequences only
  • the synchronously aligned sequences generated from the nucleic acid sequences obtained from the sample would be a linear combination of microorganisms’ genomic sequences aligned with the chimeric reference sequence.
  • the nucleic acid sequences obtained from the sample contains any human nucleic acid sequences, such human nucleic acid sequences that are not aligned with the chimeric reference sequence would be left out.
  • such human nucleic acid sequences may be appended to the end of the linear combination of microorganisms’ genomic sequences.
  • the synchronously aligned sequences generated from the nucleic acid sequences obtained from the sample would be a linear human genomic nucleic acid sequence as aligned with the reference sequence.
  • the nucleic acid sequences obtained from the sample contains any non-human genomic nucleic acid sequence, such non- human genomic nucleic acid sequence that are not aligned with the human genome reference sequence would be left out.
  • such non-human genomic nucleic acid sequence may be appended to the end of the linear human genomic nucleic acid sequence.
  • the reference sequence is a chimeric reference nucleic acids sequence having a human genomic nucleic acid sequence merged (or appended) with a plurality of microorganisms’ genomic nucleic acid sequence
  • the synchronously aligned sequences generated from the nucleic acid sequences obtained from the sample would be a linear human genomic nucleic acid sequence merged with one or more microorganisms’ genomic nucleic acid sequence as aligned with the chimeric reference sequence.
  • the synchronously aligned sequences can then be further analyzed by comparing the synchronously aligned sequences with a control nucleic acid sequence.
  • the nucleic acid sequence data set can be obtained from a database, preferably in BAM or SAM format, in which each nucleic acid sequence is accompanied with location information relative to the reference sequence in some embodiments, the control nucleic acid sequence is the same nucleic acid sequence with the reference sequence that the nucleic acid sequences from the sample are aligned.
  • control nucleic acid sequence is different nucleic acid sequence from the reference sequence that the nucleic acid sequences from the sample are aligned.
  • the control sequence can be the reference sequence having only human genomic nucleic acid sequence or a nucleic acid sequence including a plurality of microorganisms’ genomic nucleic acid sequence.
  • only a portion of the synchronously aligned sequences that were not aligned previously with the reference sequence will be analyzed against the control nucleic acid sequence (e.g., a fusion sequence of a plurality of microorganisms’ genomic nucleic acid sequence, etc.).
  • FIG. 2. shows at least three scenarios how the nucleic acid reads obtained from the sequencer can be processed via an aligner and the BAMBAM analyzer.
  • scenarios 1-3 a plurality of short reads that may contain mixed population of human genome sequence reads and microorganism sequence reads are obtained from the sequencer.
  • the short reads are aligned with a reference sequence comprising a fused human genome sequence and a plurality of microorganism sequence at one end of the human genome sequence.
  • the aligned sequence at the aligner can then be formatted in BAM file format and analyzed for further information by comparing with the control nucleic acid sequence comprising a fused human genome sequence (HG) and a plurality of microorganism sequence (MO) at one end of the human genome sequence.
  • the reference sequence used in aligner and the control nucleic acid sequence used in BAMBAM analysis can be the same or substantially similar file (e.g., having changed or altered order of microorganism sequences, etc ).
  • the short reads are aligned with a reference sequence comprising a fused microorganism sequences of a plurality of microorganisms.
  • the human genome sequence reads can be discarded from further analysis, and only aligned microorganism sequences are converted into BAM file format.
  • Such converted microorganism sequences can then he analyzed by comparing with the control nucleic acid sequence comprising a fused microorganism sequences of a plurality of microorganisms or a control sequence comprising a fused human genome sequence (HG) and a plurality of microorganism sequence (MO) at one end of the human genome sequence.
  • the short reads are aligned with a reference sequence comprising a human genome sequence (HG) only.
  • the unaligned sequences (supposedly non-human sequences) are separated out and converted into BAM file format.
  • Such converted unaligned sequences are then analyzed by comparing with the control nucleic acid sequence comprising a fused microorganism sequences of a plurality of microorganisms or a control sequence comprising a fused human genome sequence (HG) and a plurality of microorganism sequence (MO) at one end of the human genome sequence.
  • origin of the nucleic acid sequence from the sample can be identified by aligning the nucleic acid sequence with the control nucleic acid sequence having a plurality of microorganisms’ genomic nucleic acid sequences.
  • each genomic sequence merged in the control nucleic acid sequence is associated with the microorganism information and the location in the chimeric reference nucleic acid.
  • identification of the origin of the nucleic acid sequence from the sample can be rapidly and readily accomplished by determining the aligned locus in the control nucleic acid sequence and number of reads aligned in such locus
  • the origin of the nucleic acid sequence from the sample can be identified by identifying the portion of the chimeric reference nucleic acid sequence that are aligned with at least 5 reads, at least 10 reads, at least 20 reads, etc.
  • the threshold for the number of reads in such embodiments can be pre- determined based on the sequencing depth (e.g., lOx, 20x, 40x, 50x, etc.).
  • any aligned read that has less than 90% homology, less than 80% homology, or less than 70% homology with the aligned chimeric reference nucleic acid sequence can be disregarded from the further analysis.
  • any aligned reads, taken together, that covers less than 50%, less than 40%, less than 30%, less than 20%, of any organisms' genomic sequences in the control nucleic acid sequence can be disregarded from the further analysis to reduce any false positive signal.
  • Identification of the microorganism’s origin can be expanded to multiple microorganisms in the microbiome of a sample.
  • the origins of the nucleic acid sequences from the sample can be identified by identifying the portions of the control nucleic acid sequence that are aligned with at least 5 reads, at least 10 reads, at least 20 reads, etc.
  • the nucleic acid sequences from the sample include reads that are aligned to the genomic nucleic acid sequence of microorganism A, B, and C, it is likely that the sample includes microorganism A, B, and C.
  • the threshold for the number of reads in such embodiments can be also pre-determined based on the sequencing depth (e.g , lOx, 2Qx, 4Qx, 50x, etc ).
  • any aligned read that has less than 90% homology, less than 80% homology, or less than 70% homology with the aligned chimeric reference nucleic acid sequence can be disregarded from the further analysis.
  • any aligned reads, taken together, that covers less than 50%, less than 40%, less than 30%, less than 20%, of any organisms’ genomic sequences in the chimeric reference nucleic acid sequence can be disregarded from the further analysis to reduce any false positive signal.
  • any presence or emergence of mutation in the microorganism in the sample can be identified by aligning with the control nucleic acid and identifying a mismatched nucleotide sequence indicating a mutation (e.g., a deletion, a point mutation, an insertion, a duplication, etc.).
  • ratios among the multiple types of microorganisms in the sample can be determined from comparing the quantities of reads aligned with the control nucleic acid sequence. For example, once the origins of the nucleic acid sequences from the sample are identified by aligning with the control nucleic acid sequence, the quantities of reads that are aligned to each portion of the control nucleic acid sequence corresponding to distinct genomic nucleic acid sequence of a microorganism can be determined. The ratio between or among the quantities of reads is likely to reflect the amount ratio between or among the multiple organisms in the sample.
  • a relative quantity or a quantity change of a microorganism can be determined by quantifying the number of reads that are aligned in a portion of the chimeric nucleic acid sequence in a first and second condition.
  • One use of such example may include determination of quantity change of a strain of MRSA on a burn wound of a patient infected by MRSA upon antibiotic A treatment to determine the effect of antibiotic A in MRSA infection treatment.
  • genomic nucleic acid sequences are obtained from at least two skin samples (e.g., via biopsy, wiping the surface of the skin, etc.): one before the antibiotic A treatment and another after the antibiotic A treatment, and the obtained genomic nucleic acid sequences are aligned with the control nucleic acid sequence including full genomic sequence of MRSA.
  • the number of reads aligned to a portion of genomic sequence of MRSA, preferably same portion of genomic sequence of MRSA, in the chimeric reference nucleic acid sequence can be quantified for the genomic nucleic acid sequences of before antibiotic A treatment and genomic nucleic acid sequences of after antibiotic A treatment.
  • the relative quantity or changes in the quantity can be measured by either a difference in absolute number of reads in those samples or a percentile change (increase or decrease) between those samples.
  • Another use of such example may include detection of a change in microbiome balance in a wound by determining the ratio changes among a plurality of microorganisms in the injured tissue in such example, tissue samples can be obtained in different time points via biopsies, and genomic nucleic acid sequences from such tissues are obtained.
  • genomic nucleic acid sequences are aligned with the control nucleic acid sequence including full length human genomic nucleic acid (e.g., organized by individual chromosome, etc.) merged with genomic nucleic acid sequences of any potential microorganisms that may be present in the wound tissue (e.g., actinobacteria, saprospiraceae, capnocytophaga, christensenellaceae, acidobacteria, corynebacterium, etc.).
  • any potential microorganisms e.g., actinobacteria, saprospiraceae, capnocytophaga, christensenellaceae, acidobacteria, corynebacterium, etc.
  • the number of reads aligned to a portion of genomic sequence of microorganisms in the control nucleic acid sequence can be quantified for determining the ratio among the microorganisms present in the colon cancer tissue microbiome in the patient as well as changes of the microorganism quantity in the injured tissue.
  • the number of reads aligned with genomic nucleic acid sequences of a microorganism can be normalized with a number of reads aligned with a human genome sequence.
  • the nucleic acid sequence obtained from the sample can be aligned with a control nucleic acid sequence that has a human genomic nucleic acid sequence derived from a matched normal tissue from the same patient to so obtain unmatched reads with the control nucleic acid sequence that may represent ulcer- specific mutations.
  • a diseased tissue e.g., diabetic ulcer, etc.
  • the nucleic acid sequence obtained from the sample can be aligned with a control nucleic acid sequence that has a human genomic nucleic acid sequence derived from a matched normal tissue from the same patient to so obtain unmatched reads with the control nucleic acid sequence that may represent ulcer- specific mutations.
  • the nucleic acid sequence obtained from the sample can be aligned with a control nucleic acid sequence that has a human genomic nucleic acid sequence derived from a healthy individual, and further aligned with a nucleic acid sequence derived from a matched normal tissue from the same patient in order to obtain information on ulcer- specific mutation as well as individual germline variations.
  • contemplated systems and methods will be especially advantageous to identify the presence of one or more microorganisms in an injured or diseased tissue (and particularly skin), to track the changes in the microbiome of a diseased tissue, without a priori knowledge on the existence and/or types of microorganisms in the sample.
  • silico microbiome analysis can provide quick and thorough results by avoiding cumbersome amplification and/or isolation process of individual genomic nucleic acid of microorganisms using a common primer and/or a specific primer to the genomic nucleic acid of microorganisms.
  • the inventors further contemplate that based on such obtained microbiome information of the sample, various actions can be taken further. For example, where the samples are obtained from patient’s infected tissue before and after antibiotics treatment, the relative quantity and/or change of microbiome of the patient’s infected tissue can be analyzed to update and/or generate the patient’s record with respect to the effectiveness of the antibiotics treatment.
  • a treatment regimen can be generated or updated based on the effectiveness of the antibiotics treatment can be provided, for example, changing to another antibiotics if the quantities of one or two types of bacteria are not reduced or even increased after the antibiotics treatment or if the balance of the microbiome is changed due to decrease or increase of one or two types of bacteria over others, and so on.
  • the injured tissue can also be analyzed for the immune status, and obtained omics information from the injured or otherwise non-healthy tissue can be used to determine an immune cell signature, which will advantageously guide a practitioner towards a specific treatment regimen.
  • omics data from injured tissue can be analyzed to identify immune cell signatures by associating presence and/or quantity of mRNA of specific subpopulations of immune cells as is disclosed in copending US provisional application with the serial number 62/647621, which was filed 3/23/2018, and which is incorporated by reference herein.
  • suitable omics analyses also include proteomics analyses that will be able to detect specific proteins or protein fragments that are unique to a specific microorganism.
  • the human body possesses commensal bacteria, fungi, viruses and larger organisms at a ratio in the range of 1:1 to 10:1 (commensahhuman cells).
  • bacteria a human can harbor 1014 bacterial cells and 105-106 bacterial generations per human generation.
  • 19 phyla have bacteria have been detected but most bacteria fall into four different phyla: Actinobacteria, Firmicutes, Bacteroidetes, and proteobacteria (as determined by 16S RNA metagenomic sequencing).
  • Actinobacteria Firmicutes, Bacteroidetes, and proteobacteria (as determined by 16S RNA metagenomic sequencing).
  • FIG, 3 illustrates one embodiment of checkpoint inhibitors and response to immunotherapy during a wound healing process
  • Systemic Approach to Wound Healing as contemplated by the inventors comprise NGS DNA Sequencing Analysis for Microhiome Characterization: Optimal Treatment and Wound Management. NGS RNA Sequencing Analysis (Simultaneous) of Host Wound plus wound associated microbial community.
  • FIG. 4 illustrates one embodiment of three immune modulating compounds and control of cutaneous infection.
  • FIG. 5 illustrates one embodiment of nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) during the wound healing process.
  • FIG. 6 illustrates one embodiment of compounds that synergize with a TLR2 ligand to induce IL-8 secretion.
  • FIG. 7 illustrates one embodiment of tissue polypeptide specific (TPS) antigen synergy with other germ line-encoded pattern recognition receptors (PRRs).
  • FIG. 8 illustrates one embodiment of Bioluminescent S. aureus as a Tool to Identify Novel Therapeutics for MRSA.
  • FIG. 9 illustrates one embodiment of time course of neutrophil recruitment following TPS treatment.
  • FIGs. 1 illustrates one embodiment of time course of neutrophil recruitment following TPS treatment.
  • FIG. 10 and 11 illustrates one embodiment of wound progression following infection with S. aureus.
  • FIG. 12 illustrates one embodiment of bacterial enumeration - treatments significantly decrease wound size and bacterial burden.
  • FIG. 13 illustrates one embodiment of formulation development and formulation stability.
  • FIG. 14 illustrates one embodiment of TPS cream formulation. Results against surgical site infection by S. aureus NRS 384.

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Abstract

Les systèmes et les méthodes selon l'invention concernent l'analyse in silico de caractérisation du microbiome ou d'identification de la contamination d'une entité biologique ou d'un échantillon, le plus souvent un tissu malade ou endommagé. De préférence, les systèmes et les méthodes selon l'invention utilisent des séquences d'acides nucléiques chimériques qui comprennent une pluralité de séquences de génome de microorganismes fusionnées ensemble pour former un seul fichier de séquence d'acides nucléiques en format BAM de sorte à aligner les séquences d'acides nucléiques obtenues à partir de l'entité biologique ou de l'échantillon.
PCT/US2019/054875 2018-10-05 2019-10-04 Profilage du microbiome moléculaire pour la cicatrisation de plaies WO2020073007A1 (fr)

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WO2015126904A1 (fr) * 2014-02-18 2015-08-27 The Arizona Board Of Regents On Behalf Of The University Of Arizona Identification bactérienne dans des infections patentes
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WO2015126904A1 (fr) * 2014-02-18 2015-08-27 The Arizona Board Of Regents On Behalf Of The University Of Arizona Identification bactérienne dans des infections patentes
US20170159108A1 (en) * 2014-05-06 2017-06-08 Is-Diagnostics Ltd. Microbial population analysis
US20170367640A1 (en) * 2014-10-21 2017-12-28 uBiome, Inc. Method and system for microbiome-derived diagnostics and therapeutics for conditions associated with microbiome taxonomic features
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