Patent Grant
11525164
This application contains a Sequence Listing which is being submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 13, 2022, is named 38837-202_SEQUENCE-LISTING_ST25.txt and is 122,158 bytes in size.
The present invention provides for a method to determine the micron-scale spatial co-localization of genomic material within a 3-dimensional sample by microdroplet encapsulation and high-throughput sequencing of fractionations of microclusters from the sample.
The local spatial organization of the gut microbiome influences a variety of ecological properties, including colonization (see Lee, S. M. et al. Bacterial colonization factors control specificity and stability of the gut microbiota. Nature 1-6 (2013). doi:10.1038/nature12447; Pereira, F. C. & Berry, D. Microbial nutrient niches in the gut. Environ Microbiol 19, 1366-1378 (2017); Donaldson, G. P. et al. Gut microbiota utilize immunoglobulin A for mucosal colonization. Science 360, 795-800 (2018); Whitaker, W. R., Shepherd, E. S. & Sonnenburg, J. L. Tunable Expression Tools Enable Single-Cell Strain Distinction in the Gut Microbiome. Cell 169, 538-546.e12 (2017)), metabolism (see Nagara, Y., Takada, T., Nagata, Y., Kado, S. & Kushiro, A. Microscale spatial analysis provides evidence for adhesive monopolization of dietary nutrients by specific intestinal bacteria. PLoS ONE 12, e0175497 (2017)), host-microbe and inter-microbial interactions (see Wexler, A. G. et al. Human symbionts inject and neutralize antibacterial toxins to persist in the gut. Proc. Natl. Acad. Sci. U.S.A. 201525637-6 (2016). doi:10.1073/pnas.1525637113) and community stability (see Reichenbach, T., Mobilia, M. & Frey, E. Mobility promotes and jeopardizes biodiversity in rock—paper—scissors games. Nature 448, 1046-1049 (2007); Coyte, K. Z., Schluter, J. & Foster, K. R. The ecology of the microbiome: Networks, competition, and stability. Science 350, 663-666 (2015)). However, current microbiome profiling approaches such as metagenomic sequencing require homogenization of the input material and thus the physical destruction of any underlying spatial information. While imaging techniques could reveal useful spatial information, they rely on hybridization by short DNA probes of limited spectral diversity, yielding data with low taxonomic resolution and often requiring extensive empirical optimization (see Valm, A. M., Welch, J. L. M. & Borisy, G. G. CLASI-FISH: Principles of combinatorial labeling and spectral imaging. Systematic and Applied Microbiology 35, 496-502 (2012); Amann, R. & Fuchs, B. M. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nature Reviews Microbiology 6, 339-348 (2008)). Bacteria are also densely packed in communities, limiting identification and analysis of individual cells (see Mark Welch, J. L., Hasegawa, Y., McNulty, N. P., Gordon, J. I. & Borisy, G. G. Spatial organization of a model 15-member human gut microbiota established in gnotobiotic mice. Proc. Natl. Acad. Sci. U.S.A. 21, 201711596-E9114 (2017)). Imaging approaches can profile simple synthetic communities composed of a small number of cultivable species (see Geva-Zatorsky, N. et al. (2015); Whitaker, W. R., Shepherd, E. S. & Sonnenburg, J. L. (2017), but imaging techniques are challenging to scale to complex and diverse natural microbiomes. A direct and unbiased method for high-taxonomic resolution and micron-scale dissection of natural microbial biogeography is critically needed to mechanistically elucidate the role of the gut microbiome in health and disease.
In macroecology, plot sampling is used to study the spatial organization of large ecosystems, which are otherwise impractical to fully characterize. By surveying many smaller plots from a larger region, one can delineate local distributions of species and statistically infer fundamental properties of global community organization and function. The methods of the present invention provide a multiplexed sequencing technique that analyzes microbial cells in their native geographical context to statistically reconstruct the local spatial organization of the microbiome. Microbial colocalization can be shown in a variety of biological samples, including, soil, gut and biofilm. The methods of the present invention can determine which microbes are spatially associated with which other microbes and can comprise the following steps: (1) taking an intact sample and preserving its spatial structure via in-situ perfusion and polymerization of a chemical matrix, (2) processing that matrix by chemical or enzymatic steps, (3) fractioning the matrix into smaller microparticles, (4) capture each microparticle in emulsion droplets with unique molecular barcodes, (5) PCR amplification of said genetic material from microparticles in each droplet, (6) breaking up the droplets and pooling amplified material for next-generation sequencing measurements.
The present disclosure provides for a method of determining the compositions/identities and/or abundances of organisms (e.g., microbes such as microbial identities and/or abundances) in a biological sample. The method may comprise: (a) immobilizing the biological sample in a matrix; (b) fracturing/breaking the matrix (that comprises the biological sample) into clusters; and (c) determining identities and/or abundances of microbes in the clusters.
The clusters (each cluster of the clusters) may comprise co-localized cells.
In step (c), the identities and/or abundances of organisms (e.g., microbes) may be determined by sequencing DNAs (e.g., genomic DNAs) and/or RNAs.
In step (c), the identities and/or abundances of organisms (e.g., microbes) may be determined by analyzing proteins, polypeptides, carbohydrates, and/or metabolites.
The matrix may be a gel matrix.
In step (a), the biological sample may be immobilized via perfusion and polymerization of the matrix.
The matrix may comprise a polymer, such as an acrylamide polymer.
The matrix may comprise a plurality of 16S ribosomal RNA (16S rRNA) (gene) amplification primers. The plurality of 16S rRNA amplification primers may be covalently linked to the matrix. The plurality of 16S rRNA (gene) amplification primers may be linked to the matrix through photocleavable linkers, such as acrydite linkers.
The method may further comprise step (d) processing the matrix by chemical or enzymatic means after step (a) or step (b). For example, step (d) may comprise lysing cells. The method may further comprise step (e) passing the clusters through a filter for size selection. After step (e), the clusters may have a median diameter ranging from about 1 μm to about 100 μm, from about 10 μm to about 50 μm, from about 1 μm to about 20 μm, from about 1 μm to about 50 μm, from about 10 μm to about 40 μm, from about 10 μm to about 80 μm, about 1 μm, about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 170 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 80 μm, or about 900 μm.
The clusters may be microparticles.
In step (b), the matrix may be fractured through cryo-fracturing such as cryo-bead beating.
In step (c), identities and/or abundances of organisms (e.g., microbes) may be determined through droplet-based encapsulation.
The droplet-based encapsulation may be through co-encapsulating the clusters with beads in droplets (e.g., emulsion droplets), wherein each droplet comprises (consists essentially of, or consists of) a cluster and a bead, each bead comprising a unique molecular barcode.
The beads may comprise a plurality of 16S rRNA (gene) amplification primers. The plurality of 16S rRNA (gene) amplification primers linked to each bead may comprise a unique (and/or identical) molecular barcode.
The plurality of 16S rRNA (gene) amplification primers may be covalently linked to the beads.
The plurality of 16S rRNA (gene) amplification primers may be linked to the beads through photocleavable linkers, such as acrydite linkers.
The beads may comprise a polymer, such as an acrylamide polymer.
The droplet-based encapsulation may be through capturing the clusters in emulsion droplets comprising molecular barcodes, each emulsion droplet comprising identical molecular barcodes.
The (emulsion) droplets may have a diameter ranging from about 35 μm to about 45 μm, from about 1 μm to about 100 μm, from about 10 μm to about 50 μm, from about 1 μm to about 20 μm, from about 1 μm to about 50 μm, from about 10 μm to about 40 μm, or from about 10 μm to about 80 μm.
The method may further comprise step (f) cleaving the plurality of 16S rRNA (gene) amplification primers from the matrix and/or the beads.
The method may further comprise step (g) degrading the matrix. The matrix may be degraded through exposure to reducing conditions.
The method may further comprise step (h) polymerase chain reaction (PCR) amplification.
The sequencing/analysis may be deep sequencing or any sequencing or other techniques discussed herein or understood by a skilled artisan.
The biological sample may be obtained from a mammal. The biological sample may be obtained from a nervous system, a pulmonary system, a peripheral vascular system, a cardiovascular system, and/or a gastrointestinal system of a mammal. The biological sample may be obtained from the brain, a lung, a bronchus, an alveolus, an artery, a vein, a heart, an esophagus, a stomach, a small intestine, a large intestine, or combinations thereof.
The biological sample may be obtained from a tumor or may be a tumor sample.
The biological sample may be a soil sample, a gut sample, and/or a biofilm sample.
The biological sample may be an environmental sample.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The methods and systems of the present disclosure provide a Metagenomic Plot-sampling by sequencing (MaP-seq), a multiplexed sequencing technique that analyzes microbial cells in their native geographical context to statistically reconstruct the local spatial organization of the microbiome (
The present disclosure provides for a method of determining the compositions/identities and/or abundances of organisms (e.g., microbes such as microbial identities and/or abundances) in a biological sample. The method may comprise: (a) immobilizing the biological sample in a matrix; (b) fracturing/breaking the matrix (that comprises the biological sample) into clusters; and (c) determining identities and/or abundances of microbes in the clusters.
The clusters (each cluster of the clusters) may comprise co-localized cells.
In step (c), the identities and/or abundances of organisms (e.g., microbes) may be determined by sequencing DNAs (e.g., genomic DNAs) and/or RNAs.
In step (c), the identities and/or abundances of organisms (e.g., microbes) may be determined by analyzing proteins, polypeptides, carbohydrates, and/or metabolites.
The matrix may be a gel matrix.
In step (a), the biological sample may be immobilized via perfusion and polymerization of the matrix.
The matrix may comprise a polymer, such as an acrylamide polymer.
The matrix may comprise a plurality of 16S ribosomal RNA (16S rRNA) (gene) amplification primers. The plurality of 16S rRNA amplification primers may be covalently linked to the matrix. The plurality of 16S rRNA (gene) amplification primers may be linked to the matrix through photocleavable linkers, such as acrydite linkers.
The method may further comprise step (d) processing the matrix by chemical or enzymatic means after step (a) or step (b). For example, step (d) may comprise lysing cells. The method may further comprise step (e) passing the clusters through a filter for size selection. After step (e), the clusters may have a median diameter ranging from about 1 μm to about 100 μm, from about 10 μm to about 50 μm, from about 1 μm to about 20 μm, from about 1 μm to about 50 μm, from about 10 μm to about 40 μm, from about 10 μm to about 80 μm, about 1 μm, about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 170 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 80 μm, or about 900 μm.
The clusters may be microparticles.
In step (b), the matrix may be fractured through cryo-fracturing such as cryo-bead beating.
In step (c), identities and/or abundances of organisms (e.g., microbes) may be determined through droplet-based encapsulation.
The droplet-based encapsulation may be through co-encapsulating the clusters with beads in droplets (e.g., emulsion droplets), wherein each droplet comprises (consists essentially of, or consists of) a cluster and a bead, each bead comprising a unique molecular barcode.
The beads may comprise a plurality of 16S rRNA (gene) amplification primers. The plurality of 16S rRNA (gene) amplification primers linked to each bead may comprise a unique (and/or identical) molecular barcode.
The plurality of 16S rRNA (gene) amplification primers may be covalently linked to the beads.
The plurality of 16S rRNA (gene) amplification primers may be linked to the beads through photocleavable linkers, such as acrydite linkers.
The beads may comprise a polymer, such as an acrylamide polymer.
The droplet-based encapsulation may be through capturing the clusters in emulsion droplets comprising molecular barcodes, each emulsion droplet comprising identical molecular barcodes.
The (emulsion) droplets may have a diameter ranging from about 35 μm to about 45 μm, from about 1 μm to about 100 μm, from about 10 μm to about 50 μm, from about 1 μm to about 20 μm, from about 1 μm to about 50 μm, from about 10 μm to about 40 μm, or from about 10 μm to about 80 μm.
The method may further comprise step (f) cleaving the plurality of 16S rRNA (gene) amplification primers from the matrix and/or the beads.
The method may further comprise step (g) degrading the matrix. The matrix may be degraded through exposure to reducing conditions.
The method may further comprise step (h) polymerase chain reaction (PCR) amplification.
The sequencing/analysis may be deep sequencing, or any sequencing or other techniques discussed herein or understood by a skilled artisan.
The biological sample may be obtained from a mammal. The biological sample may be obtained from a nervous system, a pulmonary system, a peripheral vascular system, a cardiovascular system, and/or a gastrointestinal system of a mammal. The biological sample may be obtained from the brain, a lung, a bronchus, an alveolus, an artery, a vein, a heart, an esophagus, a stomach, a small intestine, a large intestine, or combinations thereof.
The biological sample may be obtained from a tumor or may be a tumor sample.
The biological sample may be a soil sample, a gut sample, and/or a biofilm sample.
The biological sample may be an environmental sample.
The present nucleic acids (e.g., primers such as 16S rRNA amplification primers) may or may not comprise barcode elements (e.g., a unique molecular barcode for each bead). Barcode elements may be used as identifiers for a cluster and may indicate the presence of one or more specific sequences in a cluster (e.g., DNA or RNA). Members of a set of barcode elements have a sufficiently unique nucleic acid sequence such that each barcode element is readily distinguishable from the other barcode elements of the set. Barcode elements may be of any length of nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30 or more nucleotides in length. Detecting barcode elements and determining the nucleic acid sequence of a barcode element or plurality of barcode elements are used to determine the presence of an associated DNA or RNA element. Barcode elements can be detected by any method known in the art, including sequencing or microarray methods.
In one embodiment, barcoded primers are constructed via a split-and-pool primer extension strategy with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more barcode extension rounds. Klein, A. M. et al. Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells. Cell 161, 1187-1201 (2015). Bose, S. et al. Scalable microfluidics for single-cell RNA printing and sequencing. Genome Biology 1-16 (2015). doi:10.1186/s13059-015-0684-3.
Microbial identities and/or abundances, or specific changes in microbiome or microbiota discussed herein can be detected using various methods, including, without limitation, quantitative PCR or high-throughput sequencing methods which detect over- and under-represented genes in the total bacterial population (e.g., 454-sequencing for community analysis; screening of microbial 16S ribosomal RNAs (16S rRNA), etc.), or transcriptomic or proteomic studies that identify lost or gained microbial transcripts or proteins within total bacterial populations. See, e.g., U.S. Patent Publication No. 2010/0074872; Eckburg et al., Science, 2005, 308:1635-8; Costello et al., Science, 2009, 326:1694-7; Orrice et al., Science, 2009, 324:1190-2; Li et al., Nature, 2010, 464: 59-65; Bjursell et al., Journal of Biological Chemistry, 2006, 281:36269-36279; Mahowald et al., PNAS, 2009, 14:5859-5864; Wikoff et al., PNAS, 2009, 10:3698-3703.
The composition/identities and abundance of the established microbiota can be studied by sequencing the 16S ribosomal RNA (or 16S rRNA) gene of a sample. 16S rRNA is a component of the 30S small subunit of prokaryotic ribosomes.
In additional embodiments, the determining step involves screening bacterial 16S rRNA genes using PCR.
The DNA library may be a genomic DNA or metagenomic library. A metagenomic library is a collection of the genomic DNAs of a mixture of organisms, such as a mixture of microbes.
The present method may or may not comprise a step of processing the matrix by chemical or enzymatic means after or before any suitable step, including, but not limited to, cell lysis, addition of a detergent or surfactant, addition of protease, addition of RNase, alcohol precipitation (e.g., ethanol precipitation, or isopropanol precipitation), salt precipitation, organic extraction (e.g., phenol-chloroform extraction), solid phase extraction, silica gel membrane extraction, CsCl gradient purification.
Photocleavable linkers may be cleaved by UV light. Photocleavable linkers may be a photocleavable oligonucleotide. Photocleavable linkers may be o-nitrobenzyl derivatives (Zhao et al. 2012: o-nitrobenzyl alcohol derivatives). U.S. Patent Publication No. 20080227742.
Sequencing
DNA may be amplified via polymerase chain reaction (PCR) before being sequenced.
The present method may comprise a step of analyzing DNA or RNA by sequencing or by microarray analysis. It should be appreciated that any suitable means of determining DNA sequence may be used in the present method.
The DNA may be sequenced using vector-based primers; or a specific gene is sought by using specific primers. PCR and sequencing techniques are well known in the art; reagents and equipment are readily available commercially.
Non-limiting examples of sequencing methods include Sanger sequencing or chain termination sequencing, Maxam-Gilbert sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat. Biotechnol., 16:381-384 (1998)), and sequencing by hybridization (Chee et al., Science, 274:610-614 (1996); Drmanac et al., Science, 260:1649-1652 (1993); Drmanac et al., Nat. Biotechnol., 16:54-58 (1998)), NGS (next-generation sequencing) (Chen et al., Genome Res. 18:1143-1149 (2008); Srivatsan et al. PloS Genet. 4:e1000139 (2008)), Polony sequencing (Porreca et al., Curr. Protoc. Mol. Biol. Chp. 7; 7.8 (2006), ion semiconductor sequencing (Elliott et al., J. Biomol Tech. 1:24-30 (2010), DNA nanoball sequencing (Kaji et al., Chem Soc Rev 39:948-56 (2010), single-molecule real-time sequencing (Flusberg et al., Nat. Methods 6:461-5 (2010), sequencing by synthesis (e.g., Illumina/Solexa sequencing), sequencing by ligation, sequencing by hybridization, nanopore DNA sequencing (Wanunu, Phys Life Rev 9:125-58 (2012), massively Parallel Signature Sequencing (MPSS); pyro sequencing, SOLiD sequencing (McKeman et al. 2009 Genome Res 19:1527-1541; Shearer et al. 2010 Proc Natl Acad Sci USA 107:21104-21109); shortgun sequencing; Heliscope single molecule sequencing; single molecule real time (SMRT) sequencing. U.S. Patent Publication No. 20140329705.
High-throughput sequencing, next-generation sequencing (NGS), and/or deep-sequencing technologies include, but are not limited to, Illumina/Solex sequencing technology (Bentley et al. 2008 Nature 456:53-59), Roche/454 (Margulies et al. 2005 Nature 437:376-380), Pacbio (Flusberg et al. 2010 Nature methods 7:461-465; Korlach et al. 2010 Methods in enzymology 472:431-455; Schadt et al. 2010 Nature reviews. Genetics 11:647-657; Schadt et al. 2010 Human molecular genetics 19:R227-240; Eid et al. 2009 Science 323:133-138; Imelfort and Edwards, 2009 Briefings in bioinformatics 10:609-618), Ion Torrent (Rothberg et al. 2011 Nature 475:348-352)) and more. For example, Polony technology utilizes a single step to generate billions of “distinct clones” for sequencing. As another example, ion-sensitive field-effect transistor (ISFET) sequencing technology provides a non-optically based sequencing technique. U.S. Patent Publication No. 20140329712.
Several methods of DNA analysis are encompassed in the present disclosure. As used herein “deep sequencing” indicates that the depth of the process is many times larger than the length of the sequence under study. Deep sequencing is encompassed in next generation sequencing methods which include but are not limited to single molecule realtime sequencing (Pacific Bio), Ion semiconductor (Ion torrent sequencing), Pyrosequencing (454), Sequencing by synthesis (lilumina), Sequencing by ligations (SOLID sequencing) and Chain termination (Sanger sequencing).
Sequencing reads may be first subjected to quality control to identify overrepresented sequences and low-quality ends. The start and/or end of a read may or may not be trimmed. Sequences mapping to the genome may be removed and excluded from further analysis. As used herein, the term “read” refers to the sequence of a DNA fragment obtained after sequencing. In certain embodiments, the reads are paired-end reads, where the DNA fragment is sequenced from both ends of the molecule.
The level of the DNA or RNA (e.g., mRNA) molecules may be determined/detected using routine methods known to those of ordinary skill in the art. The level of the nucleic acid molecule may be determined/detected by nucleic acid hybridization using a nucleic acid probe, or by nucleic acid amplification using one or more nucleic acid primers.
Nucleic acid hybridization can be performed using Southern blots, Northern blots, nucleic acid microarrays, etc.
Nucleic acid microarray technology, which is also known as DNA chip technology, gene chip technology, and solid-phase nucleic acid array technology, may be based on, but not limited to, obtaining an array of identified nucleic acid probes on a fixed substrate, labeling target molecules with reporter molecules (e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP, etc.), hybridizing target nucleic acids to the probes, and evaluating target-probe hybridization. Jackson et al. (1996) Nature Biotechnology, 14: 1685-1691. Chee et al. (1995) Science, 274: 610-613.
The sensitivity of the assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected.
Nucleic acid amplification assays include, but are not limited to, the polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, quantitative RT-PCR, etc.
Measuring or detecting the amount or level of mRNA in a sample can be performed in any manner known to one skilled in the art and such techniques for measuring or detecting the level of an mRNA are well known and can be readily employed. A variety of methods for detecting mRNAs have been described and may include, Northern blotting, microarrays, real-time PCR, RT-PCR, targeted RT-PCR, in situ hybridization, deep-sequencing, single-molecule direct RNA sequencing (RNAseq), bioluminescent methods, bioluminescent protein reassembly, BRET (bioluminescence resonance energy transfer)-based methods, fluorescence correlation spectroscopy and surface-enhanced Raman spectroscopy (Cissell, K. A. and Deo, S. K. (2009) Anal. Bioanal. Chem., 394:1109-1116).
The methods of the present invention may include the step of reverse transcribing RNA when assaying the level or amount of an mRNA.
Sequencing reads (e.g., the quality-corrected reads) may be mapped onto the genome of the microbe using any alignment algorithms known in the art. Non-limiting examples of such mapping algorithms include Bowtie; Bowtie2 (Langmead et al. 2009; Langmead et al., Fast gapped-read alignment with Bowtie 2. Nature methods 9(4), 357-9 (2012); Burrows-Wheeler Aligner (BWA, see, Li et al: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics, 26(5), 589-95 (2010)); SOAP2 (Li et al., SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics, 25(15), 1966-7 (2009)); GATK; SMRA; PINDEL; SNAP (Zaharia et al., Faster and More Accurate Sequence Alignment with SNAP, arXiv:1111.5572 (2011)]; TMAP1-4; SMALT; and Masai (Siragusa et al., Fast and sensitive read mapping with approximate seeds and multiple backtracking. CoRR abs/1208.4238 (2012)). A recent overview of the alignment algorithms can be found in Li et al., A survey of sequence alignment algorithms for next-generation sequencing. Briefings in Bioinformatics 2010, 11(5), 473-483. U.S. Patent Publication Nos. 20140214334, 20140108323 and 20140315726.
Mathematical algorithms that can be used for alignment also include, the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine optimum alignment. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. In another embodiment, GSNAP (Thomas D. Wu, Serban Nacu “Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics. 2010 Apr. 1; 26(7):873-81. 2010) can also be used.
Algorithms and parameters for alignment can be adjusted depending on the type of bacteria selected, the type of target sequence being characterized, etc.
Mapped reads may be post-processed by removing PCR duplicates (multiple, identical reads), etc.
Organisms
The organism may be a eukaryotic organism, including human and non-human eukaryotic organisms. The organism may be a multicellular eukaryotic organism. The organism may be an arthropod such as an insect. The organism also may be a plant or a fungus. The organism may be prokaryotic.
In one embodiment, the cell is a mammalian cell, such as a human cell. Human cells may include human embryonic kidney cells (e.g., HEK293T cells), human dermal fibroblasts, human cancer cells, etc.
In another embodiment, the cell is a yeast cell. The organism may be a yeast. In yet another embodiment, the cell is a bacterial cell. The organism may be bacteria.
Molecular Biology
In accordance with the present invention, there may be numerous tools and techniques within the skill of the art, such as those commonly used in molecular immunology, cellular immunology, pharmacology, and microbiology. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.
The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.
As used herein, the term “isolated” and the like means that the referenced material is free of components found in the natural environment in which the material is normally found. In particular, isolated biological material is free of cellular components. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, an isolated genomic DNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found. Isolated nucleic acid molecules can be inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated material may be, but need not be, purified.
The term “purified” and the like as used herein refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term “substantially free” is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
The terms “expression profile” or “gene expression profile” refers to any description or measurement of one or more of the genes that are expressed by a cell, tissue, or organism under or in response to a particular condition. Expression profiles can identify genes that are up-regulated, down-regulated, or unaffected under particular conditions. Gene expression can be detected at the nucleic acid level or at the protein level. The expression profiling at the nucleic acid level can be accomplished using any available technology to measure gene transcript levels. For example, the method could employ in situ hybridization, Northern hybridization or hybridization to a nucleic acid microarray, such as an oligonucleotide microarray, or a cDNA microarray. Alternatively, the method could employ reverse transcriptase-polymerase chain reaction (RT-PCR) such as fluorescent dye-based quantitative real time PCR (TaqMan® PCR). In the Examples section provided below, nucleic acid expression profiles were obtained using Affymetrix GeneChip® oligonucleotide microarrays. The expression profiling at the protein level can be accomplished using any available technology to measure protein levels, e.g., using peptide-specific capture agent arrays.
The terms “gene signature” and “signature genes” will be used interchangeably herein and mean the particular transcripts that have been found to be differentially expressed in some prostate cancer patients.
The terms “gene”, “gene transcript”, and “transcript” are used interchangeably in the application. The term “gene”, also called a “structural gene” means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. “Transcript” or “gene transcript” is a sequence of RNA produced by transcription of a particular gene. Thus, the expression of the gene can be measured via the transcript.
The term “genomic DNA” as used herein means all DNA from a subject including coding and non-coding DNA, and DNA contained in introns and exons.
The term “nucleic acid hybridization” refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are “hybridizable” to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under “low stringency” conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid).
The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors include, but are not limited to, plasmids, phages, and viruses.
Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A “cassette” refers to a DNA coding sequence or segment of DNA which codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a “DNA construct” or “gene construct.” A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.
A “polynucleotide” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA and RNA, and means any chain of two or more nucleotides. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.
“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5′- and 3′-non-coding regions, and the like. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, and carbamates) and with charged linkages (e.g., phosphorothioates, and phosphorodithioates). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, and poly-L-lysine), intercalators (e.g., acridine, and psoralen), chelators (e.g., metals, radioactive metals, iron, and oxidative metals), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Modifications of the ribose-phosphate backbone may be done to facilitate the addition of labels, or to increase the stability and half-life of such molecules in physiological environments. Nucleic acid analogs can find use in the methods of the invention as well as mixtures of naturally occurring nucleic acids and analogs. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, and biotin.
The term “polypeptide” as used herein means a compound of two or more amino acids linked by a peptide bond. “Polypeptide” is used herein interchangeably with the term “protein.”
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.
Tissue Fixation/Immobilization of Sample
In one embodiment, a tissue section is fixed and embedded in a gel matrix by in situ perfusion and acrylamide polymerization. Other methods of tissue fixation include using methyl methacrylate and glycol methacrylate, also referred to as Technovit® (emsdiasum.com/microscopy/technical/datasheet/14654 immunohistochemistry.aspx, retrieved, Mar. 26, 2019; see also, Hasegawa et al. Preservation of three-dimensional spatial structure in the gut microbiome, biorxiv.org/content/biorxiv/early/2017/08/11/175224.full.pdf, retrieved, Mar. 26, 2019). Tissues can also be fixed using a combination of sodium acrylate, a monomer used to produce superabsorbent materials, along with the comonomer acrylamide and the crosslinker N—N′-methylenebisacrylamide such as that used with expansion microscopy. Chen et al. Expansion Microscopy Science 347 (6221):543-548 (2015). Other techniques for tissue fixation, include nanoporous hydrogel-fixation, also referred to as CLARITY. Chung et al. Structural and molecular interrogation of intact biological systems. Nature 497:332-337 (2013).
Metagenomic Plot Sampling by Sequencing (MaP-Seq)
MaP-seq was applied to the mouse colonic microbiome. The methods and systems of the present invention could be applied to any structural, anatomic system, including, but not limited to the brain (central nervous system), the pulmonary system (the lungs, bronchi and alveoli), the genitouringary tract, including, but not limited to the kidneys, ureters, bladder, urethra, ovaries, testicles, prostate, penis and vagina, the peripheral vascular and cardiovascular systems, including, but not limited to the arteries (coronary, pulmonary, aorta, femoral, carotid, basilar), veins (pulmonary, vena cava, femoral), heart (left ventricle, right ventricle, left atrium, right atrium), the gastrointestinal system such as the esophagus, stomach (including, but not limited to the fundus and pyloric valve), the liver, gall balder, small intestines (ileum and jejunum), large intestines (colon), the eye and the skin. The methods and systems of the present invention could be applied to any mammalian or non-mammalian species, including, but not limited to, rats, mice, canines, felines, cows, sheep, horses, goats, birds, humans (cadaver material), reptiles and fish.
The methods and systems of the present invention could also be applied to any three-dimensional structure such as a solid tumor of any organ, including, but not limited to, bladder, bone, colon, esophagus, salivary glands, kidney, lung, Central Nervous System, Neuroendocrine System, ovaries, prostate, testicles, soft tissue and skin.
The methods and systems of the present invention could also be applied to biofilms.
We generated and characterized cell clusters (˜30 μm median diameter) from a segment of the distal colon (including both epithelium and digesta) of a mouse fed a plant-polysaccharide diet, yielding 1,406 clusters passing strict quality filtering across two technical replicates (
We next explored whether these observed spatial distributions reflect specific associations between individual bacterial taxa that may result from processes such as positive or negative interspecies interactions (e.g., cooperative metabolism (see Rakoff-Nahoum, S., Coyne, M. J. & Comstock, L. E. An Ecological Network of Polysaccharide Utilization among Human Intestinal Symbionts. Current Biology 24, 40-49 (2014)); contact-dependent killing (see Wexler, A. G. et al. (2016))) or local habitat filtering (see Nagara, Y., Takada, T., Nagata, Y., Kado, S. & Kushiro, A. (2017). Across abundant and prevalent OTUs (>2% abundance in >10% of clusters, n=24), we assessed whether their pairwise co-occurrences were detected more or less frequently than expected in comparison to a null model of independent, random assortment of OTUs (Methods, Fisher's exact test, p<0.05, FDR=0.05). Application of this strategy to the cluster mixing control experiment confirmed our ability to accurately detect positive and negative spatial associations that are expected (
The number of detected associations increased as more of the dataset is sampled, implying that detection of weaker relationships between less abundant taxa can be improved by analyzing more clusters
To further investigate how the spatial organization of the microbiota is influenced by their environmental context, we applied spatial metagenomics along the gastrointestinal (GI) tract. The mammalian GI tract is composed of distinct anatomical regions with different pH levels, oxygen concentrations, host-derived antimicrobials and transit times that together influence the local microbiota assemblage (see Donaldson, G. P., Lee, S. M. & Mazmanian, S. K. Gut biogeography of the bacterial microbiota. 1-13 (2015). doi:10.1038/nrmicro3552). We first performed an adapted 16S community profiling approach along the murine GI tract that could also infer absolute OTU abundances (see Ji, B. W. et al. Quantifying spatiotemporal dynamics and noise in absolute microbiota abundances using replicate sampling. biorxiv.org doi:10.1101/310649 (2018))
The distribution of OTUs per cluster was compared with the spatial organization of taxa in the three regions
To understand how the local spatial organization of the microbiome may vary within and across different gut compartments, we visualized the cell clusters data across the three gut regions using t-distributed Stochastic Neighbor Embedding (tSNE, utilizing Bray-Curtis distance of OTU relative abundance within clusters), as well as the abundance of prevalent bacterial families in cell clusters across the resulting manifold
Next, we explored whether these different spatial distributions reflect distinct spatial co-associations between taxa at each GI site (
We further investigated whether MaP-seq could identify individual taxa with unique or altered spatial patterns. While the cecum harbored the densest community and the highest degree of species mixing of the three sites
Having established the local spatial organization across the GI tract of mice fed a standard plant-polysaccharide diet, we next sought to understand the extent to which diet might influence spatial structuring. Diet is known to play a major role in shaping the variation of gut microbiota across individuals (see Carmody, R. N. et al. Diet Dominates Host Genotype in Shaping the Murine Gut Microbiota. Cell Host & Microbe 17, 72-84 (2015); Sonnenburg, E. D. et al. Diet-induced extinctions in the gut microbiota compound over generations. Nature 529, 212-215 (2016)). While diet shifts can rapidly alter microbiota composition within days (see David, L. A. et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559-563 (2014)), the detailed ecological mechanisms underlying these community-scale changes are not well understood. We thus took co-housed mice and split them into two cohorts where one was maintained on the plant-polysaccharide based diet (LF, same as in the previous cohorts) and one was switched to a high fat, high sugar diet (HF, commonly utilized in dietary-induced obesity studies) to assess microbiota changes associated with these two diets representing distinct macronutrient profiles. After 10 days on the two diets, a considerable loss of species richness in the cecum and colon was observed in HF-fed mice compared to LF-fed mice
To determine if a dietary shift could alter the spatial organization of the microbiota, which could contribute to the observed loss of species diversity, we performed MaP-seq on distal colon samples from mice fed the LF or HF diet. We found that the distribution of unique OTUs per ˜20 μm cluster was similar between both diets
Next, to compare the taxa spatial organization across the two diets, we visualized clusters using tSNE as before
The following are examples of the present invention and are not to be construed as limiting.
Spatial structuring promotes biodiversity and is important to the maintenance of natural ecological systems1,2. Many microbial communities, including the mammalian gut microbiome, display intricate spatial organization3-9. Mapping spatial distributions of bacterial species enables the detailed delineation of fundamental ecological processes and interactions that underlie community-wide behaviors10-12. However, current approaches have a limited capacity to measure the spatial organization of natural microbiomes with hundreds of species13-17. Here, we describe spatial metagenomics, a framework to dissect the organization of a microbiome at micron-scale spatial resolution and metagenomic depth through nucleic acid “plot sampling”. Intact microbiome samples are immobilized within a gel matrix and subjected to cryo-fracturing to generate clusters of co-localized cells, and the identities and abundances of taxa present in these clusters are determined via droplet-based encapsulation and deep sequencing. Analysis of thousands of microbiome clusters from the mouse intestine across three distinct regions revealed heterogeneous microbial distributions with positive and negative co-associations between specific taxa. While the murine intestinal microbiome mostly exhibited regionally distinct spatial organizations, robust associations between Bacteroidales taxa were observed across gut compartments. Analysis of a dietary perturbation revealed phylogenetically clustered regions suggesting local habitat filtering that may be important to maintenance of diversity observed on plant-polysaccharide diets, and enabled identification of spatial niches that may be shared across distinct diets. Spatial metagenomics constitutes a powerful new culture-independent technique to mechanistically study microbial biogeography in complex habitats.
To perform MaP-seq, an input sample is first physically fixed by immobilizing the microbiota via perfusion and in situ polymerization of an acrylamide polymer matrix that also contains a covalently linked reverse 16S rRNA amplification primer. The embedded sample is then fractured via cryo-bead beating, subjected to cell lysis, and passed through nylon mesh filters for size selection to yield cell clusters or particles of desired and tunable physical sizes (i.e. by utilizing different mesh filter sizes). Resulting clusters contain genomic DNA immobilized in their original arrangement, preserving local spatial information. Next, a microfluidic device is used to co-encapsulate these clusters with gel beads, each containing uniquely barcoded forward 16S rRNA amplification primers. Primers are photocleaved from the beads and clusters, genomic DNA is released from clusters by triggered degradation of the polymer matrix within droplets, and PCR amplification of the 16S V4 region is performed. Droplets are then broken apart, and the resulting library is subjected to deep sequencing. Sequencing reads are filtered and grouped by their unique barcodes, which yield the identity and abundance of bacterial operational taxonomic units (OTUs) within individual cell clusters.
To rigorously test the feasibility of this spatial metagenomics approach, we first generated separate cluster communities from either homogenized mouse fecal bacteria or E. coli (Methods) and profiled them with MaP-seq. The resulting data revealed that the majority of detected barcodes mapped uniquely to their respective initial communities with minimal mixing (
To explore the utility of spatial metagenomics to map the natural biogeography of microbiota in complex communities, we applied MaP-seq to the mouse colonic microbiome. We generated and characterized cell clusters (˜30 μm median diameter) from a segment of the distal colon (including both epithelium and digesta) of a mouse fed a plant-polysaccharide diet, yielding 1,406 clusters passing strict quality filtering across two technical replicates (
We next explored whether these observed spatial distributions reflect specific associations between individual taxa that may result from processes such as positive or negative interspecies interactions (e.g., cooperative metabolism24, contact-dependent killing20) or local habitat filtering11. Across abundant and prevalent OTUs (>2% abundance in >10% of clusters, n=24), we assessed whether their pairwise co-occurrences were detected more or less frequently than expected in comparison to a null model of independent, random assortment of OTUs (Methods, Fisher's exact test, p<0.05, FDR=0.05). Application of this strategy to the cluster mixing control experiment confirmed our ability to accurately detect positive and negative spatial associations that are expected. Out of 276 possible pairwise combinations of taxa in the murine colon, we detected 75 statistically significant associations between diverse taxa, the majority of which were positive (72/75) but relatively weak in magnitude (
The number of detected associations increased as more of the dataset is sampled, implying that detection of weaker relationships between less abundant taxa can be improved by analyzing more clusters. Nonetheless, the detected associations showed good correspondence between technical replicates. Importantly, despite high inter-host microbiome variability, the nature of the associations (i.e., sign, magnitude, and number) and some strong associations could be recapitulated in MaP-seq profiling of a second co-housed mouse, such as the co-occurrence of Bacteroidales taxa. This characterization implies that individual taxa in the colon are organized in distinct and reproducible spatial relationships.
To further investigate how the spatial organization of the microbiota is influenced by their environmental context, we applied spatial metagenomics along the gastrointestinal (GI) tract. The mammalian GI tract is composed of distinct anatomical regions with different pH levels, oxygen concentrations, host-derived antimicrobials and transit times that together influence the local microbiota assemblage9. We first performed an adapted 16S community profiling approach along the murine GI tract that could also infer absolute OTU abundances25 (
We first assessed the distribution of OTUs per cluster to compare the spatial organization of taxa in the three regions (
To understand how the local spatial organization of the microbiome may vary within and across different gut compartments, we visualized the cell clusters data across the three gut regions using t-distributed Stochastic Neighbor Embedding (tSNE, utilizing Bray-Curtis distance of OTU relative abundance within clusters), as well as the abundance of prevalent bacterial families in cell clusters across the resulting manifold (Methods,
Next, we explored whether these different spatial distributions reflect distinct spatial co-associations between taxa at each GI site (
While the spatial association networks revealed by MaP-seq differed across the three GI regions, some common co-associations (or lack of associations) were observed. For example, a positive association between Lachnospiraceae (OTU 10) and Lactobacillaceae (OTU 4) was found in both the cecum and colon; on the other hand, Coriobacteriaceae (OTU 1), an abundant taxon at all sites, lacked co-associations with other taxa and was thus randomly assorted at all sites. Together, the differing spatial architectures observed across GI sites suggest that regional environmental factors can variably shape some local spatial structuring of the microbiota, while conserved spatial patterns across sites are more likely the result of robust ecological interactions not affected by environmental variations.
We further investigated whether MaP-seq could identify individual taxa with unique or altered spatial patterns. While the cecum harbored the densest community and the highest degree of species mixing of the three sites (
Having established the local spatial organization across the GI tract of mice fed a standard plant polysaccharide diet, we next sought to understand the extent to which diet might influence spatial structuring. Diet is known to play a major role in shaping the variation of gut microbiota across individuals28,29. While diet shifts can rapidly alter microbiota composition within days30, the detailed ecological mechanisms underlying these community-scale changes are not well understood. We thus took co-housed mice and split them into two cohorts where one was maintained on the plant polysaccharide based diet (LF, same as in the previous cohorts) and one was switched to a high fat, high sugar diet (HF, commonly utilized in dietary-induced obesity studies) to assess microbiota changes associated with these two diets representing distinct macronutrient profiles. After 10 days on the two diets, a considerable loss of species richness in the cecum and colon was observed in HF-fed mice compared to LF-fed mice (
To determine if a dietary shift could alter the spatial organization of the microbiota, which could contribute to the observed loss of species diversity, we performed MaP-seq on distal colon samples from mice fed the LF or HF diet. We found that the distribution of unique OTUs per ˜20 μm cluster was similar between both diets (
Understanding the phylogenetic distribution of an ecosystem can provide important insights into ecological processes underlying community assembly31,32. To better quantify possible changes in phylogenetic diversity between the two diets, we calculated the net relatedness index (NRI) of clusters, a standardized effect size of the mean phylogenetic distance of taxa present within clusters against a null model of random sampling from the local species pool (Methods) 31. For each microbiota cluster, a positive NRI value indicates phylogenetic clustering of its taxa, whereas a negative NRI indicates phylogenetic over-dispersion. While most clusters had NRI values near 0, suggesting random phylogenetic distributions, both LF and HF diets showed a subset of clusters with high negative NRI values suggesting a high degree of phylogenetic over-dispersion. Interestingly, NRI values in LF clusters were overall significantly higher compared to HF values (Mann-Whitney U test, p<10-18), driven by a subset of LF clusters with positive NRIs not observed in HF clusters (
Next, to compare the taxa spatial organization across the two diets, we visualized clusters using tSNE as before (
Spatial metagenomics enables the high-throughput characterization of microbial biogeography through microscopic plot sampling of co-localized nucleic acids at tunable length scales. This general approach could be applied to interrogate a variety of perturbations in the gut (e.g., diet, antibiotics, fecal microbiota transplantation), other mammalian associated microbiota (e.g. skin, genital), or diverse environmental ecosystems, such as soils or biofilms. Importantly, MaP-seq enables in-depth analysis of these processes at previously inaccessible and ecologically meaningful local length scales within individual microbiomes. Improvements to further increase the throughput of the approach could better delineate weaker or rarer co-associations and help investigate structuring across many different characteristic length scales within microbiomes. A variety of established spatial ecology tools and emerging computational and analytical approaches could be applied to this new type of high-dimensional microbiome dataset. Extensions of this general framework to spatially profile other biological molecules such as RNA, proteins and metabolites will enable mapping of complex cellular systems across mechanistically important and functionally distinct axes. Plot sampling of biological structures at microscopic scales opens up new directions of research that employ spatial ecology tools to study these complex systems.
Materials and reagents. All primers and FISH probes were ordered from Integrated DNA Technologies. Primers containing any modifications were HPLC purified by the manufacturer. Photocleavable primers were protected from unnecessary light exposure throughout.
Animal procedures. All mouse procedures were approved by the Columbia University Medical Center Institutional Animal Care and Use Committee (protocol AC-AAAR1513) and complied with all relevant regulations. 6-8 week-old female C57BL6/J mice were obtained from Taconic (colonic analysis,
Microfluidic device fabrication. Devices were fabricated utilizing standard SU-8 soft lithography. Silanized SU-8 silicon wafer molds were fabricated by FlowJEM with a feature height of ˜40 μm. PDMS (Dow Corning Sylgard 184) was mixed for 5 minutes at a ratio of 10:1 base to curing agent, degassed under house vacuum for 30 minutes, and poured over the wafer. The PDMS mixture was cured at 80° C. for 1 hour, allowed to cool to room temperature and removed from the wafer. Individual devices were cut from the PDMS slab and ports were punched utilizing a 1 mm biopsy punch (World Precision Instruments 504646).
Construction of the barcoded beads followed procedures from Zilionis et al. (see Zilionis, R. et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat Protoc 12, 44-73 (2017)) with minor modification for our barcoding scheme. Briefly, acrylamide beads (6% w/w acrylamide, 0.18% w/w N,N′-methylenebisacrylamide [Sigma-Aldrich 146072], 20 μM acry_pcp_pe1 [see Table 1]) were generated using a custom microfluidic droplet device. Resulting beads were ˜20-25 μm in diameter. Batches of ˜20 million beads were then subjected to three rounds of primer extension using the three sets of 96 barcode sequences (pe1, pe2, and pe3 primer extension sets, see Table 2). For each round, beads and primers were distributed into wells of a 96 well PCR microplate and primers were annealed to the beads by incubation. A Bst polymerase reaction master mix (NEB M0537L) was then distributed to each well and incubated to allow for extension. Finally, the reaction was quenched with EDTA and pooled for cleanup steps. The beads were then subjected to denaturing of the extension primers by sodium hydroxide and washing, and the extension protocol was repeated. These procedures were automated on a Biomek 4000 liquid handling robot where possible. After the final extension step, a primer targeted to the terminal 515f primer sequence (515f RC, see Table 1) was annealed, and an Exol enzymatic cleanup (NEB M0293L) was utilized to remove extension intermediates. Resulting barcoded beads were subjected to a final denaturing and washing step and stored at 4° C. in TET (10 mM Tris HCl [pH 8.0], 1 mM EDTA, 0.1% Tween-20).
All mouse samples were collected in technical replicate (TR), a single technical replicate was collected for community mixing experiments. The procedure to remove technical artifacts (i.e. “Number clusters discarded”) was not performed on community mixing experiments given that they are composed of highly homogenous communities.
Sample fixation and in situ polymerization. Intact tissue segments (from the colon, cecum or small intestine as noted) were obtained by dissection and immediately fixed in methacarn solution (60% methanol, 30% chloroform, 10% acetic acid) for 24 hours (see Johansson, M. E. V. & Hansson, G. C. Preservation of mucus in histological sections, immunostaining of mucins in fixed tissue, and localization of bacteria with FISH. Methods Mol. Biol. 842, 229-235 (2012)). The fixed tissue was trimmed with a sterile razor into segments no larger than 3 mm in length, and segments containing digesta were selected. Thus, all input samples for MaP-seq analysis contained undisturbed epithelial tissue and lumenal digesta contents. The trimmed sample was then incubated in phosphate buffered saline (PBS) for 5 minutes and was permeabilized in PBS with 0.1% v/v Triton-X 100 for 5 minutes. Next, a matrix embedding solution (see Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332-337 (2013); Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy. Science 347, 543-548 (2015) containing a reverse sequencing primer with 16S V4 primer 806rB (see Klein, A. M. et al. (2015); Apprill, A., McNally, S., Parsons, R. & Weber, L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat. Microb. Ecol. 75, 129-137 (2015)) and acrydite and photocleavable linker groups was prepared on ice by mixing concentrated stocks of the following components in order: 1×PBS, 10% w/w acrylamide (Sigma-Aldrich A9099), 0.4% w/w N,N′-Bis(acryloyl)cystamine (BAC, Alfa Aesar 44132-03), 5 μM acry_pc_pe2_816r (see Table 1), 0.01% w/w 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Sigma-Aldrich 176141), 0.2% w/w tetramethylethylenediamine (Sigma-Aldrich T7024) and 0.2% w/w ammonium persulfate (Sigma-Aldrich A3678). The BAC crosslinker enables gel degradation upon exposure to reducing conditions. The sample was dabbed dry with a sterile Kimwipe and placed in a PCR tube with excess matrix embedding solution (˜50 μL per segment) and incubated on ice for 5 minutes. Excess embedding solution was removed by pipetting and replaced, and the sample was subsequently incubated on ice for >1 hour for perfusion. Excess embedding solution was removed, and samples were placed in a 37° C. incubator in an anaerobic chamber (Coy Laboratory Products) for >3 hours. Gel-embedded samples were removed, excess polymer matrix was trimmed from the sample with a sterile razor, and the sample was washed twice with PBS and once with TET and stored in TET at 4° C.
Sample fracturing, lysis and size-selection. Samples were placed in a stainless-steel vial (Biospec 2007) along with a 6.35 mm stainless steel bead (Biospec 11709635ss), and were sealed with a silicone rubber plug cap (Biospec 2008). The vial was placed in liquid nitrogen for >2 minutes, vigorously shaken to dislodge the sample from the vial wall, and quickly transferred to a bead beater (Biospec 112011) and subjected to beating for 10 seconds. PBS was added to the vial and vortexed; clusters in PBS were removed and washed twice with PBS via centrifugation at 15K RPM for 1 minute (Eppendorf 5424). Next, embedded cells were lysed (see Spencer, S. J. et al. Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers. 1-10 (2015). doi:10.1038/ismej.2015.124); clusters were resuspended in 500 μL lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM ethylenediaminetetraacetic acid [EDTA], 100 mM NaCl) with 75 U/μL lysozyme (Epicentre R1810M) and were incubated at 37° C. for 1 hour. Clusters were then resuspended in 500 μL digestion buffer (30 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.5% Triton X-100, 800 mM guanidine hydrochloride [Sigma-Aldrich G9284]) with 0.1 μg/μL proteinase K (Epicentre MPRK092), and were incubated at 65° C. for 15 minutes. Finally, clusters were incubated at 95° C. for 5 minutes to inactivate proteinase K and washed three times with TET.
Samples were next subjected to size-selection. Clusters were first passed through a 40 μm cell strainer (Fisher 22-363-547) to remove large particulate matter. Next, nylon mesh filters (Component Supply Company, 7 μm: U-CMN-7-A, 15 μm: U-CMN-15-A, 31 μm: U-CMN-31-A) were cut to size using a ½″ hole punch and two filter punches were placed in a holder (EMD Millipore SX0001300) for each size. Clusters were passed through the 31 μm filter, 15 μm filter, and 7 μm filter sequentially using a 3 mL syringe (BD 309657); for each filter, clusters were passed through three times, and retained clusters on filters were washed once with TET. Clusters were washed off the 15 μm filter (large, ˜30 μm median diameter) and 7 μm (medium, ˜20 μm median diameter) or collected from the pass-through from the final 7 μm filter (small, ˜7 μm median diameter). The concentration of clusters was quantified by counting on a hemocytometer (INCYTO DHC-N01) and stored at 4° C. in TET for processing within ˜2 days.
Co-encapsulation of beads and clusters. A microfluidic co-encapsulation strategy was utilized with three syringe pumps (Harvard Apparatus Pump 11 Elite) and observed under a microscope (Nikon Eclipse Ti2). First, 300 μL of HFE-7500 (3M) with 5% w/w surfactant (RAN Biotechnologies 008—FluoroSurfactant) was loaded into a 1 mL low dead volume syringe (Air-Tite Products A1), the syringe was fitted with a needle (BD 305122) and polyethylene tubing (Scientific Commodities Inc., BB31695—PE/2) and primed on a syringe pump. 30 μL of packed barcoded beads were then removed and washed twice with wash buffer (WB, 10 mM Tris HCl [pH 8.0], 0.1 mM EDTA, 0.1% Tween-20) and twice with bead buffer (10 mM Tris HCl [pH 8.0], 0.1% Tween-20, 50 mM KCl, 10 mM fresh DTT [utilized to degrade clusters within droplets]) by addition of buffer and centrifugation at 15K RPM for 1 minute. After the 4 washes, remaining buffer supernatant was removed with a gel-loading tip (Fisher 02-707-139). ˜5 μL of packed beads were loaded into polyethylene tubing and primed with a 1 mL syringe (BD 309626) backfilled with 500 μL HFE-7500. The tubing was protected from light with a black tubing sheath (McMaster-Carr 5231K31) and primed on a syringe pump with needle facing upwards.
Next, a cluster stock was vortexed for 1 minute, 2,500 clusters were removed, washed three times in WB, and the remaining buffer was removed as above. A 45 μL encapsulation mix was prepared (25 μL NEBNext Q5 Hot Start HiFi PCR Master Mix [NEB M0543L], 4 μL Nycoprep Universal [Accurate Chemical & Scientific Corp. AN1106865), 5 μL 10% w/v Pluronic F-127 [Sigma-Aldrich P2443], 1.25 μL 20 mg/mL BSA [NEB B90005], 9.75 μL nuclease-free water) and clusters were resuspended in the mix and vortexed for >10 s. A 1 mL low dead volume syringe was backfilled with 500 μL HFE-7500, and the encapsulation mix was added directly into the tip of the syringe. A needle and polyethylene tubing were fitted to the syringe, protected from light with a black tubing sheath, and primed on a syringe pump with needle facing upwards.
Tubing was connected for the carrier, bead and cluster encapsulation mix channels to a new microfluidic device. Pumps were primed for the carrier, beads and cluster encapsulation mix channels in order and once stable bead packing was observed set to final flow rates of 2 μL/min for carrier, 0.3 μL/min for beads, and 2.7 μL/min for cluster encapsulation mix. Once stable droplet formation was observed, polyethylene tubing was connected to the outlet port and emulsion was collected in a PCR tube (Axygen PCR-02-L-C) prefilled with 10 μL of 30% w/w surfactant in HFE-7500 and 50 μL of mineral oil. Under these conditions, generated droplets were ˜35-45 μm in diameter with bead occupancy of ˜25-50% (packed bead ordering enables loading beating expected Poisson encapsulation statistics (see Abate, A. R., Chen, C.-H., Agresti, J. J. & Weitz, D. A. Beating Poisson encapsulation statistics using close-packed ordering. Lab Chip 9, 2628-2631 (2009)) and extremely low cluster occupancy of <0.1% (cluster aggregation and channel clogging is a limiting factor at higher concentrations).
The resulting products were then subjected to a second PCR to add sample indexes and Illumina P5 and P7 adapters. 10 μL of cleanup product was used as template for a 50 μL reaction with 1×NEBNext Q5 Hot Start HiFi PCR Master Mix, 0.5 μM of each of the indexing primers (p5_X, p7_X, see Table 3), and 0.1×SYBR Green I (Invitrogen S7567). The PCR (98° C. for 30 s, cycle: 98° C. for 10 s, 68° C. for 20 s, 65° C. for 30 s; 65° C. for 2 m) was run on a real-time PCR machine (Bio-Rad CFX96) to stop reactions during exponential amplification (typically ˜10 cycles). Products were assessed on an agarose gel (2% E-gel, Thermo Fisher G501802) to confirm the expected ˜490 bp amplicon and were subjected to a 1×SPRI bead cleanup as above. Resulting libraries were quantified via fluorometric quantitation (Thermo Fisher Q32854), pooled, and were subjected to sequencing with an Illumina MiSeq 500 cycle v2 kit (read1: 254 bp, read2: 254 bp) at 12 pM loading concentration with 20% PhiX spike in. Sequence filtering and 16S analysis. For MaP-seq data, a custom python script was utilized to demultiplex reads based on barcode identity and strip primer sequences from reads. Reads were merged and filtered using USEARCH 9.2.64 (see Edgar, R. C. & Flyvbjerg, H. Error filtering, pair assembly and error correction for next-generation sequencing reads. Bioinformatics 31, 3476-3482 (2015) with maximum expected errors of 1. The resulting sequences were then dereplicated, de-novo clustered with a minimum cluster size of 2, and reads were mapped to OTUs at 97% identity (see Edgar, R. C. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996-998 (2013). Taxonomy was assigned to OTUs using the RDP classifier (see Wang, Q., Garrity, G. M., Tiedje, J. M. & Cole, J. R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology 73, 5261-5267 (2007). This yielded an OTU table consisting of individual barcodes (i.e., putative clusters) as samples.
Cluster mixing quality control experiment. Two bacterial communities were assembled; the first contained a single strain (e.g. E. coli NEB-beta), the second contained homogenized fecal bacteria. E. coli is not expected in the mouse gut at high abundances (see Xiao, L. et al. A catalog of the mouse gut metagenome. Nature Biotechnology 33, 1103-1108 (2015). To generate homogenized fecal bacteria, fecal pellets were subjected to bead beating (Biospec 1001) with 0.1 mm glass beads in PBS for 1 minute and passed through a 40 μm cell strainer. The two communities were fixed in methacarn, resuspended in approximately equal volume matrix embedding solution to fixed pellet volume and subjected to cluster generation as per the MaP-seq protocol above. The resulting size-selected clusters were then mixed in equal quantity and subjected to encapsulation and sequencing.
Analysis of MaP-seq data. An overview of all MaP-seq datasets generated in this study can be found in Table 5. The resulting dataset contained a large number of barcodes/clusters with varying numbers of reads. A conservative threshold cutoff for considering real clusters was set as the total number of reads in a sample divided by 2,500 (i.e., the number of clusters that were utilized as input during microfluidic encapsulation, and assuming an equal read distribution for each cluster). Reactions yielding an extremely low number of clusters passing this threshold (i.e., <50) were conservatively excluded as they may represent failed encapsulation or amplification reactions.
Clusters were first pre-processed to remove a small number of clusters displaying highly similar OTU abundance profiles within a single technical replicate that appeared to represent technical artifacts (i.e., clusters encapsulated into droplets containing multiple barcoded beads or beads erroneously containing multiple barcodes) which could confound association detection. The pairwise Pearson correlation of all clusters was calculated, and highly correlated sets of clusters (r>0.95) dominated by a single technical replicate and large in size (>90% belonging to a single technical replicate, clusters constitute>1% of the overall dataset) were removed. These artifacts constituted a low amount of the overall dataset. For analysis of presence or absence of species within a cluster, a 2% relative abundance threshold within clusters was utilized, given observation of a small amount of background read-through across clusters and to ensure that at least 2 reads (and not singletons) were required to denote presence of a species.
To determine pairwise associations, prevalent and abundant OTUs within filtered clusters (>2% relative abundance in >10% of clusters) were identified, and 2 by 2 contingency tables of appearance (>2% relative abundance) were calculated for all pairs of OTUs. Fishers exact test was then used to calculate the probability of pairs occurring more or less together than expected (i.e. a null model of random assortment of the two species, assuming equiprobable occupancy at all sites), and resulting p-values were adjusted via the Benjamini-Hochberg procedure (FDR=0.05).
For t-distributed Stochastic Neighbor Embedding (tSNE) analysis (see Maaten, L. V. D. & Hinton, G. Visualizing Data using t-SNE. Journal of Machine Learning Research 9, 2579-2605 (2008), reads for each cluster were subsampled to the lowest number for all clusters in the dataset (as specified in the text) since raw relative abundance values were analyzed (i.e. not utilizing a 2% relative abundance threshold as in other analyses). Bray-Curtis distance between taxa relative abundances within clusters was calculated, and this resulting distance matrix was utilized as the input for tSNE analysis.
The Net Relatedness Index (NRI) was calculated as previously described (see David, L. A. et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559-563 (2014) adapting code from the relatedness_library.py script from Qiime 1.9.1 (see Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335-336 (2010)) which implements the same calculation as in phylocom 4.2 (see Webb, C. O., Ackerly, D. D. & Kembel, S. W. Phylocom: software for the analysis of phylogenetic community structure and trait evolution. Bioinformatics 24, 2098-2100 (2008)). Briefly, species presence and absences across clusters were defined using the same 2% relative abundance threshold, and clusters containing only one OTU were omitted from analysis. OTU sequences were aligned and a neighbor-joining tree was constructed using MUSCLE 3.8.31 (see Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 1792-1797 (2004). The NRI was calculated as a standardized effect size for each cluster: NRI=−1*(MPD,-./012−MPD3.--)/sd(MPD3.--), where MPD,-./012 denotes the mean phylogenetic distance (MPD), and MPD-./012 & sd(MPD3.--) indicate the mean MPD, and the standard deviation of the MPD over 1000 iterations of a null mode. The null model, calculated for each cluster, was random draws for the number of OTUs present in the sample (i.e. preserving cluster OTU richness) from the sample pool (i.e. any OTU observed at least once in any cluster in the sample) without replacement. The null model therefore preserves the OTU richness of each cluster but randomizes the OTUs present from the set of OTUs occurring in the sample.
Bulk 16S sequencing and spike-in for absolute abundance calculation. The bulk sequencing protocol followed our established spike-in sequencing pipeline (see Ji, B. W. et al. (2018)). Briefly, genomic DNA (gDNA) extraction was performed using a custom liquid handling protocol on a Biomek 4000 robot based on the Qiagen MagAttract PowerMicrobiome DNA/RNA Kit (Qiagen 27500-4-EP) but adapted for lower volumes. Samples were subjected to bead beating for a total of 10 minutes. For samples processed with the spike-in sequencing approach for absolute abundance calculation, the sample added was weighed on an analytical balance, and 10 uL of a frozen spike-in strain concentrate (Sporocarcina pasteurii, ATCC 11859, an environmental bacterium not found in the gut microbiome) was added during gDNA preparation. Resulting gDNA was subjected to amplification and sequencing of the 16S V4 region following a dual indexing scheme (see Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K. & Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology 79, 5112-5120 (2013)) but utilized updated 515f and 806rB primers as in the MaP-seq technique. A 20 μL PCR amplification was performed (1 μM forward and 1 μM reverse barcoded primers, 1 μL prepared gDNA, 10 μL NEBNext Q5 Hot Start HiFi Master Mix, 0.2× final concentration SYBR Green I). The PCR (98° C. for 30 s; cycle: 98° C. for 20 s, 55° C. for 20 s, 65° C. for 60 s, 65° C. for 5 m) was run on a real-time PCR machine to stop reactions during exponential amplification. Amplicon products were quantified and pooled, the expected 390 bp product was gel-extracted, and paired-end sequencing was performed with an Illumina MiSeq 300 cycle v2 kit (read1: 154 bp, read2: 154 bp, custom sequencing primers spiked into sequencing kit) at 10 pM loading concentration with 20% PhiX spike in. Resulting sequences were processed with USEARCH as above. The absolute bacterial density for a sample (A) was calculated by utilizing the weight of sample added (w) and proportion of reads mapping to spike in strain (p/) in the following formula: A=(1−p/)/(p/*w). The absolute density of individual OTUs was calculated by rescaling the total sample absolute density by the relative abundance of sample OTUs. 16S FISH and imaging. Samples were fixed as with the MaP-seq protocol, embedded within paraffin blocks, 4 μm thick lumenal sections were cut and deparaffinized. 16S FISH was performed as previously described (see Mark Welch, J. L., Rossetti, B. J., Rieken, C. W., Dewhirst, F. E. & Borisy, G. G. Biogeography of a human oral microbiome at the micron scale. Proceedings of the National Academy of Sciences 113, E791-800 (2016); Whitaker, W. R., Shepherd, E. S. & Sonnenburg, J. L. (2017). Briefly, previously validated FISH probes targeting abundant taxa present in the sample were obtained with conjugated fluorophores suitable for multiplex imaging: Erec482_a488 or Erec482_cy3 (see Franks, A. H. et al. Variations of bacterial 710 populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Applied and Environmental Microbiology 64, 3336-3345 (1998) targeting Lachnospiraceae, Lab158_cy3 (see Harmsen, H., Elfferich, P. & Schut, F. A 16S rRNA-targeted probe for detection of lactobacilli and enterococci in faecal samples by fluorescent in situ hybridization. Microbial Ecology in Health and Disease 11, 3-12 (1999)) targeting Lactobacillaceae and Enterococcaceae, Ato291_cy5 (see Harmsen, H. et al. Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups. Applied and Environmental Microbiology 66, 4523-4527 (2000)) targeting Coriobacteriaceae, Eub338_cy5 (see Amann, R. I. et al. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Applied and Environmental Microbiology 56, 1919-1925 (1990)) targeting Bacteria, and Non338_cy5 (see Wallner, G., Amann, R. & Beisker, W. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14, 136-143 (1993)) control probe (see Table 4). Sections were incubated with probes at 10 ng/μL in FISH hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl pH 7.5, 0.01% SDS, 10% formamide) at 47° C. for 4 hours. Sections were then incubated in preheated FISH wash buffer (0.9 M NaCl, 20 mM Tris-HCl pH 7.5) for 10 minutes, washed 3× times in PBS, incubated with 10 μg/mL DAPI in PBS for 10 minutes and washed 3× times in PBS. Sections were then mounted in mounting medium (Vector Laboratories H1000).
Images were acquired on a Nikon Eclipse Ti2 epifluorescence microscope with a SOLA-SE2 illuminator and Andor Zyla 4.2 plus camera controlled by Nikon Elements AR software. DAPI, FITC/GFP, RFP and CY5 filter cubes (Nikon 96359, 96362, 96364, 96366 respectively) were utilized. Large area four-color fluorescence scans with three 0.6 μm Z-stacks within the 4 μm section were performed with a Plan Apo λ 40× objective. The extended depth of focus (EDF) module was applied to resulting Z-stacks to obtain a focused image across the stack, and images across the entire section were stitched together.
The human gut contains trillions of microorganisms (microbiota) that form a complex and unique ecosystem within our bodies. It is now clear that these bacteria have systemic effects on the host and can directly interact with many classes of pharmaceutical interventions, altering efficacy and clinical outcomes1,2. A prime example of this effect is in cancer immunotherapy, where recent studies suggest that the commensal microbiota modulate the efficacy of therapies involving monoclonal antibodies (mAbs) targeted to the PD-1 receptor, via stimulation of the immune system2-7. Importantly, it has been observed that living bacteria in gut are required to elicit this effect3. Correspondingly, approaches to alter microbiomes to improve the efficacy of cancer immunotherapy are sorely needed.
Current microbiome manipulation strategies broadly fall under two approaches: chemical perturbation and probiotic supplementation8. The abundance of bacterial species within a given microbiome can be altered by administration of chemical compounds (i.e. different diets, prebiotic compounds, antibiotics). Alternatively, new bacterial strains or combinations of strains (probiotics or fecal microbiota transplant) with functionality of interest can be administered. However, the pervasive variability of individual microbiomes limits the efficacy of these techniques. Chemical perturbations will be unsuccessful if a targeted bacterial species is not present, and their effect can be highly variable. Supplemented probiotic strains may not robustly colonize all microbiomes9. An alternative to these approaches is to completely replace a microbiome with a new defined microbiome containing specific desired functionality. Here, precision microbiome replacement, a new paradigm in manipulating microbiomes, can be used to enhance cancer immunotherapy.
Specific aims: To develop a precision microbiome replacement therapy to improve the efficacy of cancer immunotherapies, we will (1) generate a comprehensive reference collection of gut bacterial strains, (2) identify strains promoting immunotherapy efficacy using combinatorial in vivo animal model screens, and (3) develop a microbiome transplantation therapy and formulate strains into stable consortia for delivery.
Approach: (1) Generate a comprehensive reference collection of gut bacterial strains. Individual bacterial strains can act as effectors (i.e., stimulating the host immune system) in the context of complex communities10. Fecal samples will be collected from geographically and environmentally distinct individuals representing global gut microbial diversity. Samples will then be subjected to culturing and isolation in anaerobic settings, and individual strains will be isolated utilizing colony picking robots. Resulting bacterial strains will be identified and characterized using whole-genome sequencing and unique strains of interest will be subjected to long-term cryogenic storage. This sequencing characterization may be conducted by utilizing robotic liquid handling for library preparation (i.e. Labcyte Echo 550, Agilent Bravo, Formulatrix Mantis; sequence on HiSeq X Ten). This automated approach will allow for generation of a gut bacterial strain collection resource in an economic manner.
(2) Identify strains promoting immunotherapy efficacy using combinatorial in vivo animal model screens. Representative strains from the collection will be selected, revived from storage and inoculated into cohorts of germ-free mice. The mice will be subjected to standard cancer models (e.g. metastatic cutaneous squamous cell carcinoma) and given mAb checkpoint immunotherapy (e.g. cemiplimab) and efficacy and response to therapy will be measured. Importantly, the screen will be performed with different combinations of strains rather than individual strains, to enable efficient and higher throughput screens10. Strains promoting efficacy of immunotherapy will be identified.
(3) Develop a new microbiome transplantation therapy and formulate strains into stable consortia for delivery. To perform efficient microbiome transplantation, strategies utilizing oral antibiotic therapy to clear to eradicate commensal microbiota and subsequent oral delivery of new microbial strains will be tested in gnotobiotic mouse models with humanized microbiota. Combinations of antibiotics, dosing, and timing of the therapy in addition to physical clearing of the gut and dietary changes will be explored to optimize efficient elimination of endogenous microbiota and colonization of new strains. Next, the identified immunotherapy enhancing strains will be formulated into a complex microbiome consortium recapitulating the ecology and functionality of naturally occurring microbiomes. The stability of the microbiome (i.e. retention of desired strains over time, resistance to invasion by other commensal strains) will be measured in mice models and improved by iterative design.
Some species of gut bacteria may be recalcitrant to in vitro isolation. Recent studies, however, suggest that the majority of the gut microbiome is culturable12, and the cultivability of species could be further improved by systematic exploration of culture media formulation. The transplantation and resulting microbiome could differ across individuals due to interactions between the strains and the host. However, recent studies suggest that environment dominates host genotype in determining microbiota composition, implying that microbiome transplantation may be reproducible across different host backgrounds13.
Although there may be variability of microbiomes across individuals, direct therapeutic microbiomes interventions can be used. Alternatively, new microbiomes with desired functionality can be designed and replaced. Cancer immunotherapy offers a salient first application of the concept, but the pipeline could be broadly scaled to other microbiome linked human disorders.
Disruption of the normal homeostatic balance of the gut can lead to profound changes in the gut microbiome. For example, antibiotics are known to cause large-scale alterations to the gut microbiome. In general, antibiotics not only target the intended pathogens, but often cause collateral damage in wiping out native commensal microbiota that have sensitivity to the compound. Clinical administration of antibiotics not only reduces biodiversity in the gut microbiome, but also predisposes individuals to a variety of short- and long-term diseases, including antibiotic-associated C. difficile infections, diabetes, and inflammation. While it is generally believed that antibiotic exposure disrupts the state of the microbiome by increasing its fragility and susceptibility to pathogenic infections, specific mechanisms mediating this process is not understood. In large ecological systems, changes in spatial patterning can play an important role in susceptibility to invasion, for example in exotic plant invasion in river and creek ecosystems. Exposure to antibiotics c a n lead to destabilization of the natural commensal microbiota by removing key members in the community that facilitate robust interspecies interactions, which in turn is marked by a profound change in the microbial spatial architecture that reduces the microbiome's natural resistance to colonization by pathogens. We used two wild-type C57BL6/J mice that were both fed on a conventional diet and co-housed prior to normalize their gut microbiota, which was validated by bulk fecal sequencing. We then separated the mice into individual cages and introduced ciprofloxacin (0.625 mg/mL) in drinking water ad-libitum for 2 days in one cage and a sham control in the other cage. We extracted small intestinal tissues from both the control and ciprofloxacin-treated mice and applied bulk 16S sequencing and MIST-seq. As expected, exposure to antibiotics significantly shifted the gut community, leading to an overall loss in microbiome diversity and the domination of particular groups (e.g. Lactobacillales and Clostridiales) compared to the wild-type control (
The prevalent use of antibiotics both in pediatrics and adult populations and its impact on the gut microbiome is hypothesized to be a key contributor in the rise of autoimmune and metabolic disorders. However, the impact of specific antibiotics on the gut microbiome can vary significantly depending on the type (e.g. broad vs narrow spectrum, antibiotic class), therapeutic dosage and duration, resistance profiles of endogenous bacteria, and geographic location along the GI. We will explore how antibiotics can alter the spatial microbiota organization. Altered spatial patterns due to antibiotics exposure may reflect changes in microbiota function beyond simple variations in community composition or abundance. We will use antibiotics with various modes of action and varying levels of host and microbiota impact. Specifically, we will administer Ciprofloxacin (Lincoasimide; single oral gavage 10 mg/kg), Vancomycin (Glycopeptide, 0.625 mg/mL, drinking water ad libitum), Ampicillin (□-lactam, 0.5 mg/mL, drinking water ad libitum), Streptomycin (Aminoglycoside, 5 mg/mL, drinking water ad libitum) to different cohorts of 5 pre-cohoused wild-type C57BL6/J mice as previously described. Mice from each cohort will be sacrificed at day 0 (before treatment), 3, 7 and 10 (
To functionally characterize gut microbiota ecology, we will employ a classical ecology approach to introduce species into novel or perturbed environments, and tracked them longitudinally over space and time. We will introduce “mock” murine fecal transplants into wild-type and antibiotic-perturbed mice and profile the colonization process. Specifically, 5 cohorts of C57BL6/J mice will be obtained commercially (Taconic Biosciences), 4 of which will be orally treated with different antibiotics for 10 days, and the remaining will serve as a control group. We will isolate live fecal microbiota from mice obtained through another vendor (i.e. Jackson Laboratories, Charles River Laboratories) that are known to harbor highly distinct microbiomes, which we will validate by bulk 16S sequencing (
The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions and dimensions. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety. Variations, modifications and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. While certain embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not as a limitation.
This application claims priority to U.S. Provisional Application No. 62/648,716 filed on Mar. 27, 2018, which is incorporated herein by reference in its entirety.
This invention was made with government support under OD009172 and AI132403 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20090098555 | Roth et al. | Apr 2009 | A1 |
| 20100285975 | Mathies et al. | Nov 2010 | A1 |
| 20120149584 | Olle et al. | Jun 2012 | A1 |
| 20120165215 | Andersen et al. | Jun 2012 | A1 |
| 20150259728 | Cutliffe et al. | Sep 2015 | A1 |
| Number | Date | Country |
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| 2012048341 | Apr 2012 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20190300968 A1 | Oct 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62648716 | Mar 2018 | US |
