Method and System for Detection of an Organism

BACKGROUND

Detecting, identifying, and phenotyping pathogens found in healthcare settings is critical both for diagnostic and surveillance purposes. Traditional bacterial and fungal diagnostic procedures rely on culture techniques that produce a genus or species level identification after 24-48 hours. Such tests are ordered for patients demonstrating symptoms indicative of an infection. While culture has been the standard diagnostic method for over one hundred years, its slow turnaround time means that a physician must prescribe antibiotics before knowing the identity of the organism or its drug resistances.

More recently, rapid techniques such as qPCR and mass spectrometry have allowed sub-24 hour turnaround times and enabled surveillance applications. For example, many hospitals in the United States test every patient for MRSA on admission to determine an appropriate caution level (e.g., quarantine) for patients who are at a high risk for spreading an infection to other patients. qPCR offers quick results but minimal information—a typical test only detects the presence of one or a few sequences from one organism. Testing for additional organisms or the presence of drug resistance or virulence genes adds substantially to the cost of the test.

A test that offers sub-24 hour turnaround time while identifying a large number of organisms would offer many benefits in a healthcare setting including broad-range surveillance and faster prescriptions of the most appropriate antibiotic. The present application discloses compositions, kits, and methods that can be used to detect any or several of a large set of organisms present in a sample as well as a number of families of drug resistance genes.

SUMMARY

Provided herein are compositions, kits, and methods for identifying an organism. The organism can be a microbe, microorganism, or pathogen, such as a virus, bacterium, or fungus. In one embodiment, an organism is distinguished from another organism. In another embodiment, a strain, variant or subtype of the organism is distinguished from another strain, variant, or subtype of the same organism. For example, a strain, variant or subtype of a virus can be distinguished from another strain, variant or subtype of the same virus.

In some aspects, a probe set for identifying pathogenic organisms or strains in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing at least 5 different strains, variants, or subtypes of at least 3 pathogenic organisms, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence is provided.

In some embodiments, pathogen strains or organisms comprise a virus, bacterium, or fungus. In some embodiments, the at least 3 pathogenic organisms include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Acinetobacter baumanii, Clostridium difficile, Escherichia coli, Enterobacter (aerogenes, cloacae, asburiae), Enterococcus (faecium, faecalis), Klebsiella pneumoniae, Proteus mirabilis, Candida albicans, and Pseudomonas aeruginosa; or subtypes or strains thereof.

In some embodiments, the probe set can not only detect and distinguish between the at least 3 organisms but can also distinguish between common strains or subtypes of the organisms. In some embodiments, the probe set detects and distinguishes among the organisms responsible for more than 90% of the hospital acquired infections at some site.

In one aspect, a probe set for identifying the presence of drug resistance genes in the organisms in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing at least 3 classes of resistance genes, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence is provided.

In one aspect, a kit containing any probe set described herein and the reagents and protocol to capture the target sequences of the organisms present in the input sample is provided.

In some aspects, a kit for the simultaneous detection of pathogens including three or more of the organisms listed in Table 2 is provided. In some embodiments, the kit is for research use. In some embodiments, the kit is a diagnostic kit. In some aspects, a kit for the simultaneous detection of antibiotic resistance genes including three or more of the genes listed in Table 3 is provided. In some embodiments, the kits described herein can be used to prepare DNA for massively parallel sequencing. In some embodiments, the kits described herein can provide molecular barcodes for the labeling of individual samples. In some embodiments, the kits described herein can include at least 10 of the probe sequences listed in Table 1.

In some embodiments, the kits described herein can be used to circularize single-stranded DNA probes by: (i) hybridization to a complementary target DNA sequence, (ii) extension across a gap by DNA polymerase, and (iii) ligation of the extended probe to form a single stranded, covalently closed circular DNA molecule.

In one aspect, a composition comprises a probe set for identifying pathogenic organisms or strains in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing three or more of the organisms listed in Table 2, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence. In one embodiment, the plurality of probes, when implemented into an assay, allows for the substantially simultaneous detection and distinguishing of three or more of the antibiotic resistance genes listed in Table 3 is provided.

In one aspect, a composition comprises a probe set for identifying antibiotic resistance genes of pathogenic organisms or strains in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing three or more of the antibiotic resistance genes listed in Table 3, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence is provided.

In one aspect, a composition comprises a probe set for identifying pathogenic organisms or strains in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing three or more organisms that cause Hospital Associated Infections (HAIs) at some site, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence is provided. In some embodiments, the three or more organisms that cause HAIs at some site comprise organisms responsible for more than 90% of the hospital acquired infections at some site. In some embodiments, the three or more organisms that cause HAIs at some site comprise organisms responsible for more than 60% of the hospital acquired infections at some site. In some embodiments, the three or more organisms that cause HAIs at some site comprise organisms responsible for more than 30% of the hospital acquired infections at some site. In some embodiments, the site is a surgical site, catheter, ventilator, intravenous needle, respiratory tract catheter, medical device, blood, blood culture, urine, stool, fomite, wound, sputum, pure bacterial culture, mixed bacterial culture, bacterial colony, or any combination thereof.

In some embodiments, a probe set is operable to detect CARB, CMY, CTX-M, GES, IMP, KPC, NDM, ampC, OXA, PER, SHV, VEB, VIM, ermA, vanA, canB, mecA, or mexA family or classes of genes, or any combination thereof. In some embodiments, some of the genomic regions chosen as target sequences are known to be highly conserved such that each genus or species tends to contain a single version of the region, thus allowing genus or species identification. In some embodiments, some of the genomic regions chosen as target sequences are known to be highly variable such that each strain or substrain will contain a different version of the region, thus enabling strain or substrain identification and differentiation. In some embodiments, some portion of a plurality of the selected target sequences are sequenced simultaneously and then mapped to a database of reference sequences to determine the most likely identities of the organisms or genes present in the sample. In some embodiments, some portion of a plurality of the selected target sequences are sequenced simultaneously and then assembled into one or more consensus sequences. When sequencing information is gathered from the probes for antibiotic resistance genes, for plasmids, and for an organism, a distinguishing fingerprint can be derived for the pathogen, and can serve as means to identify the source and extent of an outbreak.

In one aspect, a kit comprising one or more reagents, wherein the reagents comprise a probe set according to claims 1-11, reagents for obtaining a sample, reagents for extracting nucleotides from a sample, enzymes, reagents for amplifying a region of interest, reagents for purifying nucleotides, reagents for purifying captured regions of interest, buffers, sequencing reagents, or any combination thereof, wherein the reagents allow for the capture of target sequences of three more pathogens listed in Table 2 is provided.

In one aspect, a kit comprising one or more reagents, wherein the reagents comprise a probe set according to claims 1-11, reagents for obtaining a sample, reagents for extracting nucleotides from a sample, enzymes, reagents for amplifying a region of interest, reagents for purifying nucleotides, reagents for purifying captured regions of interest, buffers, sequencing reagents, protocol or any combination thereof, wherein the reagents allow for the capture of target sequences of three or more pathogens listed in Table 2 and capture of target sequences of three or more antibiotic resistance genes listed in Table 3 is provided.

In some embodiments, the reagents allow the capture reaction to be performed in a single tube. In some embodiments, the reagents allow the capture reaction to be performed in less than three hours. In some embodiments, the reagents allow the capture reaction to be performed in less than two hours. In some embodiments, the detection of the three or more pathogens occurs substantially simultaneously.

In some embodiments, the plurality of probes comprises at least 3 of the probe sequences listed in Table 1. In some embodiments, each probe comprises the first sequence that hybridizes to a 5′ end of said target sequence and a second sequence that hybridizes to a 3′ end of said target sequence. In some embodiments, the probe set can distinguish between strains or subtypes of the organisms. In some embodiments, the detection the three or more antibiotic resistance genes occurs substantially simultaneously. In some embodiments, the detection of the three or more pathogens and the three or more antibiotic resistance genes occurs substantially simultaneously.

In some embodiments, a kit allows for preparation of DNA for massively parallel sequencing. In some embodiments, a kit further comprises molecular barcodes for the labeling of individual samples.

In some embodiments, the probe set of a kit comprises at least 10 of the probe sequences listed in Table 1. In some embodiments, the probe set of a kit comprises at least 20 of the probe sequences listed in Table 1.

In some embodiments, kit reagents can be used to circularize single-stranded DNA probes by: (i) hybridization to a complementary target DNA sequence, (ii) extension across a gap by a DNA polymerase, and (iii) ligation of the extended probe to form a single stranded, covalently closed circular DNA molecule.

In one aspect, a method of identifying an organism or pathogenic strain, variant or subtype comprising: a) contacting a sample with a plurality of probes listed in Table 1, wherein said plurality of probes detects and distinguishes at least 3 different organisms or pathogenic strains listed in Table 2, or variants or subtypes thereof; b) hybridizing a 5′ end of a target sequence of said organisms or pathogenic strains, or variants or subtypes thereof, a 3′ end of said target sequence, or said target sequence with a probe of said plurality; c) sequencing said target sequence; and d) identifying from said sequencing said organisms or pathogenic strains, or variants or subtypes thereof is provided.

In one embodiment, the method is performed in less than 12 hours. In one embodiment, the identifying is performed in less than 3 hours. In one embodiment, the identifying is performed in less than 2 hours. In one embodiment, the identifying is with at least 99% specificity or sensitivity.

In one aspect, a method of stratifying a host into a therapeutic group comprising: a) contacting a sample from said host with a plurality of probes listed in Table 1, wherein each probe specifically distinguishes different non-host organisms or pathogenic strains listed in Table 2, or variants or subtypes thereof; b) hybridizing a 5′ end of a target sequence of a non-host organism or pathogen, a 3′ end of said target sequence, or said target sequence with a probe of said plurality; c) sequencing said target sequence; d) determining an identity of said non-host organism or pathogenic strain, or variant or subtype thereof, from said sequencing; and e) stratifying said host into a therapeutic group based on said identity is provided. In one embodiment, the method further comprises determining the genotype of the host from the sample.

In some embodiments, an additional non-host organism is identified. In some embodiments, an additional strain, variant or subtype of said organism or pathogen is identified. In some embodiments, the therapeutic group differs than a therapeutic group in which only one of the non-host organisms is identified. In some embodiments, the therapeutic group differs than a therapeutic group in which only one of said strains, variants, or subtypes of said pathogen is identified.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 depicts an exemplary kit configuration, indicating the position of samples and barcoding reagents within the supplied materials within the kit.

FIG. 2 provides a matrix depiction of a subset of a probeset for discrimination of genus and species amongst many genomes of various organisms. Each column on the x-axis indicated a single probe capture region, and each row indicates a reference database genome within the genus or species labeled. Dark boxes indicate that a probe is not predicted to provide sequence for this organism, whereas white boxes indicate that this probe is predicted to bind and provide sequence enabling the detection of this organism.

FIGS. 3A-3B depict exemplary plots of data that can be used to quantify target organisms including (FIG. 3A) Acinetobacter and (FIG. 3B) S. saprophyticus. In each case, genomic DNA isolated from a culture of each organism was quantified and a dilution series of 4 orders of magnitude aliquoted. Each aliquot was sequenced in triplicate and the total sequencing reads per aliquot divided by the number of internal control reads to produce a normalized quantitation of the DNA present in the sample. The plotted results indicate a highly linear and quantitative relationship between sequenced reads detected and input DNA.

FIG. 4 depicts a graph showing that the kits described herein can resolve mixed samples containing multiple organisms. In each case, genomic DNA isolate from a culture of each organism was quantified and a aliquoted into a sample at even copy numbers, with the sample matrix indicating mixes of up to 5 distinct genomes within each sample. Each sample was sequenced in duplicate and the total sequencing reads for each individual genome per sample divided by the number of internal control reads to produce a normalized relative quantitation of the each of the genomic DNA species present in the sample. The graphed results indicate accurate detection of multiple species within a mixed DNA sample.

FIG. 5 depicts a plot demonstrating strong correlation (R²=0.98) between the log normalized counts obtained via PGM vs log normalized counts obtained via qPCR. Genomic DNA from an organism was quantified and a aliquoted as a dilution series over ˜5 orders of magnitude. Each sample was sequenced in triplicate and the total sequencing reads for the genome per sample divided by the number of internal control reads to produce a normalized relative quantitation genomic DNA species present in each aliquot. qPCR was also performed in triplicate on each sample using a genome specific primer pair, and the qPCR relative copy number then plotted against the sequencing data (PGM normalized count). The results demonstrate a linear agreement with quantitation by qPCR over >4 orders of magnitude.

FIG. 6 depicts a plot of the ratio of viral (HIV) reads to GFP against the initial template concentration in the reaction. cDNA from HIV was quantified and a aliquoted as a dilution series over ˜4 orders of magnitude. Samples were prepared in the presence of 1000 genome equivalents of human DNA isolated from cultured HEK-293 cells, or in the absence of background competing DNA. Each sample was sequenced and the total sequencing reads for the genome per sample divided by the number of GFP internal control reads to produce a normalized relative quantitation of HIV cDNA present in each aliquot. No significant difference was observed in the number of sequencing reads per sample in the presence or absence of competing background Human DNA.

FIG. 7 depicts a plot comparing the detection of cDNA from 2 HIV strains (CN009 and CN006) obtained via PGM vs. MiSeq. The plot is shown as the adjusted GFP read count against the CN009 Template count. In each case, cDNA from HIV CN009 was quantified and a aliquoted as a dilution series over ˜5 orders of magnitude. Into each CN009 aliquot, 3000 genome equivalents of CN006 genome were also added. Each sample was sequenced in duplicate and the total sequencing reads for each individual genome per sample divided by the number of internal control reads to produce a normalized relative quantitation of the each of the genomic DNA species present in the sample. The plots indicated a consistent level of CN006 detection detected per sample, and a linear detection of CN009 over >4 orders of magnitude. This also demonstrates the detection of two species at minor variant frequencies of as low as 1%.

FIG. 8 depicts a plot of sequencing counts per probe within a probeset, for replicate sample B1 against replicate sample B2. The plot demonstrates a highly linear and reproducible probeset internal performance.

FIGS. 9A-9B depict plots of the ratio of minor:major pathogen PGM reads against the percent ratio of minor:major pathogen in the reaction for (FIG. 9A) minor pathogen detected to 1% major pathogen (S. epidermidis and E. coli) and (FIG. 9B) minor pathogen detected to 10% major pathogen (S. saprophyticus and A. baumannii). In each case, genomic DNA isolated from a culture of two organisms was quantified and aliquoted into a sample at a ratio of 10:1, 1:1, 1:10 and 1:100. Each sample was sequenced in triplicate and the total sequencing reads for each individual genome per sample divided by the number of internal control reads to produce a normalized relative quantitation of the each of the genomic DNA species present in the sample. The graphed results indicate accurate detection of multiple species within a mixed DNA sample down to a minor variant level of at least 1%.

FIG. 10A describes a list of assay components within the HAI BioDetection kit.

FIG. 10B illustrates the layout of customer samples and control samples of two 8 well strips that the BioDetection assay is performed within.

FIG. 10C indicates validated performance specifications and criteria for the HAI BioDetection kit.

FIG. 11A describes the multilevel structure of the HAI BioDetection probeset.

FIG. 11B provides a matrix depiction of two probes within the BioDetection probset to illustrate discrimination of species and strain amongst many genomes of Staphlyococcus. Each column on the x-axis indicates a SNP detected by either probe 1 or probe 2 capture region, and each row indicates a reference database genome within the genus or species labeled. Black boxes indicate that a probe is not predicted to provide sequence for this organism, whereas shaded boxes indicate that this probe is predicted to bind and provide sequence enabling the detection of this organism.

FIG. 11C describes the levels of multiplexity achieved by the HAI BioDetection kit by assaying many sequence variants within a sample, compared to the single nucleotide discriminatory ability of a PCR primer.

FIG. 12 illustrates the workflow from input sample, either a purified genomic DNA from culture, e.g. DNA enriched from a swab of a patient wound site. The BioDetection kit workflow is illustrated in elapsed time (from t=12.00) and the workflow timing for each individual step is broken out to the left of the workflow. A barcoding primer set of allows 16 to 96 samples to be sequenced simultaneously in one run on a sequencing platform (in this illustration and Ion Torrent PGM, but alternatively and Illumina MiSeq or HiSeq platform). The interpretation box illustrates a computational software and graphical display of simplified data output.

FIG. 13 illustrates a graphical display of summarized sequencing results from the BioDetection kit. The graphical display is subdivided into Genus, species and strain level detection results, resistance gene information if resistance loci are detected, the readcount for samples and internal controls, and also any potential warnings due to poor sample performance. A color-coded similarity score (green=similar, yellow=moderate similarity, red=little similarity) and a similarity score absolute value, are calculated for the sequenced similarity detected by the kit and compared to the next most related organism at genus, species and strain level using a reference database of published genomic sequences, and containing previous genome sequences detected by the BioDetection kit. In the illustrated example, the sample has been demonstrated to contain both Enterococcus faecalis as the primary species, and Escherichia coli a minor species present within the sample. The samples are 65.4% and 74% homologous to the nearest neighbor strains described.

FIG. 14 illustrates a schematic comparison of the turnaround time and workflow steps to generate substrain level resolution and drug resistance typing of bacterial samples using either traditional microbiology, and combination of PCR and/or Mass spectroscopy, whole genome sequencing (WGS) or the BioDetection kit. A clear advantage is illustrated with the BioDetection kit in terms of fewer workflow steps and faster achievement of substrain resolution and drug resistance typing compared to these alternative methods.

FIG. 15A describes a collection of 38 MRSA samples were subtyped using both HAI BioDetection Kit and spa locus VNTR typing (using PCR and Sanger sequencing). Sequence regions captured using the BioDetection kit were used to construct a phylogenetic tree was constructed using sequence data, and each sample was annotated with spa-typing result for the same sample. The tree demonstrates the discrimination of samples with the same spa-type into multiple unique isolates using the BioDetection kit. Further the grouping clustering generated by the BioDetection kit largely groups according to spa-type, as would be predicted for more closely related samples.

FIG. 15B describes the number of sequence variants detected amongst 38 sequenced with the BioDetection kit, or typed by spa type, or 8 representative samples encapsulating the broad phylogenetic tree structure and then Sanger sequenced using then MLST subtyping amplicons, or the 16S ribosomal sequencing amplicon. The total number of MRSA samples uniquely discriminated by each approach is also described.

FIG. 15C describes 4 bacterial cohorts sequenced using the BioDetection kit. The table indicates the number of samples per cohort, and the number of % of the samples that were discriminated into unique isolates. The data demonstrates that the HAI kit is capable of near unique discrimination of bacterial isolates within many large cohorts.

FIG. 16A is an in silico model predicting the superiority of the present invention over VNTR approaches. Thirty-three genome sequences were extracted from references databases for which whole genome sequence assemblies were available. An in silico analysis extracted regions of these genomes that are assayed using MLVA, MLST and spa subtyping methods. The discriminatory index (defined as the % of total genomes discriminated into unique isolates) of each technique was calculated based upon the assayed regions, and compared with the regions assayed by the BioDetection kit. The BioDetection kit discriminated all samples into unique groups, and demonstrated a higher discriminatory index than other assays.

FIG. 16B is a tangelgram demonstrating better phylogeny reproduction by the BioDetection kit. The sequences extracted by the in silico analysis of Figure x2 were used to construct phylogenetic trees to describe the relationships between samples. The whole genome sequence (WGS) was used as a reference tree, and the BioDetection and MLVA constructed trees compared using a tangelgram figure. Red and pink lines represent regions of the tree that are significantly different between methods, whereas parallel grey lines illustrate relationships that are described equivalently by both methods. The figure demonstrates significant discordance between the MLVA and WGS trees, but largely comparable phylogenetic relationships described by the WGS and BioDetection trees. This data indicates that the evolutionary relationships described by the BioDetection kit are a more accurate appraisal of the whole genome relationships and evolutionary distance between samples than MLVA approaches. Similar significant discordances were observed to between WGS and spa-typing and MLST trees compared to MLVA.

FIG. 17 demonstrates detection from DNA isolated from stool, urine and sputum. Sputum, stool and urine sample derived from human individuals were spiked with genomic DNA isolated from cultured bacteria. Samples were extracted using standard DNA extraction methods, such that each sample contained some amount of gDNA plus any additional complex biomolecules that are carried through the extraction from these sample types (heme, complex polysaccharides and potential enzyme inhibitors). Each isolated DNA sample was assayed using the BioDetection kit and results are tabulated. This data demonstrates accurate species and strain detection from DNA isolated from sputum, urine and stool samples, plus identification of resistance genes present within each sample.

FIG. 18 is a summary of 707 bacterial samples sequenced and identified using the BioDetection kit. The table demonstrates the capability of the kit to detect species not listed and validated within the performance specifications, due to the broad detection and discriminatory ability of the selected sequence capture regions.

FIG. 19 shows detection of VRE from rectal swabs. A collection of n=24 positive and negative rectal swab samples screened by microbial culture were collected after primary screening at a hospital laboratory. Bound DNA released into a PBS wash solution by incubation for 1 hr at 37 degrees centrigrade, isolated using a gDNA extraction kit and then assayed using the BioDetection kit. The resulting data was tabulated to indicate the detection of organisms and drug resistance genes (plus read counts) from each sample, and comparison to the clinical surveillance by culture. This data demonstrates accurate species, strain and resistance gene detection from rectal swabs. In particular the data illustrates detection of multiple Enterococcus strains, plus vancomycin resistance, and additional co-present species on the rectal swabs, such as E. coli and K. pneumoniae. Further, sample PGCA963 demonstrates low level E. faecium and E. coli detection in a culture negative sample.

FIG. 20 describes a summary of drug resistance loci detected over n=707 clinical isolates and clinical specimens sequenced using the HAI kit. Read count for each marker exceeds a minimum of 10 reads and often incorporates detection of multiple sequences within the gene, providing high confidence detection. This table demonstrates a range of drug resistance markers confirmed for detection by the BioDetection kit.

FIG. 21 demonstrates a comparison of 2 samples sequenced using the BioDetection kit from clinical Klebsiella isolates. The sequence represents the captured sequence of a single probe within the BioDetection probeset. The pairwise sequence comparison illustrates mismatches between a single probe loci (of multiple discriminating probes) and indicates that even a single loci commonly contains multiple SNPs of high confidence discrimination between closely related species.

FIG. 22A shows high confidence SNP calling by readcount vs WGS. A cohort of 20 MRSA samples were sequenced using the BioDetection kit on an Ion Torrent PGM, and a Nextera™ whole genome sequencing approach on a MiSeq. Samples were sequenced on a Ion 316 chip (˜3.2M reads), and a single MiSeq run (˜15M reads). Reads were aligned to a reference genome and coverage compared between sequencing approaches. The plots describe the genomic coordinates (x-axis) and the log 10 sequencing read depth at each nucleotide (y-axis) for 3 individual probes. The BioDetection kit generates considerably higher readcounts (10-100 fold) at discriminatory regions between samples, enabling higher confidence SNP calling for this targeted sequencing vs the low read depth of whole genome sequencing. This also supports accurate detections for each of the SNPs by independent library constructions using different sequencing technologies.

FIG. 22B shows genomic coordinates. Two sequence alignments compare the consensus read sequence at 2 regions captured by both HAI BioDetection kit, and Nextera Nextera™ whole genome sequencing and reference genome alignment. For samples TC14, TC5 and TC4, the sequences show agreement for detection of an indel within sample TC14, and two SNPs within TC14 relative to TC4 and TC5.

DETAILED DESCRIPTION

Approximately one out of every twenty hospitalized patients will contract a nosocomial infection, more commonly known as a hospital-acquired infection (HAI). More than 70 percent of the bacteria that cause HAIs can be resistant to at least one of the antibiotics most commonly used to treat them. Early detection can be important for controlling the spread of hospital-acquired infections. After culturing for growth and isolation of pathogens, clinical microbiology laboratories may rely on observable phenotype and simple biochemical assays to determine the bacterial type and antibiotic sensitivity. Determining the most effective antibiotic treatment for the infected patient, not the causal agent of the infection, is usually the prerogative of the physician. The resolution of conventional microbiological assays may be insufficient to determine the precise genotype underlying antibiotic resistance. Consequently, the same organism can infect multiple patients, and the spread of infection can go unnoticed for long periods.

Urinary tract infection (UTI) is the most common hospital-acquired infection. UTIs account for about 40 percent of hospital-acquired infections, and an estimated 80 percent of UTIs are associated with urinary catheters. Pneumonia is the second most common HAI. In critically ill patients, ventilator-associated pneumonia (VAP) is the most common nosocomial infection. VAP can double the risk of death, significantly increase intensive care unit (ICU) length of stay, and can add to each affected patient's hospital costs.

A key problem for microbiology labs is the turnaround time from receiving a microbial sample to determining key actionable information for patient care, such as antibiotic drug resistance within the sample, or strain identification for comparison to known high-risk strains. Existing technologies such as PCR or mass spectroscopy have allowed the turnaround time to be improved relative to classical methods for some actionable information, such as species identification, or presence of a select few drug resistance genes, but there are few practical approaches to assaying the large number of drug resistance genes or key species needed to be identified to confidently predict patient treatment.

DNA microarray offers broad detection ability for genomic loci, but is complicated by slow sample preparation and false positive and false negative sample results due to the hybridization based approach. Targeted DNA sequencing using the BioDetection kit allows the greater breadth of target detection, and higher resolution and higher accuracy discrimination due to the single base accuracy of DNA sequencing.

A second competing approach to targeted sequencing is whole genome sequencing. This approach has several disadvantages relative to the targeted sequencing approach provided by the invention. First, whole genome libraries contain many uninformative regions that are identical between the majority of isolates in a species, and thus provide no information to discriminate. These worthless reads mean that many more WGS reads are required per sample to capture informative regions, and prevent higher numbers of samples to be multiplexed into a single sequencing channel to amortize sequencing costs. Second, WGS libraries contain a representative fraction of any DNA present within a sample. As such, primary samples containing human tissue, or many uninteresting bacteria from the perspective of patient health, will comprise mainly of unwanted human or commensal bacterial reads. Efficient detection of important bacteria and drug resistance genes within a sample requires a more efficient targeted approach. Thirdly, library preparation times are slower and more laborious using WGS approaches, and the data analysis time significantly longer than that of a targeted sequencing approach in which only key informative regions are analyzed. This faster analysis reduces turnaround time and costs, and allow simplified data representations for easier understanding for clinical scientists unfamiliar with next generation sequencing data.

Provided herein are compositions, methods, systems and kits for detecting an organism, such as a pathogen, such as a pathogen that causes HAIs, as well as methods for using the system to identifying and detect the organism. The system can comprise a probe or plurality of probes. Also provided herein, are compositions, methods, systems and kits for detecting an organism, such as a pathogen, such as a pathogen that causes HAIs, and detecting and identifying antibiotic resistance genes, which, in some embodiments, can be performed simultaneously.

Probes

In some embodiments, the invention provides panels of probes and methods of using them, where the panels include circularizing capture probes, such as molecular inversion probes. Basic design principles for circularizing probes, such as simple molecular inversion probes (MIPs) as well as related capture probes are known in the art and described in, for example: Nilsson et al., Science, 265:2085-88 (1994); Hardenbol et al., Genome Res.; 15:269-75 (2005); Akharas et al., PLOS One, 9:e915 (2007); Porecca et al., Nature Methods, 4:931-36 (2007); Deng et al., Nat. Biotechnol., 27(4):353-60 (2009); U.S. Pat. Nos. 7,700,323 and 6,858,412; and International Publications WO 2011/156795, WO/1999/049079 and WO/1995/022623, all of which are incorporated by reference in their entirety.

A system for detection of an organism, such as identifying a strain, variant or subtype of a pathogen, can comprise a mixture or probe set comprising a plurality of probes. The target organism for a particular probe may be any organism, such as a viral, bacterial, fungal, archaeal, or eukaryotic, organisms, including single cellular and multicellular eukaryotes. In particular embodiments, a target organism is a pathogen. In some embodiments, target organisms include organisms associated with or that cause HAIs, such as those organisms provided in Table 2.

In some embodiments, each single-stranded capture probe can hybridize to two complementary regions on a target DNA with a gap region in between. An enzyme, such as DNA polymerase, can be used to fill in the gap using the target as template, and stop adding nucleotides when it reaches the phosphorylated 5′-terminus of the hybridized probe. An enzyme, such as a thermostable ligase, can be used to covalently close the extended probe to form a circular molecule. Exonucleases can be used to digest away residual probe molecules. The filled-in, circularized probe can be resistant to exonuclease digestion, and can serve as template for preparation of the sequencing library by known methods, such as PCR. Sample-associated barcodes can be added and can enable multiple barcoded samples to be blended and analyzed together, such as on a DNA sequencer.

A probe can refer to a sequence that hybridizes to another sequence. The probe can be a linear, unbranched polynucleic acid. The probe can comprise two homologous probe sequences separated by a backbone sequence, where the first homologous probe sequence is at a first terminus of the nucleic acid and the second homologous probe sequence is at the second terminus to the nucleic acid, and where the probe is capable of circularizing capture of a region of interest of at least 2 nucleotides. Circularizing capture can refer to a probe becoming circularized by incorporating the sequence complementary to a region of interest.

In a preferred embodiment, the probes contain two arms, joined by a backbone, that hybridize to a target sequence. A polymerase molecule can extend the 3′ end of the probe by copying a target region into a probe molecule. A ligase molecule can circularize a probe molecule by joining the 3′ end of the copied target to the 5′ end of the original probe molecule.

In one embodiment, probe arms can hybridize to the target nucleic acid molecule, surrounding the capture region; a polymerase extension can fill in the gap between the arms and a ligase can create a circular molecule out of the extended probe. After an exonuclease digestion removes the original template molecules, primers can be used to amplify the captured probes. The primers can contain a 3′ end homologous to the backbone (forward) and its reverse complement (reverse primer). The 5′ of the primer may contain a sequencing adapter for a particular next generation sequencing platform and may also contain a barcode sequence between the 5′ and 3′ segments such that multiple samples, each amplified with primers containing a sample-specific barcode, can be multiplexed into a single sequencing run. As the two probe arms are linked by a backbone, on-target binding is energetically favorable, even when many (hundreds, thousands, or tens of thousands) of probes are present in a single reaction (compare to PCR, in which one primer of a pair may hybridize and extend at an off-target locus). As with PCR, each MIP can capture a well-defined region of the target sequence (compare to hybridization capture methods, which yield a variety of molecules centered around the target).

In a preferred embodiment, a backbone of a probe molecule contains the same sequence in all probes. A backbone can contain two primer binding sites that allow amplification of probe arms and a captured target sequence. In a preferred embodiment, the primers used may contain a barcode to allow multiple samples to be separated after simultaneous sequencing. In a preferred embodiment, the primers also contain 5′ ends that adapters for a next-generation sequencing platform such as the Ion Torrent PGM, Illumina MiSeq, Illumina HiSeq, Nanopore, etc (FIG. 1).

The probe set can include large number of probes, e.g., 10, 20, 30, 40, 50, 100, 200, 400, 500, 1000, 2000, 3000, 4000, 5000, 10000, 20000, 40000, 80000, or more. The probe set can include one or more probes directed to a large number of different target organisms, e.g., at least 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1250, 1500, 1750, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more different target organisms. In some embodiments, a mixture including one or more probes to a plurality of target organisms contains only one probe to a target organism. In other embodiments, the mixture contains more than one probe to a target organism, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 probes for a target organism. In certain embodiments, such as embodiments designed for use with patient test samples, the mixture further includes probes with homologous probe sequences that specifically hybridize to the host genome for applications such as host genotyping. In some embodiments, the mixtures of the invention further comprise sample internal calibration standards.

In one embodiment, the plurality of probes can detect at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, or 2000 different organisms or pathogens. In another embodiment, the plurality of probes can detect at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1250, 1500, 1750, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more different strains, variants or sub-types of a pathogen or different strains or sub-types of different pathogens. In one embodiment, the probe set identifies detects at least 2 different bacterial or fungal strains. In another embodiment, the probe set identifies at least 50 different organisms, such as 50 different pathogens, or 50 different strains or subtypes of a pathogen, such as Staphylococcus aureus.

In another embodiment, the probe set can comprise probes capable of detecting a single molecule of a pathogen, thereby detecting, distinguishing or identifying the pathogen.

Each probe in the probe set can comprise the same or different backbone size, sequence, chemistries, configuration of barcodes and sequences, specific sequences for probe enrichment, target sites for probe cleavage, hybridization arm physical and chemical properties, probe identification regions, low structure optimized design, or any combination thereof. A probe may be selected to screen key loci for pathogenicity and/or drug susceptibility, and a genetic fingerprint or genotype for each sub-strain that contains key phenotypic information is generated.

In another embodiment, the probe comprises a first sequence that hybridizes to a 5′ end of a target sequence and a second sequence that hybridizes to a 3′ end of a target sequence, wherein the target sequence can be used to identify, detect, or distinguish an organism, such as pathogen. In some embodiments, the probes in the mixture each comprise a first and second homologous probe sequence—separated by a backbone sequence—that specifically hybridize to a first and second sequence (such as sequences 3′ and/or 5′ to a target sequence, respectively) in the genome of at least one target organism. In some embodiments the first and second homologous probe sequences are not complementary to the target sequence, but ligate to the 5′ and 3′ termini of a target nucleic acid, e.g. a microRNA, and possess appropriate chemical groups for compatibility with a nucleic acid-ligating enzyme, such as phosphorylated or adenylated 5′ termini, and free 3′ hydroxyl groups. The probe can be capable of circularizing capture of a region of interest.

In some embodiments, the homologous probe sequences or the sequences of the probe that hybridize or are homologous to the 3′ and/or 5′ region of a target sequence specifically hybridizes to target sequences in the genome of their respective target organism, but do not specifically hybridize to any sequence in the genome of a predetermined set of sequenced organisms—the exclusion set. In embodiments related to probes that do not hybridize directly to the capture target, the ‘homologous probe sequences’ are designed specifically to not substantially hybridize to any sequence within a defined set of genomes, i.e., an exclusion set. In the case of biological samples from a subject, the exclusion set includes the host's genome. In particular embodiments, the exclusion set also includes a plurality of viral, eukaryotic, prokaryotic, and archaeal genomes. In more particular embodiments, the plurality of viral, eukaryotic, prokaryotic, and archaeal genomes in the exclusion set may comprise sequenced genomes from commensal, non-virulent, or nonpathogenic organisms. In still more particular embodiments, the exclusion set for all probes in a mixture share a common subset of sequenced genomes comprising, for example, a host genome and commensal, non-virulent, or non-pathogenic organisms. In general, the exclusion set varies between probes in the mixture so that each probe in the mixture does not specifically hybridize with the target sequence of any other probe in the mixture.

In some embodiments, the sequences 3′ and/or 5′ to a target sequence are separated by a region of interest (e.g., the target sequence) of at least two nucleotides. In particular embodiments, they are separated by at least 5, 6, 7, 8, 9, 10, 12, 14, 18, 20, 25, 30, 50, 75, 100, 150, 200, 300, 400, 600, 1200, 1500, 2500, or more nucleotides. In some embodiments, the first and second target sequences are separated by no more than 5, 6, 7, 8, 9, 10, 12, 14, 18, 20, 25, 30, 50, 75, 100, 150, 200, 300, 400, 600, 1200, 1500, or 2500 nucleotides.

In some embodiments, probes can be designed to capture conserved regions, and upon DNA sequencing, can reveal polymorphisms and genetic aberrations that allow for the resolution of known or novel variants or closely related strains of organisms. In some embodiments two or more probes can be used for one or more or every organism wished to be tested for, which can permit discrimination of closely related organisms, even when a sample comprises more than one organism.

In one aspect, the probes in the probe set each comprising homologous probe sequences which are substantially free of secondary structure, do not contain long strings of a single nucleotide (e.g., they have fewer than 7, 6, 5, 4, 3, or 2 consecutive identical bases), are at least about 8 bases (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 30, or 32 bases in length), and have a T_min the range of 50-72° C. (e.g., about 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62° C.). In some embodiments the first and second homologous probe sequences are about the same length and have the same T_m. In other embodiments, length and T_mof the first and second homologous probe sequences differ. The homologous probe sequences in each probe may also be selected to occur below a certain threshold number of times in the target organism's genome (e.g., fewer than 20, 10, 5, 4, 3, or 2 times).

The backbone sequence of the probes may include a detectable moiety and a primer-binding sequence. In some embodiments, the backbone sequence of the probes comprises a second primer. In particular embodiments, the detectable moiety is a barcode. In certain embodiments the backbone further comprises a cleavage site, such as a restriction endonuclease recognition sequence. In certain embodiments, the backbone contains non-WatsonCrick nucleotides, including, for example, abasic furan moieties, and the like.

In another aspect, the invention provides a kit comprising one or more sets of probes, such as one or more sets of probes from the probes provided in Table 1. In one embodiment, a kit comprises one or more reagents for obtaining a sample (e.g., swabs), reagents for extracting DNA, enzymes (such as polymerase and/or ligase to capture a region of interest), reagents for amplifying the region of interest, reagents for purifying the DNA or amplified or captured regions of interest (e.g., purification cartridge), buffers, sequencing reagents, or any combination thereof. In one embodiment, the kit may be a low throughput kit, such as a kit for a small number of samples. For example, a kit may be a low throughput kit, such as a kit for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 20, 24, 28, 32, 36, 40, 42, 48, or between 8-48 samples. In another embodiment, the kit may be a high-throughput kit, such as a kit for a large number of samples. For example, a kit may be a high-throughput kit, such as a kit for 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, or more samples. For example, a kit may be a high-throughput kit, such as a kit for between 50-96, 50-384, 50-1536, 96-384, 96-1536, or 384-1536 samples. In some embodiments, a kit as described herein can comprise enough reagents to prepare one or more specimens for sequencing. For example, a kit as described herein can comprise enough reagents to prepare 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 48, 50, 60, 70, 80, 90, 96, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 384, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1536, 1750, 2000 or more specimens for sequencing.

Method of Using Probe

Also provided herein is a method of using one or more probes disclosed herein, such as one or more probe set, for detecting, identifying, or distinguishing one or more organisms. The method can comprise identifying a an organism with a plurality of probes can detect at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, or 2000 different pathogens. In another embodiment, the plurality of probes can detect at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1250, 1500, 1750, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more different strains, variants or sub-types of a pathogen or different strains or sub-types of different pathogens.

The method can comprise detecting or distinguishing different organisms, different pathogens, different strains, variants or sub-types of a pathogen or different strains, variants or sub-types of different pathogens, with at least 70% sensitivity, specificity, or both, such as with at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89% sensitivity, specificity, or both, such as with at least 90% sensitivity, specificity, or both. Each probe may detect or distinguish different organisms, different pathogens, different strains or sub-types of a pathogen or different strains or sub-types of different pathogens with at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sensitivity, specificity, or both, in an assay. Alternatively, a combination of probes may be used for detecting or distinguishing different organisms, different pathogens, different strains, variants or sub-types of a pathogen or different strains, variants or sub-types of different pathogens, with at least 70% sensitivity, specificity, or both, such as with at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89% sensitivity, specificity, or both, such as with at least 90% sensitivity, specificity, or both. Furthermore, the confidence level for determining the specificity, sensitivity, or both, may be with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% confidence.

In one embodiment, a method for detecting the presence of one or more target organisms is by contacting a sample suspected of containing at least one target organism with any of the probe set disclosed herein, capturing a region of interest of the at least one target organism (e.g., by polymerization and/or ligation) to form a circularized probe, and detecting the captured region of interest, thereby detecting the presence of the one or more target organisms.

In certain embodiments, the captured region of interest may be amplified to form a plurality of amplicons (e.g., by PCR). In some embodiments the sample is treated with nucleases to remove the linear nucleic acids after probe-circularizing capture of the region of interest. In some embodiments, the circularized probe is linearized, e.g., by nuclease treatment. In other embodiments the circularized probe molecule is sequenced directly by any means known in the art, without amplification. In certain embodiments, the circularized probe is contacted by an oligonucleotide that primes polymerase-mediated extension of the molecules to generate sequences complementary to that of the circularized probe, including from at least one to as many as 1 million or more concatemerized copies of the original circular probe.

In particular embodiments, the circularized probe molecule is enriched from the reaction solution by means of a secondary-capture oligonucleotide capture probe. A secondary-capture oligonucleotide capture probe may comprise a moiety designed to be captured, such as a biotin molecule, and a nucleic acid sequence designed to hybridize to at least 6 nucleotides of the circularized probe. The nucleic acid sequence designed to hybridize to at least 6 nucleotides of the circularized probe may include 1, 2, 4, 8, 16, 32 or more nucleotides of the polymerase-extended capture product.

In certain embodiments, the probe and/or captured region of interest is sequenced by any means known in the art, such as polymerase-dependent sequencing (including, dideoxy sequencing, pyrosequencing, and sequencing by synthesis) or ligase based sequencing (e.g., polony sequencing). The sequencing can be by Sanger sequencing or massive parallel sequencing, such as “next generation” (Next-gen) sequencing, second generation sequencing, or third generation sequencing. For example, sequencing can be by second generation or third generation sequencing methods, such as using commercial platforms such as Illumina, 454 (Roche), Solid, Ion Torrent PGM (Life Technologies), PacBio, Oxford, Life Technologies QDot, Nanopore, or any other available sequencing platform. Massive parallel sequencing can allow for the simultaneous sequencing of one million to several hundred millions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 48, 50, 60, 70, 80, 90, 96, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 million, of reads from amplified DNA clones. The reads can read any number of bases, such as 50-400 bases.

An internal nucleotide control, such as DNA at a known concentration, can be used with the methods and samples described herein. In one embodiment, an internal nucleotide control can serve as an internal calibrator, such as for determining copy number. In some embodiments, a sequencing read that aligns to the calibrator can also serve as a positive control for the performance of the assay, such as in the context of every sample.

In one aspect, the probes, methods, and kits described herein can be used to test for the presence of one or more organisms, such as those in Table 2. In one embodiment, the probes, methods, and kits described herein can be used to test for the presence of one or more antibiotic resistance genes, such as those in Table 3. In a preferred embodiment, the probes, methods, and kits described herein can be used to test for the presence of one or more organisms, such as those in Table 2, and test for the presence of one or more antibiotic resistance genes, such as those in Table 3, in parallel, such as in one sample tube, in the same sample, simultaneously, or any combination thereof. In some embodiments, in a single reaction tube, a kit can be used to test for the two or more microbes most commonly associated with hospital-acquired infections, and simultaneously tests for the presence of two or more antibiotic resistance genes. For example, a kit can be used to test for the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more microbes most commonly associated with hospital-acquired infections, and simultaneously tests for the presence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more antibiotic resistance genes simultaneously. For example, in a single reaction tube, a kit can be used to test for the 12 microbes most commonly associated with hospital-acquired infections, and simultaneously tests for the presence of 18 antibiotic resistance genes.

In one embodiment, one or more organisms can be identified from a sample, such as a sample form a host and the organism being identified is a pathogen. In one embodiment, the sample is a biological sample, such as from a mammal, such as a human. In another embodiment, a genotype of the host is identified or detected from the sample or another sample from the host. The identification of one or more organisms (such as one or more pathogens, such as different pathogens or subtypes or strains of pathogens), can be used to select one or more therapeutics or treatments for the host. In another embodiment, the identification of one or more organisms (such as one or more pathogens, such as different pathogens or subtypes or strains of pathogens), can be used to stratify the host into a therapeutic group, such as for a particular drug treatment or clinical trial. In one embodiment, HPV strain identification can be used to stratify a host into a cancer therapeutic group or to select a cancer treatment.

The yet another embodiment identification of one or more organisms (such as one or more pathogens, such as different pathogens or subtypes or strains of pathogens) and the genotype of a host can be used to select one or more therapeutics or treatments for the host. In another embodiment, the identification of one or more organisms (such as one or more pathogens, such as different pathogens or subtypes or strains of pathogens) and the genotype of the host can be used to stratify the host into a therapeutic group, such as for a particular drug treatment or clinical trial.

Also provided herein is a method for identifying an organism, such as a genetic signature of an organism, a subtype or strain of a pathogen in a short timeframe or with a fast turnaround time. In another embodiment, a genotype of an individual or host can also be identified within the short time frame. For example, the identification of a pathogen in a sample or the genotype of a host can completed in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In one embodiment, from contacting the sample with one or more probes to identifying the organism by sequencing can be performed in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In yet another embodiment, from contacting the sample with the probe to identifying the organism (such as one or more pathogens) by sequencing, and transmitting the results to a health care professional (such as a clinician or physician) can be performed in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In yet another embodiment, from contacting the sample with the probe to identifying the organism (such as one or more pathogens) by sequencing, transmitting the results to a health care professional (such as a clinician or physician), and selection of a therapeutic can be performed in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

Also provided herein is a method for simultaneous quantification and identification of an organism, such as identifying one or more subtypes or substrains of a pathogen. Multiplexing is also provided herein, wherein a multiple pathogens, substrains or subtypes of pathogens, can be detected simultaneously or in a single reaction tube.

In one embodiment, conversion of sequence data to quantitative report can be performed by using selected validated parameters. Any software known in the arts can be used for any of the methods disclosed herein.

In some embodiments, an organism identified and/or quantified using the methods described herein can be the cause of an infection in a subject, such as a nosocomial infection (also known as a hospital-acquired infection (HAI)) which is an infection whose development is favored by a hospital environment. In some embodiments, an infection can be acquired by a patient during a hospital visit or one developing among hospital staff. Such infections can include, for example, fungal and bacterial infections and can be aggravated by a reduced resistance of individual patients. Organisms responsible for HAIs can survive for a long time on surfaces in the hospital and can enter or be transmitted to the body through wounds, catheters, and ventilators. In some embodiments, the route of transmission can be contact transmission (direct or indirect), droplet transmission, airborne transmission, common vehicle transmission, vector borne transmission, or any combination thereof.

People in hospitals can already be in a poor state of health, impairing their defense against bacteria. Advanced age or premature birth along with immunodeficiency, due to, for example, drugs, illness, or irradiation, present a general risk. Other diseases can present specific risks, for example, chronic obstructive pulmonary disease can increase chances of respiratory tract infection. Invasive devices, for example, intubation tubes, catheters, surgical drains, and tracheostomy tubes can bypass the body's natural lines of defense against pathogens and can provide an easy route for infection. Patients already colonized on admission can be put at greater risk when they undergo a procedure, such as an invasive procedure. A patient's treatment itself can leave the patient vulnerable to infection, for example, immunosuppression and antacid treatment can undermine the body's defenses, while antimicrobial and recurrent blood transfusions can also be risk factors.

Non-limiting examples of HAIs include Ventilator associated pneumonia (VAP), Staphylococcus aureus, Methicillin resistant Staphylococcus aureus (MRSA), Candida albicans, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Clostridium difficile, Tuberculosis, Urinary tract infection, Hospital-acquired pneumonia (HAP), Gastroenteritis, Vancomycin-resistant Enterococcus (VRE), and Legionnaires' disease. In some embodiments, HAIs can be caused by one or more of the organisms provided in Table 2.

Nucleotides, such as DNA and RNA, can be isolated from any suitable sample and detected using the probes described herein. Non-limiting examples of sample sources include catheters, medical devices, blood, blood cultures, urine, stool, fomites, wounds, sputum, pure bacterial cultures, mixed bacterial cultures, and bacterial colonies.

In some embodiments, the probe sets described herein can be used to detect and distinguish among the organisms responsible for more than 10% of the hospital acquired infections at a site. For example, the probe sets described herein can be used to detect and distinguish among the organisms responsible for more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the hospital acquired infections at a site. In some embodiments, a site can be a surgical site, wound, tract, urinary catheter, ventilator, intravenous needle, syringe, respiratory tract, invasive device, intubation tube, catheter, surgical drain, tracheostomy tube, saline flush syringe, vial, bag, tube or any combination thereof.

Method of Generating Probe

A further aspect of the invention provides methods of making the mixtures of probes provided by the invention. The methods comprise providing a set of reference genomes and an exclusion set of genomes. The sequence of the reference genomes can be partitioned (in silico) into n-mer strings of about 18-50 nucleotides. The partitioned n-mer strings can be screened to eliminate redundant sequences, sequences with secondary structure, repetitive sequences (e.g., strings with more than 4 consecutive identical nucleotides), and sequences with a T_moutside of a predetermined range (e.g., outside of 50-72° C.). The screened n-mers can be further screened to identify homologous probe sequences by eliminating n-mers that specifically hybridize to a sequence in the genome in the exclusion set of genomes (e.g., if a pairwise alignment contains 19 of 20 matches in an n-mer, such as a 25-mer) or occurs in the genome of the target organism more than a specified number of times. The screening may also remove n-mers that are present in more than or less than a specified number of the reference genomes. The screening may also remove n-mers that will not interact favorably with enzymes to be used with the probe sequences. For example, a particular polymerase may work with higher efficiency if the last 3′ base of the probe is a G or C. Similarly, a particular ligase may work more efficiently on certain bases at the ligation junction. For example, Ampligase (Epicentre) will ligate a gap between AG and GT at least 10 times more efficiently than a gap between TC and CC.

In particular embodiments, a homologous probe sequence may occur only once in the genome of the target organism. For target organisms with a single-stranded genome, the homologous probe sequence may occur only once in the complement of the genome of the target organism. In one embodiment, where a sequenced variant of the target organism is available (e.g., the same species, genus, or serovar), the homologous probe sequences can be filtered so as to specifically hybridize to the genome of the additional sequenced variant(s) resulting in a probe that groups related organisms. In an alternate embodiment, the homologous probe sequences can be filtered so as to not specifically hybridize to the genome of the sequenced variant (e.g., the sequenced variant is part of the exclusion set), resulting in a probe that discriminates between related organisms. These filter processes can be iterated for each target organism to be detected by the particular mixture. In some embodiments, the candidate homologous probe sequences can be screened to eliminate those that will specifically hybridize with other probes in the mixture.

Probe selection can be based on a database of different pathogens, strains of a pathogen, or both, such as a database comprising more than 10 different pathogens, strains of a pathogen, or both. For example, probe selection can be based a database comprising more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, or more different pathogens, strains of a pathogen, or both. In some embodiments, probe selection can be based on a database of different pathogens, strains of a pathogen, or both, that are known to cause HAIs, such as a database comprising more than 10 different pathogens, strains of a pathogen, or both, that are known to cause HAIs. For example, probe selection can be based a database comprising more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, or more different pathogens, strains of a pathogen, or both, that are known to cause HAIs, and optionally with additional strains or sub-types of other pathogens. In one embodiment, probes for organisms associated with HAIs are selected by partitioning all available genomes of organisms associated with HAIs into one or more subsets based on sequence similarity. For each subset candidate probe sets are generated that capture all strains. A filter can then be applied for specificity against human/microbial/viral/fungal genomes.

Some of clinical tests based on the methods disclosed herein rely on the ability to determine or approximate the number of input template molecules (genomes) in a sample. A two step method can be used to calculate the number of template molecules in a sample from the sequencing read counts. 1) Each sample sequenced can have a known quantity of a control sequence added to it. One embodiment employs GFP as the control sequence. It is contemplated to use several control sequences added in different quantities. The first step in analyzing sequencing reads can be to normalize the counts based on the number of reads that came from the control sequence. This normalization accounts for the fact that more material from sample A than from sample B may have been put into the sequencing reaction. 2) Since different MIPs (or primer pairs or hybridization capture probes) might work with different efficiencies, the second step of the quantification process can be to normalize between probes. In one embodiment, this normalization relies on experiments in which fixed amounts of different templates were sequenced and might reveal, e.g., that a probe against one strain or organism produces 2 circularized MIPs per template but a probe against anther strain or organism produces 3. Thus, the count for the first probe might be multiplied by 33.3 and the count for the second probe divided by 50 to produce comparable load counts for the two strains.

Some embodiments use a mixed quantity of GFP as the control sequence and a variable quantity of one or more organisms or strains. Some samples may contain only GFP and template DNA while others also included a human background. After the sequencing reads are separated by sample, the method can calculate the ratio of reads, such as viral (HPV-18, HIV-CN006, and HIV-CN009) reads, to GFP and plots that ratio against the number of template molecules in the reaction. Those plots indicate generally excellent agreement between the viral/GFP ratio and the input template quantity.

Compared to other assays, high throughput sequencing offers a relatively unique ability to detect and genotype the pathogen DNA and the human DNA in a sample from a single reaction. In current clinical practice, genotyping the pathogen and human may require multiple tests, potentially doubling (or more) the expense compared to simply detecting a pathogen. The methods disclosed herein enable simultaneous genotyping with minimal added cost and often no added labor. Other selection/enrichment technologies would also enable these tests.

The methods disclosed herein provide for simultaneously detecting or genotyping multiple pathogens.

For example, the methods provide for: coinfection of HIV and HCV, simultaneously genotyping/quantifying HIV while testing for diseases common in immunocompromised patients. Doctors typically only test for diseases like Candida, CMV, etc upon presentation of some other symptom. However, if the tests can be added at minimal cost, this might be a unique market and feature for Pathogenica's product, for example, HPV and other STIs. There is an interest in testing for HPV and other STIs, primarily chlamydia and gonorrhea to simplify screening, especially in patient populations with limited access to doctors. There is also an interest in testing for these diseases as additional risk factors for cervical cancer.

Probe Panel

Table 1 lists the probe arm sequences in one embodiment of the present invention designed to detect a variety pathogenic organisms, such as those provided in Table 2, from a sample. Non limiting examples include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Acinetobacter baumanii, Clostridium difficile, Escherichia coli, Enterobacter (aerogenes, cloacae, asburiae), Enterococcus (faecium, faecalis), Klebsiella pneumoniae, Proteus mirabilis, Candida albicans, and Pseudomonas aeruginosa. The probe set can also be used to detect many common drug resistance genes, including, but not limited to CARB, CMY, CTX-M, GES, IMP, KPC, NDM, Other ampC, OXA, PER, SHV, VEB, VIM, ermA, vanA, vanB, mecA, and mexA,

Tables 1 and 3-14 provide regions of interest (leftmost columns, using the format of descriptor (e.g., organism or gene, if applicable)_reference accession number (if applicable)_first nucleotide of capture region_last nucleotide of capture region. For example, the probe “acinetobacter_NC_—010611_—627997_—628164” is directed to acineobacter, and is predicted to be capable of capturing nucleotides corresponding to nucleotides 627997 to 628164 of the reference sequence NC_—010611. Reference accession sequences can be obtained from, for example, the NCBI Entrez portal. Tables 3, 5, 7, 9, 11, and 13 provide the regions of interest and corresponding annotated genes within that region. Tables 4, 6, 8, 10, 12, and 14, in turn, provide particular exemplary oligonucleic acid sequences—provided as pairs that can be used in a MIP or adapted for use as conventional PCR primers—predicted to capture the region of interest listed in the first column of the. “Binding region 1” in Tables 4, 6, 8, 10, 12, and 14 correspond to the 5′, or ligation arm, of a MIP probe and “Binding region 2” corresponds to the 3′, or extension arm of a MIP probe. In some embodiments, substantially similar sequences to the regions of interest provided in Tables 1 and 3-14 can be used. In some embodiments, the substantially similar sequences wherein the substantially similar sequences are 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to the sequence of the regions of interest. In other embodiments, the substantially similar sequences have endpoints within 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides upstream or downstream of either of the endpoints of the regions of interest. In still other embodiments, the substantially similar sequences are 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to the sequence of the regions of interest and have endpoints within 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides upstream or downstream of either of the endpoints of the regions of interest. In still more particular embodiments, the particular exemplified endpoints and binding regions are use, e.g., as pairs of binding regions in either a single MIP capture probe, or as pairs of conventional PCR primers, e.g., using the reverse complement of the ligation arm.

Subsets of the regions of interest or particular exemplary binding regions in tables Tables 1 and 3-14 can be used concordant with the present invention, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the regions of interest or binding regions in the tables, e.g.:

oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of 1, 2, 3, 4, 5, 10, 15, 16, or all 17, of the regions of interest provided in column 1 of Table 3, or substantially similar sequences;

oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of 1, 2, 3, 4, 5, 10, 15, 20, 30, 50, 100, or all 134, of the regions of interest provided in column 1 of Table 5, or substantially similar sequences, such as:

oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of, 1, 2, 3, 4, 5, 10, or all 13, of the regions of interest provided in column 1 of Table 7, or substantially similar sequences;

oligonucleic acid molecules capable amplifying, geometrically by polymerase chain reaction, or circularizing capture of, 1, 2, 3, 4, 5, 10, 20, 40, 60, 80, or all 85, of the regions of interest provided in column 1 of Table 9, or substantially similar sequences;

oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of, 1, 2, 3, 4, 5, 10, 20, 25, or all 29 of the regions of interest provided in column 1 of Table 11, or substantially similar sequences;

oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of, 1, 2, 3, 4, 5, 10, 15, or all 20, of the regions of interest provided in column 1 of Table 13, or substantially similar sequences;

oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, or all 34 of the sequences, or reverse complements thereof, provided in the second or third column of table 4;

oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 20, 50, 100, 150, 200, 250, or all 268 of the sequences, or reverse complements thereof, provided in the second or third column of table 6;

oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 15, 20, 25, or all 26 of the sequences, or reverse complements thereof, provided in the second or third column of table 8;

oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 20, 50, 100, 150, or all 170 of the sequences, or reverse complements thereof, provided in the second or third column of table 10;

oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 20, 30, 40, 50, or all 56 of the sequences, or reverse complements thereof, provided in the second or third column of table 12;

oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 20, 30, or all 40 of the sequences, or reverse complements thereof, provided in the second or third column of table 14, as well as any combinations of the foregoing.

Table 1 provides particular probes assembled as molecular inversion probes (MIPs) capable of circularizing capture of the indicated region of interest in the leftmost column. These exemplary probes share a common backbone sequence of GTTGGAGGCTCATCGTTCCTATATTCCACACCACTTATTATTACAGATGTTATGCT CGCAGGTC, except for the peGFP_N1_—730_—925 probe, which uses the backbone GTTGGAGGCTCATCGTTCCTATATTCCTGACTCCTCATTGATGATTACAGATGTTA TGCTCGCAGGTC. Alternative backbone sequences can readily be used. Conventional PCR primer pairs can be adapted from these MIP probes by omitting the intervening backbone sequence and providing the reverse complement of the ligation arm (5′) probe. Tables 4, 6, 8, 10, 12, and 14 provide subsets of the probes in Table 1 where the individual arms are provided in the second and third columns, respectively. Tables 4, 6, 8, 10, 12, and 14 collectively provide the same probe arms that are present in Table 1.

TABLE 1

A particular embodiment of the probe sets provided by the invention

Name
Sequence

peGFP_N1_730_925
/5Phos/GTGGTATGGCTGATTATGATCTAGAGTGTTGGAGGCTCATCGTTCCTATA

TTCCTGACTCCTCATTGATGATTACAGATGTTATGCTCGCAGGTCGAGTTTGGACAA

ACCACAACTAGAA

plasmids_NC_010660_187035_187205
/5Phos/GCTGTCACCGTCCAGACGCTGTTGGCGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCCGTGCCTTCAAGCGCG

plasmids_NC_014232_5501_5677
/5Phos/GACTCCGCAGAATACGGCACCGTGCGCAGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCGTACAGGCCAGTC

AGC

plasmids_NC_011980_58308_58487
/5Phos/GCAGTCGGTAACCTCGCGCGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCGCGCTATCTCTGCTCTCACTGC

plasmids_NC_011838_178818_178996
/5Phos/GCTGTCCTGGCTGCAAGCCTGGGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCGAACTGCTGATGGACGT

plasmids_FN554767_13017_13190
/5Phos/GACAGCAGACTCACCGGCTGGTTCCGCTGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAAGATGCTGCTGG

CCACACTG

plasmids_NC_013655_115365_115542
/5Phos/GACAGAACAAGTTCCGCTCCGGGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACGGATACGCCGCGCAT

plasmids_NC_013950_90185_90338
/5Phos/GAGGACCGAAGGAGCTAACCGGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGCCGCATACACTATTCTC

plasmids_NC_015599_37281_37455
/5Phos/GCTGTAATGCAAGTAGCGTATGCGCTCGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAACAGCAAGGCCGCC

AATGCCTGACG

plasmids_NC_013951_69899_70067
/5Phos/GAACGTCTGGCGCTGGTCGCCTGCCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCACAGGTGCTGACGTGGT

plasmids_NC_007351_37979_38146
/5Phos/CGCATATGCTGAATGATTATCTCGTTGCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTTGCTCAATGAG

GTTATTCA

plasmids_FN822749_1846_2009
/5Phos/GACGACAGATGCAGGTTGAGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCCGCATCGCCGATGCTCATC

plasmids_NC_004851_143949_144109
/5Phos/CGCCTGCTCCAGTGCATCCAGCACGAATGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATGCTCTCCGCCATC

GCGTTGTCA

plasmids_NC_010558_156799_156957
/5Phos/AGTGCGTTCACCGAATACGTGCGCAGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAGGTTATGCCGCTCAAT

TC

plasmids_NC_007635_38395_38566
/5Phos/AATCCAGGTCCTGACCGTTCTGTCCGTGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCTCCGTTGAGCTGA

TGGA

plasmids_NC_009787_17946_18116
/5Phos/GAGGTGGCCAACACCATGTGTGACCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGACGCCGGTATATCGGTA

TCGAGCTGCT

plasmids_NC_012547_53585_53752
/5Phos/CGCATATGCTGAATGATTATCTCGTTGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGGTGATCTTGCTCA

ATGAGGTTATTC

plasmids_NC_006671_56259_56438
/5Phos/GAAGTGCCGGACTTCTGCAGAGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCACGGCCTGATGGAGGCCGC

plasmids_NC_014385_53151_53310
/5Phos/GCTAATCGCATAACAGCTACGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCCATCACGTAACTTATTGATGATA

TT

plasmids_FN649418_57169_57339
/5Phos/GCTGCGGTATTCCACGGTCGGCCGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAGGAACGCTGCCTGTGGTC

plasmids_NC_005011_8620_8785
/5Phos/GAATCAATTATCTTCTTCATTATTGATGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTGCGGCTCAACTCAA

GCA

plasmids_NC_014843_98413_98578
/5Phos/GTCACACGTCACGCAGTCCGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCGCATTCATGGCGCTGATGGC

plasmids_NC_008490_5165_5334
/5Phos/GTGTTACTCGGTAGAATGCTCGCAAGGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTAGATGACATATCA

TGTAAGTT

plasmids_NC_015963_147516_147686
/5Phos/CGGAACTGCCTGCTCGTATGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCAACGATATAGTCCGTTAT

plasmids_NC_007365_100545_100708
/5Phos/GCTCTCCGACTCCTGGTACGTCAGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCGCGCATTAATGAAGCAC

plasmids_NC_009838_104163_104332
/5Phos/GATGTTGCGATTACTTCGCCAACTATTGGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTGTAATTATGACG

ACGCCG

plasmids_NC_013452_4052_4209
/5Phos/CTCATTCCAGAAGCAACTTCTTCTTGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGATAGCCATGGCTACAA

GAATA

plasmids_NC_010409_39768_39935
/5Phos/GCAATACCAGGAAGGAAGTCTTACTGGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCATTGGAGAACAGAT

GATTGATGT

plasmids_NC_014233_50337_50492
/5Phos/GTATCGCCACAATAACTGCCGGAAGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAACGATATAGTCCGTTATG

plasmids_NC_013950_91008_91174
/5Phos/GCTGTGGCACAGGCTGAACGCCGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGTGATGTCATTCTGGTTAA

GA

plasmids_NC_002698_168967_169123
/5Phos/ACATAATCTGAATCTGAGACAACATCGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGCACTCTGGCCACAC

TGG

plasmids_NC_013362_56651_56805
/5Phos/GTGAAGCGCATCCGGTCACCGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCATGGCATAGGCCAGGTCAATAT

plasmids_NC_014208_52313_52469
/5Phos/GGTTCTGGACCAGTTGCGTGAGCGCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTAACATCGTTGCTGCT

CCAT

betalactamase_AB372224_738_905
/5Phos/CGCTGGATTTCACGCCATAGGCGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTCGCTACCGTTGATGATT

betalactamase_EF685371_398_548
/5Phos/CGTATAGGTGGCTAAGTGCAGCGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAACTCATTCCTGAGGGTTTC

betalactamase_DQ149247_231_371
/5Phos/GTACATACTCGATCGAAGCACGAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCGGAATAGCGGAAGCTTTC

betalactamase_AY750911_244_414
/5Phos/AAGGTCGAAGCAGGTACATACTCGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGACATGAGCTCAAGTCCA

AT

betalactamase_DQ519087_417_575
/5Phos/GAAGCTTTCATAGCGTCGCCTAGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTAGCTAGCTTGTAAGCAAA

TTG

betalactamase_AM231719_379_537
/5Phos/GAAGCTTTCATGGCATCGCCTAGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCTAGCTTGTAAGCAAACTG

betalactamase_Y14156_663_819
/5Phos/CGCTACCGGTAGTATTGCCCTTGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGAATATCCCGACGGCTTTC

betalactamase_JN227085_763_931
/5Phos/ATCGCCACGTTATCGCTGTACTGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTTACCCAGCGTCAGATTCC

betalactamase_EU259884_1030_1170
/5Phos/CAAGTACTGTTCCTGTACGTCAGCGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCGCCAGTAACTGGTCTAT

TC

betalactamase_HQ913565_578_730
/5Phos/CAACGTCTGCGCCATCGCCGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCCGCAATATCATTGGTGGTGC

betalactamase_AY524988_385_552
/5Phos/GCCGCCCGAAGGACATCAACGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCCAGACGGGACGTACACAAC

CARB_AF030945_646_795
/5Phos/CGTGCTGGCTATTGCCTTAGGGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAATACTCCTAGCACCAAATC

CARB_U14749_1227_1390
/5Phos/CATTAGGAGTTGTCGTATCCCTCAGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATACTCCGAGCACCAAATC

CARB_AF313471_2731_2906
/5Phos/AAATTGCAGTTCGCGCTTAGCGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTCCATAGCGTTAAGGTTTC

CMY_DQ463751_613_790
/5Phos/GCGCCAAACAGACCAATGCTGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCGATTTCACGCCATAGGCTC

CMY_EF685371_397_552
/5Phos/GTATAGGTGGCTAAGTGCAGCAGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCGTAACTCATTCCTGAGGG

CMY_EU515251_583_733
/5Phos/GTCATCGCCTCTTCGTAGCTCGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCATATCGATAACGCTGG

CMY_X92508_126_301
/5Phos/AGTATCTTACCTGAAATTCCCTCACGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCTCTCGTCATAAGTCGA

ATG

CMY_AB061794_343_489
/5Phos/CATCACGAAGCCCGCCACAGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCGCCCTTGAGCGGAAGTATC

CMY_JN714478_1882_2055
/5Phos/ACCAATACGCCAGTAGCGAGAGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAACGTAGCTGCCAAATC

CMY_X91840_1872_2046
/5Phos/CAATCAGTGTGTTTGATTTGCACCGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACCCGGAATAGCCTGCTC

CTXM_EF219134_13713_13858
/5Phos/CGGATAACGCCACGGGATGAGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCACCGGGTCAAAGAATTCCTC

CTXM_HQ398215_802_947
/5Phos/GCGGCGTGGTGGTGTCTCGTTGGAGGCTCATCGTTCCTATATTCCACACC

ACTTATTATTACAGATGTTATGCTCGCAGGTCCGCTGCCGGTCTTATCAC

CTXM_AM982522_639_788
/5Phos/GCCACGTCACCAGCTGCGGTTGGAGGCTCATCGTTCCTATATTCCACACC

ACTTATTATTACAGATGTTATGCTCGCAGGTCCGGCTGGGTGAAGTAAGTC

GES_HM173356_1163_1321
/5Phos/GCTCGTAGCGTCGCGTCTCGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCTTGACCGACAGAGGCAAC

GES_AF156486_1754_1905
/5Phos/CAGCAGGTCCGCCAATTTCTCGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGTGGACGTCAGTGCGC

GES_HQ874631_571_748
/5Phos/CCATAGAGGACTTTAGCCACAGTGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACACCGCTACAGCGTAAT

GES_FJ820124_1174_1338
/5Phos/CATATGCAGAGTGAGCGGTCCGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCAATTCTTTCAAAGACCAGC

IMG_DQ361087_489_645
/5Phos/CCATTAACTTCTTCAAACGATGTATGGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCCGTGCTGTCGCTAT

IMG_JN848782_301_475
/5Phos/GTGCTGTCGCTATGGAAATGTGGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCAACCAAACCACTAGGTTATCTT

IMG_EF192154_182_328
/5Phos/GTCAGTGTTTACAAGAACCACCAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATGCATACGTGGGAATAGATT

IMG_AY033653_1343_1500
/5Phos/CGGAAGTATCCGCGCGCCGTTGGAGGCTCATCGTTCCTATATTCCACACC

ACTTATTATTACAGATGTTATGCTCGCAGGTCTTCGATCACGGCACGATC

IMG_AF318077_871_1047
/5Phos/CGAACCAGCTTGGTTCCCAAGGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCACTGCGTGTTCGCTC

IMG_AF318077_515_657
/5Phos/GATGCTGTACTTTGTGATGCCTAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGCTTGGCAAGTACTGTTC

KPC_HM066995_226_375
/5Phos/GCAAGAAAGCCCTTGAATGAGCGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCGTTATCACTGTATTGCAC

KPC_GQ140348_624_799
/5Phos/AATCAACAAACTGCTGCCGCTGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTGTACTTGTCATCCTTGT

KPC_EU729727_683_840
/5Phos/CCAGTCTGCCGGCACCGCGTTGGAGGCTCATCGTTCCTATATTCCACACC

ACTTATTATTACAGATGTTATGCTCGCAGGTCTCGAGCGCGAGTCTAGC

KPC_FJ234412_691_839
/5Phos/CCGACTGCCCAGTCTGCCGGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCCGAGCGCGAGTCTAGCC

NDM_JN104597_64_211
/5Phos/GTAAATAGATGATCTTAATTTGGTTCACGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTGCTGGCCAATCGT

CG

NDM_FN396876_2744_2885
/5Phos/CACAGCCTGACTTTCGCCGCGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCCAAGCAGGAGATCAACCTGC

NDM_FN396876_2958_3117
/5Phos/GGTGGTCGATACCGCCTGGGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCGTGAAATCCGCCCGACG

NDM_JN104597_314_465
/5Phos/CATGTCGAGATAGGAAGTGTGCGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGATGCGCGTGAGTCAC

NDM_FN396876_2382_2548
/5Phos/CAATCTGCCATCGCGCGATTGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCCGGCAATCTCGGTGATGC

OXA_EF650035_239_388
/5Phos/CGAAGCAGGTACATACTCGGTCGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGAGCTAAATCTTGATAAAC

TT

OXA_EU019535_389_537
/5Phos/TAGAATAGCGGAAGCTTTCATGGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCTAGCTTGTAAGCAAACTG

OXA_EF650035_423_594
/5Phos/CAAGTCCAATACGACGAGCTAAAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAATAGCATGGATTGCACTTC

OXA_DQ309276_232_380
/5Phos/GGTACATACTCGGTCGAAGCACGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATCTTGATAAACTGAAATAG

CG

OXA_DQ445683_232_380
/5Phos/GGTACATACTCGGTCGATGCACGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCTTGATAAACCGGAATAGCG

OXA_X75562_201_366
/5Phos/GTAATTGAACTAGCTAATGCCGTACGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTATGACACCAGTTTCTA

GGC

OXA_M55547_995_1154
/5Phos/CAAGTACTGTTCCTGTACGTCAGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCCAGTTGTGATGCATTC

OXA_AY445080_313_469
/5Phos/TCTCTTTCCCATTGTTTCATGGCGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGCGGAAATTCTAAGCTGAC

PER_Z21957_217_371
/5Phos/GTAGGTTATGCAGTTATTAGGTTCAGGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGACTCAGCCGAGTCAAGC

PER_HQ713678_6002_6167
/5Phos/GCAGTACCAACATAGCTAAATGCGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAATAACAAATCACAGGCCAC

PER_GQ396303_667_844
/5Phos/GGTCCTGTGGTGGTTTCCACCGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGCGATAATGGCTTCATTGG

PER_X93314_954_1122
/5Phos/TAACCGCTGTGGTCCTGTGGGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCTGCGCAATAATAGCTTCATTG

PER_HQ713678_4517_4674
/5Phos/GGAAGCGTTGCTTGCCATAGTGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAACCGAAGCACCATGTAATT

PER_HQ713678_5074_5219
/5Phos/GTTCGGTGCAAAGACGCCGGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCTCGCAGACTTCAATATCAATATT

PER_GQ396303_254_399
/5Phos/CACCTGATGCAGAACCAGCATGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGGCCACGTTATCACTGTG

SHV_AY661885_656_806
/5Phos/CAGCTGCCGTTGCGAACGGTTGGAGGCTCATCGTTCCTATATTCCACACC

ACTTATTATTACAGATGTTATGCTCGCAGGTCCGCAGATAAATCACCACAATC

SHV_AF535128_587_761
/5Phos/GCTCAGACGCTGGCTGGTCGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCCCGCAGATAAATCACCACG

SHV_U92041_406_579
/5Phos/GCCAGTAGCAGATTGGCGGCGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCGAACGGGCGCTCAGACG

SHV_AY288915_617_764
/5Phos/CCACTGCAGCAGATGCCGTGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCGTATCCCGCAGATAAATCACC

SHV_HQ637576_88_245
/5Phos/TTAATTTGCTTAAGCGGCTGCGGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCAGCTGTTCGTCACCG

SHV_AF535128_188_362
/5Phos/GGGAAAGCGTTCATCGGCGGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCTCGCTCATGGTAATGGCG

SHV_X98102_763_913
/5Phos/TCTTATCGGCGATAAACCAGCCGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTTGCCAGTGCTCGAT

TEM_X64523_2037_2191
/5Phos/CAGTCCCTCGATATTCAGATCAGAGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTAACAATTTCGCAACCGTC

TEM_J01749_2068_2239
/5Phos/CAGCTGCGGTAAAGCTCATCAGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATAGTTAAGCCAGTATACACTC

TEM_GQ149347_3605_3747
/5Phos/GTCGGAAAGTTGACCAGACATTAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATACTAGGAGAAGTTAATAA

ATACG

TEM_U36911_4374_4551
/5Phos/CATTCTCTCGCTTTAATTTATTAACCTGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCGACCTTCTGGACA

TTATC

TEM_AF091113_1529_1699
/5Phos/GTAACAACTTTCATGCTCTCCTAAAGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGGTAACTGATGCCGTAT

TT

TEM_GU371926_11801_11944
/5Phos/GTGAAGTGAATGGTCAGTATGTTGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGTGCGCAGGAGATTAGC

TEM_J01749_766_908
/5Phos/CCTGTCCTACGAGTTGCATGATGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCATAATGGCCTGCTTCTCGC

TEM_J01749_1634_1783
/5Phos/CGTTTCCAGACTTTACGAAACACGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGTTGTGAGGGTAAACAAC

TEM_U36911_7596_7762
/5Phos/CGTTGCTTACGCAACCAAATATCGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGATCTTGCTCAATGAGGTTA

TEM_U36911_6901_7069
/5Phos/CATCATGTTCATATTTATCAGAGCTCGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTAGATTTCATAAAGTCT

AACACAC

TEM_GU371926_33909_34082
/5Phos/GTTTCCACATGGTGAACGGTGGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAACCTGTCACTCTGAATGTT

VEB_EU259884_6947_7094
/5Phos/CAAATACTAAATTATACAGTATCAGAGAGGTTGGAGGCTCATCGTTCCTA

TATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATGCAAAGCGTTAT

GAAATTTC

VEB_EF136375_596_738
/5Phos/GTTCTTATTATTATAAGTATCTATTAACAGTTGTTGGAGGCTCATCGTTC

CTATATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATTAGTGGCT

GCTGCAAT

VEB_EF420108_234_380
/5Phos/CATCGGGAAATGGAAGTCGTTATGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTCAATCGTCAAAGTTGTTC

VEB_AF010416_89_230
/5Phos/CGTGGTTTGTGCTGAGCAAAGGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAAAGTTAAGTTGTCAGTTTGAG

VIM_AY524988_385_552
/5Phos/GCCGCCCGAAGGACATCAAGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCAGACGGGACGTACACAAC

VIM_Y18050_3464_3614
/5Phos/GCAACTCATCACCATCACGGAGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGATGCGTACGTTGCCAC

VIM_AY635904_58_203
/5Phos/GCGACAGCCATGACAGACGCGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCGGACAATGAGACCATTGGAC

VIM_HM750249_275_454
/5Phos/AAACGACTGCGTTGCGATATGGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTCCGAAGGACATCAACGC

VIM_AJ536835_313_481
/5Phos/ATGCGACCAAACGCCATCGCGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCATCGTCATGGAAGTGCGTA

VIM_EU118148_131_300
/5Phos/GAACAGGCTTATGTCAACTGGGGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATAACATCAAACATCGACCC

VIM_DQ143913_921_1063
/5Phos/ACGAACCGAACAGGCTTATGTCGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTAACGCGCTTGCTGCTT

VIM_EU118148_2821_2961
/5Phos/GCTGTAATTATGACGACGCCGGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCGGTGAGATTCAGAATGC

VIM_EU118148_1060_1229
/5Phos/CATCATAGACGCGGTCAAATAGAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTCATCACCATCACGGAC

van_DQ018710.1_6481_6652
/5Phos/GTGTATGTCAGCGATTTGTCCATGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTCATATTGTCTTGCCGATT

van_DQ018710.1_6764_6926
/5Phos/GTCCACCTCGCCAACAATCAAGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCATATCAACACGGGAAAGACCT

van_AY926880.1_3640_3785
/5Phos/GCGTGATTATCACGTTCGGCAGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTGCAGATTTAACCGACAC

van_FJ545640.1_517_690
/5Phos/GGCTCGACTTCCTGATGAATACGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGAAACCGGGCAGAGTATT

van_AE017171.1_34715_34859
/5Phos/CAACGATGTATGTCAACGATTTGTGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATTGCGTAGTCCAATTCGTC

van_NC_008821.1_11898_12045
/5Phos/CAGGCTGTTTCGGGCTGTGAGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCGGGTTATTAATAAAGATGATAGGC

van_FJ349556.1_5601_5765
/5Phos/GGCTCGGCTTCCTGATGAATACGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGGCATGGTATTGACTTCATT

mecA_AY820253.1_1431_1608
/5Phos/TAATTCAAGTGCAACTCTCGCAAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTTATTCTCTAATGCGCTAT

ATATT

mecA_AY952298.1_130_302
/5Phos/GGATAGTTACGACTTTCTGCTTCAGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTATTGCTATTATCGTCA

ACG

mecA_AM048806.2_1574_1720
/5Phos/CAGTATTTCACCTTGTCCGTAACCGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTTACGACTTGTTGCATGC

mecA_EF692630.1_239_405
/5Phos/AATGTTTATATCTTTAACGCCTAAACTGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATGCTTTGGTCTTTCT

GCAT

mex_AF092566.1_371_520
/5Phos/CTGGCCCTTGAGGTCGCGGGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCCGGTCTTCACCTCGACAC

mex_AF092566.1_50_193
/5Phos/GACGTAGATCGGGTCGAGCTGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCACGGAAACCTCGGAGAATT

mex_CP000438.1_487178_487357
/5Phos/GGCGTACTGCTGCTTGCTCAGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCTGACGTCGACGTAGATCG

mex_NZ_AAQW01000001.1_461304_461466
/5Phos/CCTGTTCCTGGGTCGAAGCCGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTCGGTCACCGCGGA

erm_NC_002745.2_871803_871973
/5Phos/GTCAGGCTAAATATAGCTATCTTATCGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCAGTTACTGCTATAG

AAATTGAT

erm_NC_002745.2_871666_871841
/5Phos/CATCCTAAGCCAAGTGTAGACTCGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAGATATATGGTAATATTCC

TTATAAC

erm_EU047809.1_79_229
/5Phos/GTTTATAAGTGGGTAAACCGTGAATGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAAACGAGCTTTAGGTTT

GC

acinetobacter_NC_010611_627997_628164
/5Phos/GCAGCACTTGACCGCCATGAGTGACCAGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATCGCACCAACAACA

ATAATCG

acinetobacter_NC_010611_2417580_2417755
/5Phos/GTGATCACTGATGCACCAGATGAAGTGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTTGATATTCAAGTC

TATGACG

acinetobacter_CP002522_11753_11931
/5Phos/GATATTATTGATCATGGTGCCAAGCCAAGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAATATGAAGCTGAC

GACGCG

acinetobacter_NC_011586_3908329_3908508
/5Phos/GCTGAGCGTGAAGGTTCATGGATTATTAGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGTAAGGCTTACGGT

CTCAT

acinetobacter_NC_010611_145181_145340
/5Phos/GCATCTTGTGCAGCCTGAATAGCAGCGTGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCACGTTGAATATC

ACCTTCGGCAT

acinetobacter_NC_010611_3854494_3854662
/5Phos/AAGTCCATAATTGCTTGAGTGTAGTCATGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTTCGCACTGAAT

AATAAGAACAT

acinetobacter_NC_010400_56216_56383
/5Phos/GCTTGCTGGTTCTGCACGTAGCTTACTGGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAGATGAACAGGCTA

CTGCAA

acinetobacter_NC_010611_1454960_1455136
/5Phos/GCAGCGCTGTGCAAGTTCAATGTATTCTGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCGTGCGAGTATTC

CTTAAGTGT

acinetobacter_NC_009085_255964_256143
/5Phos/GTATAACACTCGGCCAGCGCCAAGGTTCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTCACACATCGCCA

CAATATGAT

clostridium_NC_013974_3097606_3097772
/5Phos/ACCATGCAGATACAATGAACCAGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGATGATAAGACACATCCAAT

TC

clostridium_FN665653_103469_103631
/5Phos/CATCAACAGCTTCTTGAAGCATTCGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCCAACAACTATAACAGA

ACGTC

clostridium_NC_013974_117188_117346
/5Phos/AACATATCACCTGATATTCTAGTATCGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATTCCATTATATTCAAC

AGGATTGTGA

clostridium_NC_013316_3012882_3013047
/5Phos/GCTGTTGCTTGCGGATACTGGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTATATGTAGCTCAAGTTGC

clostridium_FN668375_1212250_1212413
/5Phos/AAGAGCTAATGCAGCTATTGCACTTATGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATACACTTCAGCTAT

AAGACCAT

clostridium_NC_013315_3754484_3754640
/5Phos/AACAAGAGCAGAAGTTACAGACGTGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTATAATGGTGGCTAGAGG

TGA

clostridium_FN665654_3239860_3240039
/5Phos/ACTCGTGAAGACCATGCAGATACAAGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATACTTACAATGCCTGA

GGA

clostridium_FN668941_3228320_3228491
/5Phos/ACCATGCAGATACAATGAACCGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCTGAGGATGATAAGACACATC

clostridium_NC_013974_1962664_1962825
/5Phos/GCATCTGCTGCTTCTATTGCTCCTACTGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATGAACTGATATTA

GTTCTCCAA

clostridium_NC_003366_2769687_2769851
/5Phos/GCACAAGCTGGAGATAACATCGGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAGAGGACGTATTCACAAT

CACT

clostridium_FN665653_127741_127918
/5Phos/CTCTATCAGCTTCTACTGCTTCTTCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCATCTCATCCACAGTTA

ATATATC

clostridium_NC_013316_2259929_2260107
/5Phos/AGATGAGATTCATACTATCGTTGGAGCTGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCAGAGAGAATAGT

AAGAGGAGA

clostridium_NC_009089_94774_94937
/5Phos/CATCAACAGCTTCTTGAAGCATTGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCCAACAACTATAACAGAA

CG

clostridium_NC_013315_2044225_2044389
/5Phos/GTCAGCAATACGCCACCAAGCTCCTATGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTGGTGGATATCCTGT

TACC

clostridium_NC_013315_2299408_2299586
/5Phos/GCGCAATAGAGTTGTATAAGAGTGCTGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCATTAATTATAGAT

TATAATGTATAA

clostridium_FN668941_3244255_3244408
/5Phos/GGCATAATAGGATGGATAGATGAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTAATCCAACTTCTACTGC

TAT

clostridium_NC_013316_3610909_3611065
/5Phos/GTACATTCACATATAGACCATCTTAAGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATAGGTGCAGGTAGA

ATAGTATA

clostridium_FN665653_1104859_1105031
/5Phos/CCATACCAGTATCTTGGCATATTGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATAATGAATAACAGCAGGT

GTATTA

clostridium_NC_03366_2753681_2753838
/5Phos/AGATGAAGCACAAGCTGGAGATAAGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGGACGTATTCACAATCAC

TG

clostridium_FN665653_710906_711080
/5Phos/ATAATCATTCACCTCCATCATTCATAAGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTGAATATGGTTCGT

CTCA

clostridium_NC_009089_3706562_3706720
/5Phos/GTACATTCACATATAGACCATCTTAGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATAGGTGCAGGTAGAA

TAGT

clostridium_NC_013316_137282_1372968
/5Phos/ACTCCACCAGGATGTTGTCCGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAGGACCGTCGTGTCCAAG

clostridium_FN665652_676696_676895
/5Phos/GCAATATCAATGGTATCGAAGGCACTATGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTATTGAAGGTACTA

TTAGCGATATGC

clostridium_NC_013316_2641651_2641808
/5Phos/GTGCCGGTCTCGGTTACTCAATGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGATTATTATAATGCAGCTA

GAAG

clostridium_FN668375_3595870_3596026
/5Phos/GTACATTCACATATAGACCATCTTGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATAGGTGCAGGTAGAAT

AGTA

clostridium_FN668941_1105700_1105868
/5Phos/AGTTCCTTCATATGACTCAGTTGATTGAGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTATATCTTCAATT

ATACATTCCTGC

clostridium_NC_013974_2505182_2505359
/5Phos/CAGCAGTTGTTGCTAGAGGTATGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCATCACCAGGTGCAGCAAGT

clostridium_NC_013315_1077126_1077298
/5Phos/GCAATTCTCTGTTGTTGTCCTCCACTCAGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGTAAGAGCCTCTTC

TTGGTCATGA

clostridium_NC_009089_2182303_2182482
/5Phos/CTATTCCTGATAATAAGTGTGTCCTCATGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGGCATCATCTAACA

ATTCTTCT

clostridium_FN665652_1909777_1909942
/5Phos/GTAATTCCAATTACTTCTAGCTCTGGTGGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACCATCTTCTCCAT

GTGTAT

clostridium_NC_013316_3300896_3301062
/5Phos/CCATGCAGATACAATGAACCAGGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATGATAAGACACATCCAATT

CC

clostridium_NC_013316_871338_871499
/5Phos/CCTTCTGCCATTGTAGAACAAGCTCCATGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCTGTAACTGTCCAC

TGAGC

clostridium_NC_013316_3608873_3609047
/5Phos/CAATCATGATAGAATTAGATGGAACGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCAATAGTTCCATCAGG

AGCATC

clostridium_FN665654_3717059_3717221
/5Phos/AGTGGTGAAGGTGTTCAACAAGGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTGAAGCTGGATATGTTGGAG

clostridium_NC_013315_2010489_2010657
/5Phos/CGCCTCTTCAGAAGCGGATATCAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCAGACTTCCGCCACAACCT

clostridium_NC_013315_3236301_3236474
/5Phos/GGCATAATAGGATGGATAGATGAGCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAGCAGTTGTACCTACA

ACTAA

clostridium_NC_013315_1095924_1096090
/5Phos/AGTTCCTTCATATGACTCAGTTGATTGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTATATCTTCAATTA

TACATTCCTGCG

enterobacter_NC_014121_4735453_4735632
/5Phos/GCATGGTAGTTCGCCAGCCGCTGGAACGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACAGCAACCGCAAGTT

CTTGACAT

enterobacter_NC_015663_1014187_1014345
/5Phos/AATATCATGGTCGTGTCCAGGCACTGGCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTCTGGTAGCTGCT

TCTACTGTA

enterobacter_FP929040_3448334_3448513
/5Phos/AACTTACAACTACGCGCACTTGAATCGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAGTGTTGTATGATAG

TCTCGGT

enterobacter_NC_009436_4051820_4051985
/5Phos/GCAAGTTGAGGAGATGCTGGCATGATTCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATGGCTCTGGAAG

ATGTGCTGATC

enterococcus_FP929058_1738439_1738606
/5Phos/GCGATAATTGTAATGATTCGTGGTGTTAGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCGTTGTCAATCCAG

TTAGTAGACT

enterococcus_CP002621_1819224_1819388
/5Phos/ACTGTGGCAGTCTATGTTCCAATTGTAGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTATCGACATAATCC

TGATAATC

enterococcus_FP929058_904007_904173
/5Phos/GCGTCGCTTCTTGCGCTCGCCGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATGTATTCATACCGTCAAGT

enterococcus_FP929058_551757_551920
/5Phos/GCCTTCACAACTACGTTGGAAGGTCTTCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTAACAGTCCTGCCG

ACTAC

enterococcus_NC_004668_1122345_1122507
/5Phos/GCCTTCACAACTACGTTGGAAGGTCTTGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTAACAGTCCTGCCGA

CTACT

klebsiella_NC_009648_2885456_2885620
/5Phos/GCCGCTGAGCGGCGGCAAGCCGATGGCGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAATGGCAGGCCAAGC

TGAAGGCG

klebsiella_NC_009648_3899012_3899182
/5Phos/GCCAAGCGGCATTCTGGCGCCAGTGGAGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCAGACCGGAGTGGAC

AACGTCGAGGCG

klebsiella_NC_009648_4980596_4980757
/5Phos/GCCGTATATCATCGGCAATAACCGCACGGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCATGATGGTCAACA

AGGTGC

klebsiella_NC_009648_3266359_3266519
/5Phos/ACGAGCCGAGATAGGTCTGCAGCGTACGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTACTGATATTCACCA

TACTGCCG

klebsiella_NC_012731_2557467_2557634
/5Phos/GCAATATCTTCACCGGCAGCCACCGCGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGTATATGGCACGCCA

ATCGC

klebsiella_NC_012731_4857136_4857315
/5Phos/AATAACCTTAACGTCGCCAACACGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCGGTGAACACCTCCTGG

CACG

proteus_NC_010554_547938_548117
/5Phos/GCGGAACTGCTTGGCGTAGTAAGCGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATGTAGTGCCGTAGACCT

TCACCA

pseudomonas_NC_008463_658500_658676
/5Phos/GCGAGACCGGCGGCACCATCGTCTCCAGGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTCTGCCTGATGGAC

GTCTCCGGCTCG

pseudomonas_NC_008463_753931_754099
/5Phos/GCGGTTCACCTGTTCGCCTTCGAACACGGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCGCAGCATCTGACG

CAGGATGGTCTCG

pseudomonas_NC_009656_6431649_6431828
/5Phos/ACTCCATCGCCATCAAGGACATGGCCGGGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCGACGTGTTCCGC

ATCTTCGACGCG

pseudomonas_NC_008463_560357_560534
/5Phos/GCCTGATGCACTACAGCGCCTGGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACCACATGGTCGATCTCGA

CGACTGC

pseudomonas_NC_010322_5224859_5225023
/5Phos/GCGCATCCAGGACGGCGAGTACGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTCGAGTGCCTGCACGAGC

TGAA

pseudomonas_NC_008463_4839746_4839924
/5Phos/GCTGGAGAACGTCAAGGTGGTGATCATCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCGATAACGACGAC

CGCATCAA

staph_FN433596_2844085_2844263
/5Phos/ACGATTGGAGAAGGCAGTGTGATTGGGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGACAGATTACAATTGG

CG

staph_NC_009632_1198350_1198529
/5Phos/GCCGCAATACCGATATTCCAGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCCCATTGTCCACCAGCTGAACCG

staph_FN433596_2521244_2521419
/5Phos/GTGAAGGTCGTGCTCCTATCGGTGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGATCTGGTGAAGTTCGTAT

GAT

staph_NC_009487_430842_431017
/5Phos/GCTGGTACTTGTACTTATATCGAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCAGAAGATGATATCGTTA

CGTCAT

staph_NC_009782_2086681_2086849
/5Phos/GCGCATATTGCATTAATGGCTATAGATGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCAGCAGGTTATACA

CTCG

staph_NC_009782_58256_58423
/5Phos/GCAATTCTTACCACAGCACGAAGAACAGGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTAGATGAAGATA

ATGAAGTCG

staph_NC_013450_991049_991222
/5Phos/GCATCTTCATACAATACTTCTAGCTTACGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACAATACCAGTTGT

ATTACG

staph_NC_013450_1360842_1361008
/5Phos/GCTTCAGCGCCATTACCGCCACCAGCTGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTCTTGATATATTCT

TGTAAGCG

staph_AM990992_2526026_2526192
/5Phos/GTTCACACAACGCGCCGACTAGAATCCGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACGATATCCAAGATA

ATGATTGGCTA

staph_NC_010079_361284_361447
/5Phos/GCGCACCTACAATCGCCATTACTACACGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTCATTATCGACTGT

TACATCGACTGA

staph_NC_007795_2085723_2085901
/5Phos/AGCGCACATGTGACAGCGTGTAGGTTAGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTGCCTTAGATTGTTC

AGAACAAT

staph_NC_009641_23125_23297
/5Phos/CGAATGGATATGTACCATGGTCGATATCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCTCTAATATGATG

TCCAT

staph_FN433596_2144570_2144734
/5Phos/ACTACAACAGCAACCGCATTACAATGGCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGTGCTAAGAGGTCA

TCGGA

staph_NC_009782_54857_55020
/5Phos/AGCTTCAGATAAGTACCTATCTGAGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGAAGAATAGTTATTCTTG

ATAATGTAT

staph_AM990992_1656616_1656789
/5Phos/CGTATTGCTCGAATACATGATAGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCACAATGTATCAAGGCCAGCT

staph_NC_007793_44227_44395
/5Phos/GCGACCAGTTGTTATCGACCGTGTGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAGAACGATACGGTGCTGT

ATA

staph_NC_009641_1102949_1103116
/5Phos/CAATTACATTGTCTGTTGCGTAGATACCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTGTGGCTAATGTG

CCAGTT

staph_NC_009641_1137731_1137898
/5Phos/GCACCACTCTATAGCAGTAGCGTATTGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACAGCCAATGTCACCT

AAGTCAACA

staph_FN433596_2715713_2715871
/5Phos/ACAGTCCGAATAAGATACGACTATTCGAGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTTGTAACGTATAT

GAATAGTTGA

staph_NC_009782_606652_606825
/5Phos/AGATGCAATAACAGGTCGAATATTAATTGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCATAGTGAGAGTA

GTGAA

staph_FN433596_657625_657803
/5Phos/AGATGCAATAACAGGTCGAATATTAAGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACACATACGGCCATAGT

GAGAG

species_NC_004741_4338803_4338982
/5Phos/GAACATAACGCGACGTTCCAGCTGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTTCAGAGGTGTTGTAGT

CG

species_NC_009648_4535521_4535683
/5Phos/GCGCTGGCGCAGTATCGTGAACTGGGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCAACGTAATCTCTATT

ACCG

species_NC_010410_3677607_3677782
/5Phos/GCTGTAATGCAAGTAGCGTATGCGCTCAGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAGGCCGCCAATGCC

TGACG

species_CP001844_589057_589217
/5Phos/GCCTGTAGCAACAGTACCACGACCAGTGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACCACGTAATAATGC

ACCAA

species_CP002110_2761329_2761492
/5Phos/ACTACGCTGAAGCTGGTGACAACATTGGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTGAGGACGTATTCT

CAATC

species_NC_010473_3546640_3546818
/5Phos/GCTGGTACTTACGTTCAGATGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCACGGTGAACGCCGTTACATCC

species_CP001844_57304_57465
/5Phos/GCAATTCTTACCACAGCACGAAGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTAGATGAAGATAATGAAG

TCG

species_NC_012731_1975396_1975559
/5Phos/GCGGCGGCAGGCGGTAACGCCAGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGCGGTTATCTACCACGGCG

species_NC_003923_198857_199024
/5Phos/GCACCTACTTGTCCAGCACCAGCCATGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATACCACCACCAATAC

AAGCA

species_NC_010400_52102_52263
/5Phos/GCGCGGTAACATGCCATATTCTGCGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCTGAATGACATCACAGTCG

species_NC_010473_3310005_3310164
/5Phos/AATCAGGTCAAGGAACTGCAAGCGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCTCAATCATATGCACCGG

AATAC

species_FP929058_3022053_3022226
/5Phos/GAACATATGTGTATGACGATGCGCGGGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTACATGTCGCTTATCT

GCCAGAAGGT

species_NC_009085_1010393_1010556
/5Phos/CGTGTGCGTAGTGACGAGTTGGAGAGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGAATACGATGATGTAAG

GTACACCTA

species_CP002621_172633_172802
/5Phos/CAGGAGTTACTTCTGTTCCATGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTGAACAATTAGATCACCTCG

species_FP929040_442484_442653
/5Phos/CGTAATCTCCATTACCGATGGTCAGATCGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGTATTCTACCTCC

ACTCTCGTCT

species_NC_003923_1334345_1334501
/5Phos/CATTCGACGTTCTGGTATTACTTGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACGCTCCGCATCAGCAGCA

CCACGTT

species_NC_009085_1010678_1010853
/5Phos/CTGAACCACGGATTACTGGAGTGTCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCTGTTACTACTGTACC

ACGAC

pseudomonas_NC_008463_4756080_4756240
/5Phos/GAATCGAACGGTCTCATTAACAGATGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTTTCCAGGGATATAAG

ACGC

pseudomonas_NC_002516_1063894_1064077
/5Phos/CCCGCAGAGTCACACTCGGAGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCACTCTTGGTACTACTCACTAGC

pseudomonas_NC_008463_3182693_3182865
/5Phos/GAGTCTCTTTCAACCTGGATTAGATATGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAGATTAATAGCGTAC

TTTACTCC

pseudomonas_NC_009656_2819490_2819655
/5Phos/ATCCCGCAGATACTAGGTTCTTAATGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAACTATTCATATTACAC

CCTAAGG

pseudomonas_NC_008463_3184022_3184185
/5Phos/CAGTGGGCTATCCTAAGCCAAAGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATAAGCGAACTAACTATCA

CTTA

pseudomonas_NC_002516_1065937_1066093
/5Phos/ACAAAGCGTTCTAAACGATTAGAACTGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGAGAAAGGAAACAGGA

TAGTAC

pseudomonas_NC_002516_1067833_1068007
/5Phos/CCAATGGAGAAGTCTAAATGTCCAAGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTATCAGAGATACATGAC

TCTTAGG

pseudomonas_NC_008463_3182351_3182508
/5Phos/CGAATCACTGGACTACATTTATATTTCTGTTGGAGGCTCATCGTTCCTAT

ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCGAACCTTTATAT

TTGACCAT

pseudomonas_NC_008463_3184314_3184473
/5Phos/CTCAAGTCTTGCCCTGATAGAATTATGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCACGACTTATCTACTT

TAGAAATC

pseudomonas_AP012280_3765216_3765383
/5Phos/GGTGATCGTTATTATGATAGTACGGCGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCGGTTAAGGGAATTA

CGAC

pseudomonas_AP012280_3765033_3765192
/5Phos/ACTCGGATGGTAGGTTTATTAAAGCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTGATCGTTATTATGATA

GTACGG

enterococcus_NZ_GG703715_13422_13573
/5Phos/ACAATCGTTGTCGCACTGCATAGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAACTTGGTCTACCGTACCAC

enterococcus_NZ_GG703582_76982_77140
/5Phos/GGATAATACAATCCTAATACGTACGGAGTTGGAGGCTCATCGTTCCTATA

TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTGCTGTAACTAGGG

TAGC

enterococcus_NZ_GL455004_28219_28381
/5Phos/CTATATTCAACGGGTCACGGGTAGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCATTGATTCGATCTCGTA

ACTC

enterococcus_NZ_GG703720_94699_94852
/5Phos/AATGTTATTGTGGTTGCGTGTTCGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACTTTGGAAGTGCCCTGAC

enterococcus_NZ_GG703715_15795_15951
/5Phos/CATGTCTTCTAGTACAGGTTTGCCGGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTAAGAGGCCGCTAACT

TC

enterococcus_NZ_GL455899_32848_32984
/5Phos/CTCTGGCTCGTGGGCTCGGGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCTTCTTGAGATAGTCCGGTATAATC

enterococcus_NZ_GG692918_325104_325257
/5Phos/ATTCGATCACGATGGGCTGGGGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATTTCCTGTGTCATACACGC

enterococcus_NC_004668_920608_920750
/5Phos/CAATTGATTTAGCCACTACACCTTACGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACTATTCTGGCGACCA

CC

enterococcus_NZ_GG703575_78829_78963
/5Phos/GATAAAGAAGCGTCTTGACCCAGTGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTGGTGCTCCTTGACGC

enterococcus_NZ_GL455931_26355_26493
/5Phos/GCAAATTTAGAGAGTGCATGCATGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGAAGAGGACGGCATACAAC

enterococcus_NZ_GG669058_207026_207172
/5Phos/CATTTCATCTAGACCGCTCGTGTGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTTGAAGTGTATGTTGGGAC

proteus_NZ_GG661998_111187_111342
/5Phos/GTCGCCCTCGTGCTAACGTGTTGGAGGCTCATCGTTCCTATATTCCACAC

CACTTATTATTACAGATGTTATGCTCGCAGGTCGGTTCTTTGATGTACCGGTT

proteus_NC_010554_2037943_2038091
/5Phos/GCTGATGACGGTGAAGTTTATCAGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATTATCGCACATATTGACC

AC

proteus_NZ_GG668576_810893_811054
/5Phos/GAAATTAGCTAAAGGGATATCGCGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAACTTTCCGCCAATCCTGC

proteus_NZ_GG668594_760_939
/5Phos/CACCTACGTTCTCACCTGCACGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCATTCGATAGTACCAGTTACGTC

proteus_NZ_GG668579_22072_22234
/5Phos/GTTGCTTATAGCGTCGCTGCTGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTGGTTATCGAGAAGATAAAGG

proteus_NC_010554_2448957_2449119
/5Phos/GTAAGCGTAGCGATACGTTGAGGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAGTGAACGCACCACTGG

proteus_NC_010554_3033758_3033936
/5Phos/TCAGGTAGAGAATACTCAGGCGCGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGGAGAAGGCTAGGTTGTC

proteus_NC_010554_454391_454540
/5Phos/GCAACCCACTCCCATGGTGTGTTGGAGGCTCATCGTTCCTATATTCCACA

CCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTTCTTCATCAGACAATCTG

gyrB_NC_015663_1455472_1455621
/5Phos/GCCCTTTCAGGACTTTGATACTGGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTACGGAGACGGAGTTAT

CG

gyrB_NC_010410_4215_4366
/5Phos/ACACTGACCGATTCATCCTCGTGGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTGAAAGTGCGTTAACAACC

gyrB_NC_005773_4904_5052
/5Phos/CGGAAGCCCACCAAGTGAGTACGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGAAACCAGTTTGTCCTTAGTC

gyrB_NC_016514_5343_5487
/5Phos/ACCAGCTTGTCTTTAGTCTGAGAGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTTACGACGGGTCATTTC

AC

gyrB_NC_016603_2631439_2631616
/5Phos/CATTGGTTTGTTCTGTTTGAGAGGCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATTCATCTTCGTGAATT

GTGAC

gyrB_NC_009436_4366_4524
/5Phos/GGACTTTGATACTGGAGGAGTCATAGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTACGGAAACGGAGTTA

TCG

gyrB_NC_009512_4203_4373
/5Phos/ATGCTGGAGGAGTCGTACGTTTGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCGCGCACACTAATAGATTC

pseudomonas_NC_009085_307050_307218
/5Phos/AACTAAACCTACACGGAATTGGTTCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAGATACACGACGTTTA

TGT

pseudomonas_NC_009085_308225_308377
/5Phos/GCCGCTTCACCTACGTTAGGAAGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTAAAGATGAGTCTTTAACG

TC

pseudomonas_NC_016612_1674334_1674490
/5Phos/GACGTTTGTGCGTAATCTCAGACGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAGGAAACCGTATTCGTTCGT

pseudomonas_NC_016603_3425179_3425337
/5Phos/ACAACACTTTACCACTTGAGTGGGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAACTGCCCATGTCAAGA

TAC

pseudomonas_NC_016603_3427629_3427808
/5Phos/CCACGTTTAGTTGAACCACCGCGTTGGAGGCTCATCGTTCCTATATTCCA

CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCAATACGCCAGTTGTTAGTTC

pseudomonas_NC_010410_3543925_3544088
/5Phos/AATCGATAATAAGTACGGTGCATCCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAAGAATACATTCGCGTA

CATC

pseudomonas_NC_005966_304936_305079
/5Phos/AAGCAAGATCGAGTCTTCATAGTTGGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATATACACGATACCTGA

TTCGT

pseudomonas_NC_008593_226005_226171
/5Phos/CCGATATTCATACGAGAAGGTACACGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAGTAACTCTATTGTCAA

ACGGT

pseudomonas_NC_016514_213592_213738
/5Phos/GTAGTGAGTCGGGTGTACGTCTCGTTGGAGGCTCATCGTTCCTATATTCC

ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCTTCGATAGCAGACAGATA

GT

pseudomonas_NC_005966_303883_304054
/5Phos/ACCTACACGGAATTGGTTCTCAGTGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATACACGACGTTTGTGTG

TA

enterobacter_NC_014618_3997909_3998085
/5Phos/CAACATCATTAGCTTGGTCGTGGGGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTGCGTGTTACCAACTCGTC

enterobacter_NZ_GL892086_61549_615324
/5Phos/CGGCACGTCCGAATCGTATCAGTTGGAGGCTCATCGTTCCTATATTCCAC

ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCGTGTCCCGTATATGTTGG

enterobacter_NZ_GL892086_1664663_1664834
/5Phos/AATAGAGGCCCACAAGTCTTGTTCGTTGGAGGCTCATCGTTCCTATATTC

CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGCTCTCCACTATGGGTAGT

enterobacter_NZ_GG704865_427821_427978
/5Phos/GCTACATTAATCACTATGGACAGACAGTTGGAGGCTCATCGTTCCTATAT

TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATGGTCGATCTATCGT

CTCT

enterobacter_NZ_GL892087_1610708_1610874
/5Phos/GAAGTGTTATTCAAACTTTGGTCCCGTTGGAGGCTCATCGTTCCTATATT

CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTGAACCCTTGGTTCAA

GGT

TABLE 2

A list of organisms for which the methods and kits described herein have been

validated to detect using the compositions described herein

Acinetobacter baumannii 1656-2

Acinetobacter baumannii AB0057

Acinetobacter baumannii AB307-0294

Acinetobacter baumannii ACICU

Acinetobacter baumannii ATCC 17978

Acinetobacter baumannii AYE

Acinetobacter baumannii MDR-ZJ06

Acinetobacter baumannii SDF

Acinetobacter baumannii TCDC-AB0715

Acinetobacter calcoaceticus PHEA-2

Acinetobacter sp. ADP1

Acinetobacter sp. DR1

Clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum DSM 1731

Clostridium acetobutylicum EA 2018

Clostridium beijerinckii NCIMB 8052

Clostridium botulinum A2 str. Kyoto

Clostridium botulinum A3 str. Loch Maree

Clostridium botulinum A str. ATCC 19397

Clostridium botulinum A str. ATCC 3502

Clostridium botulinum A str. Hall

Clostridium botulinum B1 str. Okra

Clostridium botulinum Ba4 str. 657

Clostridium botulinum BKT015925

Clostridium botulinum B str. Eklund 17B

Clostridium botulinum E3 str. Alaska E43

Clostridium botulinum F str. 230613

Clostridium botulinum F str. Langeland

Clostridium botulinum H04402 065

Clostridium cellulolyticum H10

Clostridium cellulovorans 743B

Clostridium clariflavum DSM 19732

Clostridium difficile 630

Clostridium difficile BI1

Clostridium difficile BI9

Clostridium difficile CD196

Clostridium difficile strain 2007855

Clostridium difficile strain CF5

Clostridium difficile strain M120

Clostridium difficile M68

Clostridium difficile R20291

Clostridium kluyveri DSM 555

Clostridium kluyveri NBRC 12016

Clostridium lentocellum DSM 5427

Clostridium ljungdahlii DSM 13528

Clostridium novyi NT

Clostridium perfringens ATCC 13124

Clostridium perfringens SM101

Clostridium perfringens str. 13

Clostridium phytofermentans ISDg

Clostridium saccharolyticum-like K10

Clostridium saccharolyticum WM1

Clostridium sp. SY8519

Clostridium sticklandii DSM 519

Clostridium tetani E88

Clostridium thermocellum ATCC 27405

Clostridium thermocellum DSM 1313

Enterobacter aerogenes KCTC 2190

Enterobacter asburiae LF7a

Enterobacter cloacae SCF1

Enterobacter cloacae subsp.cloacae ATCC 13047

Enterobacter cloacae subsp. cloacae NCTC 9394

Enterobacter sp. 638

Enterococcus faecalis 62

Enterococcus faecalis OG1RF

Enterococcus faecalis V583

Enterococcus sp. 7L76

Escherichia coli 042

Escherichia coli 536

Escherichia coli 55989

Escherichia coli ABU 83972

Escherichia coli APEC O1

Escherichia coli ATCC 8739

Escherichia coli BL21(DE3)

Escherichia coli ‘BL21-Gold(DE3)pLysS AG'

Escherichia coli B str. REL606

Escherichia coli BW2952

Escherichia coli CFT073

Escherichia coli DH1 (ME8569)

Escherichia coli E24377A

Escherichia coli ED1a

Escherichia coli ETEC H10407

Escherichia coli HS

Escherichia coli IAI1

Escherichia coli IAI39

Escherichia coli IHE3034

Escherichia coli KO11

Escherichia coli LF82

Escherichia coli NA114

Escherichia coli O103: H2 str. 12009

Escherichia coli O111:H-str. 11128

Escherichia coli O127:H6 str. E2348/69

Escherichia coli O157:H7 str. EC4115

Escherichia coli O157:H7 str. EDL933

Escherichia coli O157:H7 str. Sakai

Escherichia coli O157:H7 str. TW14359

Escherichia coli O26:H11 str. 11368

Escherichia coli O55:H7 str. CB9615

Escherichia coli O7:K1 str. CE10

Escherichia coli O83:H1 str. NRG 857C

Escherichia coli S88

Escherichia coli SE11

Escherichia coli SE15

Escherichia coli SMS-3-5

Escherichia coli str. ‘clone D i14’

Escherichia coli str. ‘clone D i2’

Escherichia coli str. K-12 substr. DH10B

Escherichia coli str. K-12 substr. MDS42

Escherichia coli str. K-12 substr. MG1655

Escherichia coli str. K12 substr. W3110

Escherichia coli UM146

Escherichia coli UMN026

Escherichia coli UMNK88

Escherichia coli UTI89

Escherichia coli W

Escherichia fergusonii ATCC 35469

Klebsiella pneumoniae 342

Klebsiella pneumoniae KCTC 2242

Klebsiella pneumoniae NTUH-K2044

Klebsiella pneumoniae subsp. pneumoniae MGH

78578

Klebsiella variicola At-22

Proteus mirabilis HI4320

Pseudomonas aeruginosa LESB58

Pseudomonas aeruginosa M18

Pseudomonas aeruginosa NCGM2.S1

Pseudomonas aeruginosa PA7

Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa UCBPP-PA14

Pseudomonas brassicacearum subsp. brassicacearum

NFM421

Pseudomonas entomophila L48

Pseudomonas fluorescens F113

Pseudomonas fluorescens Pf0-1

Pseudomonas fluorescens Pf-5

Pseudomonas fluorescens SBW25

Pseudomonas fulva 12-X

Pseudomonas mendocina NK-01

Pseudomonas mendocina ymp

Pseudomonas putida BIRD-1

Pseudomonas putida F1

Pseudomonas putida F1

Pseudomonas putida GB-1

Pseudomonas putida KT2440

Pseudomonas putida S16

Pseudomonas putida W619

Pseudomonas stutzeri A1501

Pseudomonas stutzeri ATCC 17588 = LMG 11199

Pseudomonas stutzeri DSM 4166

Pseudomonas syringae pv. phaseolicola 1448A

Pseudomonas syringae pv. syringae B728a

Pseudomonas syringae pv. tomato str. DC3000

Shigella boydii CDC 3083-94

Shigella boydii Sb227

Shigella dysenteriae Sd197

Shigella flexneri 2002017

Shigella flexneri 2a str. 2457T

Shigella flexneri 2a str. 301

Shigella flexneri 5 str. 8401

Shigella sonnei Ss046

Staphylococcus aureus

Staphylococcus carnosus subsp. carnosus

Staphylococcus epidermidis

Staphylococcus haemolyticus JCSC1435

Staphylococcus lugdunensis

Staphylococcus pseudintermedius

Staphylococcus saprophyticus subsp.

Staphylococcus aureus

Staphylococcus saprophyticus

Staphylococcus epidermis

Acinetobacter baumannii

Enterococcus faecalis

Enterobacter cloacae

Enterobacter aerogenes

Enterococcus faecium

Candida albicans

Klebsiella pneumoniae

Escherichia coli

Clostridium difficile

Proteus mirabilis

Pseudomonas aeruginosa

TABLE 3

Genus level regions can be used for coarse discrimination of organisms.

Probe Coordinates
Gene

species_NC_004741_43388
rpsC, S4416, 30S ribosomal protein S3

03_4338982

species_NC_009648_45355
atpA, KPN_04139, F0F1 ATP synthase subunit alpha

21_4535683

species_NC_010410_36776
int, ABAYE3575, integrase/recombinase (E2 protein)

07_3677782

species_CP001844_589057_
tufA, SA2981_0525, Translation elongation factor Tu

589217

species_CP002110_276132
tuf, HMPREF0772_12641, elongation factor EF1A

9_2761492

species_NC_010473_35466
rp1B, ECDH10B_3492, 50S ribosomal protein L2

40_3546818

species_CP001844_57304_
tnpB, SA2981_0055, Transposase B from transposon

57465
Tn554 tnpB, SA2981_1617, Transposase B from

transposon Tn554

species_NC_012731_19753
putA, KP1_2030, trifunctional transcriptional

96_1975559
regulator/proline dehydrogenase/pyrroline-5-

carboxylate dehydrogenase

species_NC_003923_19885
MW0166, hypothetical protein

7_199024

species_NC_010400_52102_
rph, ABSDF0051, ribonuclease PH

52263

species_NC_010473_33100
rpoD, ECDH10B_3242, RNA polymerase sigma factor RpoD

05_3310164

species_FP929058_302205
ENT_30090, GTP cyclohydrolase I

3_3022226

species_NC_009085_10103
A1S_0279, elongation factor Tu

93_1010556

species_CP002621_172633_
rplO, OG1RF_10170, 50S ribosomal protein L15

172802

species_FP929040_442484_
ENC_04200, proton translocating ATP synthase, F1

442653
alpha subunit

species N0_003923_13343
katA, MW1221, catalase

45_133401

species_NC 009085_10106
A1S_0279, elongation factor Tu

78_101053

TABLE 4

Genus level probes

Probe Coordinates
Binding region 1
Binding region 2

species_NC_004741_4338803_4338982
GAACATAACGCGACGTTCCAGCTG
GCTTCAGAGGTGTTGTAGTCG

species_NC_009648_4535521_4535683
GCGCTGGCGCAGTATCGTGAACTGG
ACCAACGTAATCTCTATTACCG

species_NC_010410_3677607_3677782
GCTGTAATGCAAGTAGCGTATGCGCTCA
AAGGCCGCCAATGCCTGACG

species_CP001844_589057_589217
GCCTGTAGCAACAGTACCACGACCAGT
CACCACGTAATAATGCACCAA

species_CP002110_2761329_2761492
ACTACGCTGAAGCTGGTGACAACATTG
GTTGAGGACGTATTCTCAATC

species_NC_010473_3546640_3546818
GCTGGTACTTACGTTCAGAT
ACGGTGAACGCCGTTACATCC

species_CP001844_57304_57465
GCAATTCTTACCACAGCACGAA
ATCTAGATGAAGATAATGAAGTCG

species_NC_012731_1975396_1975559
GCGGCGGCAGGCGGTAACGCCAG
ACGCGGTTATCTACCACGGCG

species_NC_003923_198857_199024
GCACCTACTTGTCCAGCACCAGCCAT
AATACCACCACCAATACAAGCA

species_NC_010400_52102_52263
GCGCGGTAACATGCCATATTCTGC
CCTGAATGACATCACAGTCG

species_NC_010473_3310005_3310164
AATCAGGTCAAGGAACTGCAAGC
GTCTCAATCATATGCACCGGAATAC

species_FP929058_3022053_3022226
GAACATATGTGTATGACGATGCGCGG
GTACATGTCGCTTATCTGCCAGAAG

GT

species_NC_009085_1010393_1010556
CGTGTGCGTAGTGACGAGTTGGAGA
AGAATACGATGATGTAAGGTACACC

TA

species_CP002621_172633_172802
CAGGAGTTACTTCTGTTCCAT
TTGAACAATTAGATCACCTCG

species_FP929040_442484_442653
CGTAATCTCCATTACCGATGGTCAGATCC
ACGTATTCTACCTCCACTCTCGTCT

species_NC_003923_1334345_1334501
CATTCGACGTTCTGGTATTACTT
CACGCTCCGCATCAGCAGCACCACG

TT

species_NC_009085_1010678_1010853
CTGAACCACGGATTACTGGAGTGTC
GCCTGTTACTACTGTACCACGAC

TABLE 5

Species/strain level regions can be used for discrimination at the level of

species and strains.

Probe Coordinates
Gene

acinetobacter_NC_010
ACICU_00572, pyridine nucleotide transhydrogenase

611_627997_628164
(proton pump) subunit alpha (part2)

acinetobacter_NC_010
pepN, ACICU_02288, aminopeptidase N

611_2417580_2417755
trpC, ACICU_02557, indole-3-glycerol-phosphate

synthase

acinetobacter_CP0025
recF, ABTW07_0010, recombination protein F

22_11753_11931

acinetobacter_NC_011
gshB, AB57_3788, glutathione synthetase

586_3908329_3908508

acinetobacter_NC_010
ACICU_00129, NAD-dependent aldehyde dehydrogenase

611_145181_145340

acinetobacter_NC_010
ACICU_03630, A/G-specific DNA glycosylase

611_3854494_3854662

acinetobacter_NC_010
nadC, ABSDF0056, nicotinate-nucleotide

400_56216_56383
pyrophosphorylase (quinolinate

phosphoribosyltransferase)

acinetobacter_NC_010
near ACICU_01347, carbonic anhydrase

611_1454960_1455136

acinetobacter_NC_009
A1S_0230, phosphoglyceromutase

085_255964_256143

clostridium_NC_01397

4_3097606_3097772

clostridium_FN665653_

103469_103631

clostridium_NC_01397

4_117188_117346

clostridium_NC_01331
nifJ, CDR20291_2570, pyruvate-flavodoxin

6_3012882_3013047
oxidoreductase

clostridium_FN668375_

1212250_1212413

clostridium_NC_01331
pykF, CD196_3170, pyruvate kinase

5_3754484_3754640

clostridium_FN665654_

3239860_3240039

clostridium_FN668941_

3228320_3228491

clostridium_NC_01397

4_1962664_1962825

clostridium_NC_00336
tuf, CPE2407, elongation factor Tu

6_2769687_2769851

clostridium_FN665653_

127741_127918

clostridium_NC_01331
clpB, CDR20291_1933, chaperone

6_2259929_2260107

clostridium_NC_00908
rpoC, CD0067, DNA-directed RNA polymerase subunit

9_94774_94937
beta'

clostridium_NC_01331
CD196_1764, cell surface protein

5_2044225_2044389

clostridium_NC_01331
near CD196_1987, multiprotein-complex assembly

5_2299408_2299586
protein

clostridium_FN668941_

3244255_3244408

clostridium_NC_01331
gpmI, CDR20291_3027, phosphoglyceromutase

6_3610909_3611065

clostridium_FN665653_

1104859_1105031

clostridium_NC_00336
tuf, CPE2407, elongation factor Tu

6_2753681_2753838

clostridium_FN665653_

710906_711080

clostridium_NC_00908
gpmI, CD3171, phosphoglyceromutase

9_3706562_3706720

clostridium_NC_01331
dnaF, CDR20291_1146, DNA polymerase III PolC-type

6_1372812_1372968

clostridium_FN665652_

676696_676895

clostridium_NC_01331
CDR20291_2249

6_2641651_2641808

clostridium_FN668375_

3595870_3596026

clostridium_FN668941_

1105700_1105868

clostridium_NC_01397

4_2505182_2505359

clostridium_NC_01331
potA, CD196_0900, spermidine/putrescine ABC

5_1077126_1077298
transporter ATP-binding protein

clostridium_NC_00908
CD1878A

9_2182303_2182482

clostridium_FN665652_

1909777_1909942

clostridium_NC_01331
ntpB, CDR20291_2788, V-type ATP synthase subunit B

6_3300896_3301062

clostridium_NC_01331
spoVAD, CDR20291_0703, stage V sporulation protein AD

6_871338_871499

clostridium_NC_01331
bclA2, CDR20291_3090, exosporium glycoprotein

6_3608873_3609047
eno, CDR20291_3026, enolase

clostridium_FN665654_

3717059_3717221

clostridium_NC_01331
CD196_1739, hypothetical protein

5_2010489_2010657

clostridium_NC_01331
adhE, CD196_2753, bifunctional acetaldehyde-

5_3236301_3236474
CoA/alcohol dehydrogenase CD196_2095, sodium:solute

symporter spoVD, CD196_2497, stage V sporulation

protein D (sporulation specific penicillin-binding

protein)

clostridium_NC_01331
CD196_0911, N-acetylmuramoyl-L-alanine amidase

5_1095924_1096090

enterobacter_NC_0141
ECL_04612, 50S ribosomal subunit protein L13

21_4735453_4735632

enterobacter_NC_0156
EAE_24795, hemagluttinin domain-containing

63_1014187_1014345
protein, rp1R, EAE_04875, 50S ribosomal protein L18

enterobacter_FP92904

0_3448334_3448513

enterobacter_NC_0094
rplD, Ent638_3750, 50S ribosomal protein L4

36_4051820_4051985

enterococcus_FP92905
ENT_17660, hypothetical protein

8_1738439_1738606

enterococcus_CP00262
OG1RF_11736, group 2 glycosyl transferase

1_1819224_1819388

enterococcus_FP92905
near ENT_09350, Uncharacterized protein conserved in

8_904007_904173
bacteria

enterococcus_FP92905

8_551757_551920

enterococcus_NC_0046

68_1122345_1122507

klebsiella_NC_009648_
mqo, KPN_02629, malate:quinone oxidoreductase

2885456_2885620

klebsiella_NC_009648_
garL, KPN_03538, alpha-dehydro-beta-deoxy-D-glucarate

3899012_3899182
aldolase

klebsiella_NC_009648_
frdB, KPN_04552, fumarate reductase iron-sulfur

4980596_4980757
subunit

klebsiella_NC_009648
KPN_02970, integral transmembrane protein; acridine

3266359_3266519
resistance

klebsiella_NC_012731_
KP1_2672, putative malate dehydrogenase

2557467_2557634

klebsiella_NC_012731_
glpR, KP1_5123, DNA-binding transcriptional repressor

4857136_4857315
GlpR

proteus_NC_010554_54
PMI0497, phage terminase large subunit

7938_548117

pseudomonas_NC_00846
PA14_07660, hypothetical protein

3_658500_658676

pseudomonas_NC_00846
rpoC, PA14_08780, DNA-directed RNA polymerase subunit

3_753931_754099
beta'

pseudomonas_NC_00965
oadA, PSPA7_6223, pyruvate carboxylase subunit B

6_6431649_6431828

pseudomonas_NC_00846
PA14_06330, serine/threonine protein kinase

3_560357_560534

pseudomonas_NC_01032
PputGB1_0612, arginine decarboxylase

2_5224859_5225023
PputGB1_4676, ketol-acid reductoisomerase

pseudomonas_NC_00846
dadA, PA14_70040, D-amino acid dehydrogenase small

3_4839746_483924
subunit ung, PA14_54590, uracil-DNA glycosylase

staph_FN433596_28440
SATW20_26770, putative acetyltransferase

85_2844263

staph_NC_009632_1198
SaurJH1_1177, branched-chain alpha-keto acid

350_1198529
dehydrogenase subunit E2

staph_FN433596_25212
rplB, SATW20_23810, 50S ribosomal protein L2

44_2521419

staph_NC_009487_4308
SaurJH9_0396, hypothetical protein

42_431017

staph_NC_009782_2086
SAHV_1928, truncated amidase

681_2086849

staph_NC_009782_5825
tnpB, SAHV_1645, transposase B

6_58423

staph_NC_013450_9910
SAAV_0970, ribosomal large subunit pseudouridine

49_991222
synthase D

staph_NC_013450_1360
opuD1, SAAV_1329, BCCT family osmoprotectant

842_1361008
transporter

staph_AM990992_25260
SAPIG2450, nitrate reductase, alpha subunit

26_2526192

staph_NC 010079_3612
near USA300HOU_0330, PfoR family transcriptional

84_361447
regulator

staph_NC_007795_2085
SAOUHSC_02251, hypothetical protein

723_2085901

staph_NC_009641_2312
purA, NWMN_0016, adenylosuccinate synthetase

5_23297

staph_FN433596_21445
hlb, SATW20_19320, phospholipase C precursor

70_2144734
(pseudogene), SATW20_19830, phage protein

staph_NC_009782_5485
SAHV_0049, hypothetical protein

7_55020

staph_AM990992_16566
proC, SAPIG1569, pyrroline-5-carboxylate reductase

16_1656789

staph_NC_007793_4422
SAUSA300_0036, hypothetical protein

7_44395

staph_NC_009641_1102
NWMN_0995, phage anti-repressor protein

949_1103116

staph_NC_009641_1137
NWMN_0310, phage tail fiber

731_1137898

staph_FN433596_27157
SATW20_25670, putative amino acid permease

13_2715871

staph_NC_009782_6066
rpoB, SAHV_0540, DNA-directed RNA polymerase subunit

52_606825
beta

staph_FN433596_65762
rpoB, SATW20_06120, DNA-directed RNA polymerase beta

5_657803
chain protein

pseudomonas_NC_00846

3_4756080_4756240

pseudomonas_NC_00251

6_1063894_1064077

pseudomonas_NC_00846
PA14_35780, hypothetical protein

3_3182693_3182865

pseudomonas_NC_00965
PSPA7_0044, filamentous hemagglutinin

6_2819490_2819655

pseudomonas_NC_00846
PA14_35790, homospermidine synthase

3_3184022_3184185

pseudomonas_NC_00251
PA0984, colicin immunity protein

6_1065937_1066093

pseudomonas_NC_00251
near pyoS5, PA0985, pyocin S5

6_1067833_1068007

pseudomonas_NC_00846
PA14_35780, hypothetical protein

3_3182351_3182508

pseudomonas_NC_00846
PA14_35790, homospermidine synthase

3_3184314_3184473

pseudomonas_AP012280_

3765216_3765383

pseudomonas_AP012280_

3765033_3765192

enterococcus_NZ_GG70

3715_13422_13573

enterococcus_NZ_GG70

3582_76982_77140

enterococcus_NZ_GL45

5004_28219_28381

enterococcus_NZ_GG70

3720_94699_94852

enterococcus_NZ_GG70

3715_15795_15951

enterococcus_NZ_GL45

5899_32848_32984

enterococcus_NZ_GG69

2918_325104_325257

enterococcus_NC_0046
EF0957, maltose phosphorylase

68_920608_920750

enterococcus_NZ_GG70

3575_78829_78963

enterococcus_NZ_GL45

5931_26355_26493

enterococcus_NZ_GG66

9058_207026_207172

proteus_NZ_GG661998_

111187_111342

proteus_NC_010554_20
lepA, PMI1890, GTP-binding protein LepA

37943_2038091

proteus_NZ_GG668576_

810893_811054

proteus_NZ_GG668594_

760_939

proteus_NZ_GG668579_

22072_22234

proteus_NC_010554_24
PMIr002

48957_2449119

proteus_NC_010554_30
PMIr002

33758_3033936

proteus_NC_010554_45
PMIr006

4391_454540

pseudomonas_NC_00908
rpoB, A1S_0287, DNA-directed RNA polymerase subunit

5_307050_307218
beta

pseudomonas_NC_00908
rpoB, A1S_0287, DNA-directed RNA polymerase subunit

5_308225_308377
beta

pseudomonas_NC_01661
rpoB, KOX_07910, DNA-directed RNA polymerase subunit

2_1674334_1674490
beta

pseudomonas_NC_01660
rpoB, BDGL_003192, RNA polymerase subunit B

3_3425179_3425337

pseudomonas_NC_01660
rpoB, BDGL_003193, DNA-directed RNA polymerase subunit

3_3427629_3427808
beta

pseudomonas_NC_01041
rpoB, ABAYE3489, DNA-directed RNA polymerase subunit

0_3543925_3544088
beta

pseudomonas_NC_00596
rpoB, ACIAD0307, DNA-directed RNA polymerase subunit

6_304936_305079
beta

pseudomonas_NC_00859
rpoB, NT01CX_1107, DNA-directed RNA polymerase subunit

3_226005_226171
beta

pseudomonas_NC_01651
rpoB, EcWSU1_00211, DNA-directed RNA polymerase

4_213592_213738
subunit beta

pseudomonas_NC_00596
rpoB, ACIAD0307, DNA-directed RNA polymerase subunit

6_303883_304054
beta

enterobacter_NC_0146
Entcl_3718, two component transcriptional regulator,

18_3997909_3998085
winged helix family

enterobacter_NZ_GL89

2086_615149_615324

enterobacter_NZ_GL89

2086_1664663_1664834

enterobacter_NZ_GG70

4865_427821_427978

enterobacter_NZ_GL89

2087_1610708_1610874

TABLE 6

Species/strain level probes

Probe Coordinates
Binding region 1
Binding region 2

acinetobacter_NC_010611_627997_628164
GCAGCACTTGACCGCCATGAGTGACCA
CATCGCACCAACAACAATAATCG

acinetobacter_NC_010611_2417580_2417755
GTGATCACTGATGCACCAGATGAAGT
ATCTTGATATTCAAGTCTATGACG

acinetobacter_CP002522_11753_11931
GATATTATTGATCATGGTGCCAAGCCAA
CAATATGAAGCTGACGACGCG

acinetobacter_NC_011586_3908329_3908508
GCTGAGCGTGAAGGTTCATGGATTATTA
GGTAAGGCTTACGGTCTCAT

acinetobacter_NC_010611_145181_145340
GCATCTTGTGCAGCCTGAATAGCAGCGT
ACCACGTTGAATATCACCTTCGG

CAT

acinetobacter_NC_010611_3854494_3854662
AAGTCCATAATTGCTTGAGTGTAGTCAT
ATCTTCGCACTGAATAATAAGAA

CAT

acinetobacter_NC_010400_56216_56383
GCTTGCTGGTTCTGCACGTAGCTTACTG
AAGATGAACAGGCTACTGCAA

acinetobacter_NC_010611_1454960_1455136
GCAGCGCTGTGCAAGTTCAATGTATTCT
CTCGTGCGAGTATTCCTTAAGTGT

acinetobacter_NC_009085_255964_256143
GTATAACACTCGGCCAGCGCCAAGGTTC
GTTCACACATCGCCACAATATGAT

clostridium_NC_013974_3097606_3097772
ACCATGCAGATACAATGAACCA
GGATGATAAGACACATCCAATTC

clostridium_FN665653_103469_103631
CATCAACAGCTTCTTGAAGCATTCC
GTCCAACAACTATAACAGAACGTC

clostridium_NC_013974_117188_117346
AACATATCACCTGATATTCTAGTATC
ATTCCATTATATTCAACAGGATT

GTGA

clostridium_NC_013316_3012882_3013047
GCTGTTGCTTGCGGATACTG
CGTATATGTAGCTCAAGTTGC

clostridium_FN668375_1212250_1212413
AAGAGCTAATGCAGCTATTGCACTTAT
CATACACTTCAGCTATAAGACCAT

clostridium_NC_013315_3754484_3754640
AACAAGAGCAGAAGTTACAGACGT
GTATAATGGTGGCTAGAGGTGA

clostridium_FN665654_3239860_3240039
ACTCGTGAAGACCATGCAGATACAA
AATACTTACAATGCCTGAGGA

clostridium_FN668941_3228320_3228491
ACCATGCAGATACAATGAACC
CCTGAGGATGATAAGACACATC

clostridium_NC_013974_1962664_1962825
GCATCTGCTGCTTCTATTGCTCCTACT
ACATGAACTGATATTAGTTCTCC

AA

clostridium_NC_003366_2769687_2769851
GCACAAGCTGGAGATAACATCGG
GTAGAGGACGTATTCACAATCACT

clostridium_FN665653_127741_127918
CTCTATCAGCTTCTACTGCTTCTTC
CCATCTCATCCACAGTTAATATA

TC

clostridium_NC_013316_2259929_2260107
AGATGAGATTCATACTATCGTTGGAGCT
AGCAGAGAGAATAGTAAGAGGAGA

clostridium_NC_009089_94774_94937
CATCAACAGCTTCTTGAAGCATT
GTCCAACAACTATAACAGAACG

clostridium_NC_013315_2044225_2044389
GTCAGCAATACGCCACCAAGCTCCTAT
GTGGTGGATATCCTGTTACC

clostridium_NC_013315_2299408_2299586
GCGCAATAGAGTTGTATAAGAGTGCTG
AGCATTAATTATAGATTATAATG

TATAA

clostridium_FN668941_3244255_3244408
GGCATAATAGGATGGATAGATGA
ACTAATCCAACTTCTACTGCTAT

clostridium_NC_013316_3610909_3611065
GTACATTCACATATAGACCATCTTAA
ACATAGGTGCAGGTAGAATAGTA

TA

clostridium_FN665653_1104859_1105031
CCATACCAGTATCTTGGCATATTG
ATAATGAATAACAGCAGGTGTAT

TA

clostridium_NC_003366_2753681_2753838
AGATGAAGCACAAGCTGGAGATAA
AGGACGTATTCACAATCACTG

clostridium_FN665653_710906_711080
ATAATCATTCACCTCCATCATTCATAA
ACTGAATATGGTTCGTCTCA

clostridium_NC_009089_3706562_3706720
GTACATTCACATATAGACCATCTTA
ACATAGGTGCAGGTAGAATAGT

clostridium_NC_013316_1372812_1372968
ACTCCACCAGGATGTTGTCC
GTAGGACCGTCGTGTCCAAG

clostridium_FN665652_676696_676895
GCAATATCAATGGTATCGAAGGCACTAT
GTATTGAAGGTACTATTAGCGAT

ATGC

clostridium_NC_013316_2641651_2641808
GTGCCGGTCTCGGTTACTCAATG
GGATTATTATAATGCAGCTAGAAG

clostridium_FN668375_3595870_3596026
GTACATTCACATATAGACCATCTT
ACATAGGTGCAGGTAGAATAGTA

clostridium_FN668941_1105700_1105868
AGTTCCTTCATATGACTCAGTTGATTGA
GTTATATCTTCAATTATACATTC

CTGC

clostridium_NC_013974_2505182_2505359
CAGCAGTTGTTGCTAGAGGTATG
GCATCACCAGGTGCAGCAAGT

clostridium_NC_013315_1077126_1077298
GCAATTCTCTGTTGTTGTCCTCCACTCA
AGTAAGAGCCTCTTCTTGGTCAT

GA

clostridium_NC_009089_2182303_2182482
CTATTCCTGATAATAAGTGTGTCCTCAT
CGGCATCATCTAACAATTCTTCT

clostridium_FN665652_1909777_1909942
GTAATTCCAATTACTTCTAGCTCTGGTG
TACCATCTTCTCCATGTGTAT

clostridium_NC_013316_3300896_3301062
CCATGCAGATACAATGAACCAG
GATGATAAGACACATCCAATTCC

clostridium_NC_013316_871338_871499
CCTTCTGCCATTGTAGAACAAGCTCCAT
CCTGTAACTGTCCACTGAGC

clostridium_NC_013316_3608873_3609047
CAATCATGATAGAATTAGATGGAAC
AGCAATAGTTCCATCAGGAGCATC

clostridium_FN665654_3717059_3717221
AGTGGTGAAGGTGTTCAACAAG
ACTGAAGCTGGATATGTTGGAG

clostridium_NC_013315_2010489_2010657
CGCCTCTTCAGAAGCGGATATCA
GCCAGACTTCCGCCACAACCT

clostridium_NC_013315_3236301_3236474
GGCATAATAGGATGGATAGATGAGC
GCAGCAGTTGTACCTACAACTAA

clostridium_NC_013315_1095924_1096090
AGTTCCTTCATATGACTCAGTTGATTG
GTTATATCTTCAATTATACATTC

CTGCG

enterobacter_NC_014121_4735453_4735632
GCATGGTAGTTCGCCAGCCGCTGGAAC
ACAGCAACCGCAAGTTCTTGACAT

enterobacter_NC_015663_1014187_1014345
AATATCATGGTCGTGTCCAGGCACTGGC
GTTCTGGTAGCTGCTTCTACTGTA

enterobacter_FP929040_3448334_3448513
AACTTACAACTACGCGCACTTGAATCG
GAGTGTTGTATGATAGTCTCGGT

enterobacter_NC_009436_4051820_4051985
GCAAGTTGAGGAGATGCTGGCATGATTC
ACATGGCTCTGGAAGATGTGCTG

ATC

enterococcus_FP929058_1738439_1738606
GCGATAATTGTAATGATTCGTGGTGTTA
CCGTTGTCAATCCAGTTAGTAGA

CT

enterococcus_CP002621_1819224_1819388
ACTGTGGCAGTCTATGTTCCAATTGTA
CTTATCGACATAATCCTGATAATC

enterococcus_FP929058_904007_904173
GCGTCGCTTCTTGCGCTCGCC
AATGTATTCATACCGTCAAGT

enterococcus_FP929058_551757_551920
GCCTTCACAACTACGTTGGAAGGTCTTC
CTAACAGTCCTGCCGACTAC

enterococcus_NC_004668_1122345_1122507
GCCTTCACAACTACGTTGGAAGGTCTT
CTAACAGTCCTGCCGACTACT

klebsiella_NC_009648_2885456_2885620
GCCGCTGAGCGGCGGCAAGCCGATGGC
GAATGGCAGGCCAAGCTGAAGGCG

klebsiella_NC_009648_3899012_3899182
GCCAAGCGGCATTCTGGCGCCAGTGGA
CCAGACCGGAGTGGACAACGTCG

AGGCG

klebsiella_NC_009648_4980596_4980757
GCCGTATATCATCGGCAATAACCGCACG
GCATGATGGTCAACAAGGTGC

klebsiella_NC_009648_3266359_3266519
ACGAGCCGAGATAGGTCTGCAGCGTAC
GTACTGATATTCACCATACTGCCG

klebsiella_NC_012731_2557467_2557634
GCAATATCTTCACCGGCAGCCACCGCG
GGTATATGGCACGCCAATCGC

klebsiella_NC_012731_4857136_4857315
AATAACCTTAACGTCGCCAACACG
CTCGGTGAACACCTCCTGGCACG

proteus_NC_010554_547938_548117
GCGGAACTGCTTGGCGTAGTAAGC
CATGTAGTGCCGTAGACCTTCAC

CA

pseudomonas_NC_008463_658500_658676
GCGAGACCGGCGGCACCATCGTCTCCAG
TTCTGCCTGATGGACGTCTCCGG

CTCG

pseudomonas_NC_008463_753931_754099
GCGGTTCACCTGTTCGCCTTCGAACACG
GCGCAGCATCTGACGCAGGATGG

TCTCG

pseudomonas_NC_009656_6431649_6431828
ACTCCATCGCCATCAAGGACATGGCCGG
ATCGACGTGTTCCGCATCTTCGA

CGCG

pseudomonas_NC_008463_560357_560534
GCCTGATGCACTACAGCGCCTGG
TACCACATGGTCGATCTCGACGA

CTGC

pseudomonas_NC_010322_5224859_5225023
GCGCATCCAGGACGGCGAGTACG
CTTCGAGTGCCTGCACGAGCTGAA

pseudomonas_NC_008463_4839746_4839924
GCTGGAGAACGTCAAGGTGGTGATCATC
ACCGATAACGACGACCGCATCAA

staph_FN433596_2844085_2844263
ACGATTGGAGAAGGCAGTGTGATTGG
GGACAGATTACAATTGGCG

staph_NC_009632_1198350_1198529
GCCGCAATACCGATATTCCA
CCATTGTCCACCAGCTGAACCG

staph_FN433596_2521244_2521419
GTGAAGGTCGTGCTCCTATCGGT
AGATCTGGTGAAGTTCGTATGAT

staph_NC_009487_430842_431017
GCTGGTACTTGTACTTATATCGA
ATCAGAAGATGATATCGTTACGT

CAT

staph_NC_009782_2086681_2086849
GCGCATATTGCATTAATGGCTATAGAT
GCCAGCAGGTTATACACTCG

staph_NC_009782_58256_58423
GCAATTCTTACCACAGCACGAAGAACAG
ATCTAGATGAAGATAATGAAGTCG

staph_NC_013450_991049_991222
GCATCTTCATACAATACTTCTAGCTTAC
CACAATACCAGTTGTATTACG

staph_NC_013450_1360842_1361008
GCTTCAGCGCCATTACCGCCACCAGCT
ACTCTTGATATATTCTTGTAAGCG

staph_AM990992_2526026_2526192
GTTCACACAACGCGCCGACTAGAATCC
CACGATATCCAAGATAATGATTG

GCTA

staph_NC_010079_361284_361447
GCGCACCTACAATCGCCATTACTACAC
ACTCATTATCGACTGTTACATCG

ACTGA

staph_NC 007795_2085723_2085901
AGCGCACATGTGACAGCGTGTAGGTTA
GTGCCTTAGATTGTTCAGAACAAT

staph_NC_009641_23125_23297
CGAATGGATATGTACCATGGTCGATATC
CTCTCTAATATGATGTCCAT

staph_FN433596_2144570_2144734
ACTACAACAGCAACCGCATTACAATGGC
GGTGCTAAGAGGTCATCGGA

staph_NC_009782_54857_55020
AGCTTCAGATAAGTACCTATCTGA
GGAAGAATAGTTATTCTTGATAA

TGTAT

staph_AM990992_1656616_1656789
CGTATTGCTCGAATACATGATA
ACAATGTATCAAGGCCAGCT

staph_NC_007793_44227_44395
GCGACCAGTTGTTATCGACCGTGT
CAGAACGATACGGTGCTGTATA

staph_NC_009641_1102949_1103116
CAATTACATTGTCTGTTGCGTAGATACC
GTTGTGGCTAATGTGCCAGTT

staph_NC_009641_1137731_1137898
GCACCACTCTATAGCAGTAGCGTATTG
ACAGCCAATGTCACCTAAGTCAA

CA

staph_FN433596_2715713_2715871
ACAGTCCGAATAAGATACGACTATTCGA
CGTTGTAACGTATATGAATAGTT

GA

staph_NC_009782_606652_606825
AGATGCAATAACAGGTCGAATATTAATT
GCCATAGTGAGAGTAGTGAA

staph_FN433596_657625_657803
AGATGCAATAACAGGTCGAATATTAA
ACACATACGGCCATAGTGAGAG

pseudomonas_NC_008463_4756080_4756240
GAATCGAACGGTCTCATTAACAGAT
GCTTTCCAGGGATATAAGACGC

pseudomonas_NC_002516_1063894_1064077
CCCGCAGAGTCACACTCGGA
ACTCTTGGTACTACTCACTAGC

pseudomonas_NC_008463_3182693_3182865
GAGTCTCTTTCAACCTGGATTAGATAT
AAGATTAATAGCGTACTTTACTCC

pseudomonas_NC_009656_2819490_2819655
ATCCCGCAGATACTAGGTTCTTAAT
GAACTATTCATATTACACCCTAA

GG

pseudomonas_NC_008463_3184022_3184185
CAGTGGGCTATCCTAAGCCAAAG
CATAAGCGAACTAACTATCACTTA

pseudomonas_NC_002516_1065937_1066093
ACAAAGCGTTCTAAACGATTAGAACT
CGAGAAAGGAAACAGGATAGTAC

pseudomonas_NC_002516_1067833_1068007
CCAATGGAGAAGTCTAAATGTCCAA
TTATCAGAGATACATGACTCTTA

GG

pseudomonas_NC_008463_3182351_3182508
CGAATCACTGGACTACATTTATATTTCT
AGCGAACCTTTATATTTGACCAT

pseudomonas_NC_008463_3184314_3184473
CTCAAGTCTTGCCCTGATAGAATTAT
TCACGACTTATCTACTTTAGAAA

TC

pseudomonas_AP012280_3765216_3765383
GGTGATCGTTATTATGATAGTACGGC
CTCGGTTAAGGGAATTACGAC

pseudomonas_AP012280_3765033_3765192
ACTCGGATGGTAGGTTTATTAAAGC
GTGATCGTTATTATGATAGTACGG

enterococcus_NZ_GG703715_13422_13573
ACAATCGTTGTCGCACTGCATAG
GAACTTGGTCTACCGTACCAC

enterococcus_NZ_GG703582_76982_77140
GGATAATACAATCCTAATACGTACGGA
GCTGCTGTAACTAGGGTAGC

enterococcus_NZ_GL455004_28219_28381
CTATATTCAACGGGTCACGGGTAG
TCATTGATTCGATCTCGTAACTC

enterococcus_NZ_GG703720_94699_94852
AATGTTATTGTGGTTGCGTGTTCG
TACTTTGGAAGTGCCCTGAC

enterococcus_NZ_GG703715_15795_15951
CATGTCTTCTAGTACAGGTTTGCCG
TGTAAGAGGCCGCTAACTTC

enterococcus_NZ_GL455899_32848_32984
CTCTGGCTCGTGGGCTCGG
TTCTTGAGATAGTCCGGTATAATC

enterococcus_NZ_GG692918_325104_325257
ATTCGATCACGATGGGCTGGG
AATTTCCTGTGTCATACACGC

enterococcus_NC_004668_920608_920750
CAATTGATTTAGCCACTACACCTTAC
CACTATTCTGGCGACCACC

enterococcus_NZ_GG703575_78829_78963
GATAAAGAAGCGTCTTGACCCAGT
ATCTGGTGCTCCTTGACGC

enterococcus NZ_GL455931_26355_26493
GCAAATTTAGAGAGTGCATGCATG
GGAAGAGGACGGCATACAAC

enterococcus_NZ_GG669058_207026_207172
CATTTCATCTAGACCGCTCGTGT
GCTTGAAGTGTATGTTGGGAC

proteus_NZ_GG661998_111187_111342
GTCGCCCTCGTGCTAACGT
GGTTCTTTGATGTACCGGTT

proteus_NC_010554_2037943_2038091
GCTGATGACGGTGAAGTTTATCA
CATTATCGCACATATTGACCAC

proteus_NZ_GG668576_810893_811054
GAAATTAGCTAAAGGGATATCGCG
AACTTTCCGCCAATCCTGC

proteus NZ_GG668594_760_939
CACCTACGTTCTCACCTGCAC
ATTCGATAGTACCAGTTACGTC

proteus_NZ_GG668579_22072_22234
GTTGCTTATAGCGTCGCTGCT
CTGGTTATCGAGAAGATAAAGG

proteus NC_010554_2448957_2449119
GTAAGCGTAGCGATACGTTGAG
GAGTGAACGCACCACTGG

proteus_NC_010554_3033758_3033936
TCAGGTAGAGAATACTCAGGCGC
CGGAGAAGGCTAGGTTGTC

proteus_NC_010554_454391_454540
GCAACCCACTCCCATGGTGT
CGTTCTTCATCAGACAATCTG

pseudomonas_NC_009085_307050_307218
AACTAAACCTACACGGAATTGGTTC
GCAGATACACGACGTTTATGT

pseudomonas_NC_009085_308225_308377
GCCGCTTCACCTACGTTAGGAA
CGTAAAGATGAGTCTTTAACGTC

pseudomonas_NC_016612_1674334_1674490
GACGTTTGTGCGTAATCTCAGAC
GAGGAAACCGTATTCGTTCGT

pseudomonas_NC_016603_3425179_3425337
ACAACACTTTACCACTTGAGTGGG
GTAACTGCCCATGTCAAGATAC

pseudomonas_NC_016603_3427629_3427808
CCACGTTTAGTTGAACCACCGC
TCAATACGCCAGTTGTTAGTTC

pseudomonas_NC_010410_3543925_3544088
AATCGATAATAAGTACGGTGCATCC
GAAGAATACATTCGCGTACATC

pseudomonas_NC_005966_304936_305079
AAGCAAGATCGAGTCTTCATAGTTG
GATATACACGATACCTGATTCGT

pseudomonas_NC_008593_226005_226171
CCGATATTCATACGAGAAGGTACAC
CAGTAACTCTATTGTCAAACGGT

pseudomonas_NC_016514_213592_213738
GTAGTGAGTCGGGTGTACGTCTC
TCTTCGATAGCAGACAGATAGT

pseudomonas_NC_005966_303883_304054
ACCTACACGGAATTGGTTCTCAGT
GATACACGACGTTTGTGTGTA

enterobacter_NC_014618_3997909_3998085
CAACATCATTAGCTTGGTCGTGGG
TTGCGTGTTACCAACTCGTC

enterobacter_NZ_GL892086_615149_615324
CGGCACGTCCGAATCGTATCA
TCGTGTCCCGTATATGTTGG

enterobacter_NZ_GL892086_1664663_1664834
AATAGAGGCCCACAAGTCTTGTTC
CGCTCTCCACTATGGGTAGT

enterobacter_NZ_GG704865_427821_427978
GCTACATTAATCACTATGGACAGACA
GATGGTCGATCTATCGTCTCT

enterobacter_NZ_GL892087_1610708_1610874
GAAGTGTTATTCAAACTTTGGTCCC
CTTGAACCCTTGGTTCAAGGT

TABLE 7

Marker regions are highly polymorphic regions (like, e.g., VNTRs) that provide

fine resolution.

Probe Coordinates
Gene

plasmids_NC_011980_58308_58487
insA, MM1_0111, IS1 protein InsA, MM1_0112, IS1

protein InsB

plasmids_NC_015599_37281_37455
intIl, pN3_046, integrase

plasmids_NC_007351_37979_38146
SSPP128, IS431 transposase

plasmids_FN822749_1846_2009
ETEC1392/75_p75_00003, putative IS100

transposase

plasmids_NC_004851_143949_144109
CP0039, IS629 ORF2

plasmids_NC_010558_156799_156957
IS1-insB, IPF_205, IS1-insB

plasmids_NC_012547_53585_53752
tnpA, PGO1_p15, putative transposase TnpA

plasmids_NC_013950_91008_91174
tnpR, pKF94_116, TnpR

plasmids_NC_002698_168967_169123
insB, pWR501_0054, IS1 transposase

CMY_AB061794_343_489
intI1, DNA integrase

IMG_AY033653_1343_1500
intI1, DNA integrase

TEM_U36911_4374_4551

TEM_U36911_7596_7762

TABLE 8

Marker probes

Probe Coordinates
Binding region 1
Binding region 2

plasmids_NC_011980_58308_58487
GCAGTCGGTAACCTCGCGC
GCGCTATCTCTGCTCTCACTGC

plasmids_NC_015599_37281_37455
GCTGTAATGCAAGTAGCGTATGCGCTC
GAACAGCAAGGCCGCCAATGCCTGACG

plasmids_NC_007351_37979_38146
CGCATATGCTGAATGATTATCTCGTTGC
ATCTTGCTCAATGAGGTTATTCA

plasmids_FN822749_1846_2009
GACGACAGATGCAGGTTGA
CGCATCGCCGATGCTCATC

plasmids_NC_004851_143949_144109
CGCCTGCTCCAGTGCATCCAGCACGAAT
ATGCTCTCCGCCATCGCGTTGTCA

plasmids_NC_010558_156799_156957
AGTGCGTTCACCGAATACGTGCGCA
CAGGTTATGCCGCTCAATTC

plasmids_NC_012547_53585_53752
CGCATATGCTGAATGATTATCTCGTTG
ACGGTGATCTTGCTCAATGAGGTTATTC

plasmids_NC_013950_91008_91174
GCTGTGGCACAGGCTGAACGCCG
GGTGATGTCATTCTGGTTAAGA

plasmids_NC_002698_168967_169123
ACATAATCTGAATCTGAGACAACATC
ACGCACTCTGGCCACACTGG

CMY_AB061794_343_489
CATCACGAAGCCCGCCACA
GCCCTTGAGCGGAAGTATC

IMG_AY033653_1343_1500
CGGAAGTATCCGCGCGCC
TTCGATCACGGCACGATC

TEM_U36911_4374_4551
CATTCTCTCGCTTTAATTTATTAACCT
ATCGACCTTCTGGACATTATC

TEM_U36911_7596_7762
CGTTGCTTACGCAACCAAATATC
TGATCTTGCTCAATGAGGTTA

TABLE 9

Resistance regions can be used to detect one or more genes associated with

resistance to antimicrobial compounds, such as antibiotic resistance genes.

Probe Coordinates
Gene

plasmids_NC_013950_90185_90338
pKF94_115, beta-lactamase

plasmids_NC_013452_4052_4209
SAAV_b4 tetracycline resistance protein

plasmids_NC_014208_52313_52469
pKOX105p23, VIM-1, pKOX105p24, IntIA

pKOX105p67, truncated AadA

betalactamase_AB372224_738_905
blaCMY-39, class C beta-lactamase CMY-39

betalactamase_EF685371_398_548
beta-lactamase CMY-29

betalactamase_DQ149247_231_371
bla-OXA-86, OXA-86

betalactamase_AY750911_244_414
bla-oxa-69, beta-lactamase OXA-69

betalactamase_DQ519087_417_575
blaOXA-93, beta-lactamase OXA-93

betalactamase_AM231719_379_537
blaOXA-90, class D beta lactamase

betalactamase_Y14156_663_819
CTX-M-4, beta lactamase

betalactamase_JN227085_763_931
blaCTX-M-117, CTX-M-117 beta-lactamase

betalactamase_EU259884_1030_11
aacA4, AacA4 aminoglycoside (6')

70
acetyltransferase

betalactamase_HQ913565_578_730
blaCTX-M-106, beta-lactamase CTX-M-106

betalactamase_AY524988_385_552
blaVIM-9, VIM-9

CARB_AF030945_646_795
CARB-6, class A beta-lactamase

CARB_U14749_1227_1390
blaCARB-4, CARB-4 precursor

CARB_AF313471_2731_2906
aadA1a, AAD(3″) aminoglycoside (3″)

adenylyltransferase

CMY_DQ463751_613_790
blaCMY-23, hypothetical CMY-23 protein

precursor

CMY_EF685371_397_552
beta-lactamase CMY-29

CMY_EU515251_583_733
blaCMY-40, AmpC beta-lactamase

CMY_JN714478_1882_2055
blaCMY-66, AmpC beta-lactamase CMY-66

CMY_X91840_1872_2046
bla CMY-2, extended spectrum beta-lactamase

CTXM_EF219134_13713_13858
AadA2 aminoglycoside adenylytransferase;

confers resistance to streptomycin and

spectinomycin

CTXM_HQ398215_802_947
blaCTX-M-98, beta-lactamase CTX-M-102

CTXM_AM982522_639_788
blaCTX-M-78, CTX-M-78 beta-lactamase

GES_HM173356_1163_1321
blaGES-16, carbapenem-hydrolyzing extended-

spectrum beta lactamase GES-16

GES_AF156486_1754 1905
ges-1, beta-lactamase GES-1

GES_HQ874631_571_748
extended-spectrum beta-lactamase GES-17

GES_FJ820124_1174_1338
beta-lactamase GES10

IMG_DQ361087_489_645
blaIMP-22, metallo-beta-lactamase IMP-22

IMG_JN848782_301_475
blaIMP-33, metallo-beta-lactamase IMP-33

IMG_EF192154_182_328
blaIMP-24, metallo-beta-lactamase IMP-24

IMG_AF318077_871_1047
aacC4, aminoglycoside-N-

acetyltransferase

IMG_AF318077_515_657
aacC4, aminoglycoside 6'-N-

acetyltransferase

KPC_HM066995_226_375
b1aKPC, beta-lactamase KPC-11

KPC_GQ140348_624_799
KPC-10, beta-lactamase KPC-10

KPC_EU729727_683_840
carbapenem-hydrolyzing beta-lactamase KPC-

7

KPC_FJ234412_691_839
blaKPC-8, beta-lactamase KPC-8

NDM_JN104597_64_211
blaNDM-5, NDM-5 metallo-beta-lactamase

NDM_FN396876_2744_2885
blaNDM-1, metallo-beta-lactamase

NDM_FN396876_2958_3117
blaNDM-1, metallo-beta-lactamase

NDM_JN104597_314_465
blaNDM-5, NDM-5 metallo-beta-lactamase

NDM_FN396876_2382_2548
blaNDM-1, metallo-beta-lactamase

OXA_EF650035_239_388
bla-OXA-109, beta-lactamase OXA-109

OXA_EU019535_389_537
bla-OXA-80, beta-lactamase OXA-80

OXA_EF650035_423_594
bla-OXA-109, beta-lactamase OXA-109

OXA_DQ309276_232_380
bla-OXA-84, beta-lactamase OXA-84

OXA_DQ445683_232_380
bla-OXA-89, oxacillinase OXA-89

OXA_X75562_201_366
OXA-7, beta lactamase OXA-7

OXA_M55547_995_1154
tnpR, aac, Aac

OXA_AY445080_313_469
blaOXA-56, restricted-spectrum beta-

lactamase OXA-56

PER_Z21957_217_371
PER-1, extended-spectrum beta-lactamase

PER-1

PER_HQ713678_6002_6167
blaPER-7, blaPER-7

PER_GQ396303_667_844
blaPER-6, extended-spectrum beta-lactamase

PER-6

PER_X93314_954_1122
bla(per-2), extended-spectrum beta-

lactamase

PER_HQ713678_4517_4674
transposase

PER_HQ713678_5074_5219
transposase

PER_GQ396303_254_399
blaPER-6, extended-spectrum beta-lactamase

PER-6

SHV_AY661885_656_806
blaSHV-30, beta-lactamase SHV-30

SHV_AF535128_587_761
blaSHV-40, beta-lactamase SHV-40

SHV_U92041_406_579
SHV-8, beta-lactamase

SHV_AY288915_617_764
blaSHV-50, beta-lactamase SHV-50

SHV_HQ637576_88_245
blaSHV-135, beta-lactamase SHV-135

SHV_AF535128_188_362
blaSHV-40, beta-lactamase SHV-40

SHV_X98102_763_913
blaSHV-2a, beta-lactamase SHV-2a

TEM_GQ149347_3605_3747
near kanamycin resistance protein

TEM_GU371926_11801_11944
traN, TraN

TEM_J01749_766_908
tet, tetracycline resistance protein

VEB_EU259884_6947_7094
blaVEB-6, VEB-6 extended-spectrum beta-

lactamase

VEB_EF136375_596_738
blaVEB-4, extended-spectrum beta-lactamase

VEB-4

VEB_EF420108_234_380
blaVEB-5, extended spectrum beta-lactamase

VEB-5

VEB_AF010416_89_230
veb-1, extended spectrum beta-lactamase

VIM_AY524988_385_552
blaVIM-9, VIM-9

VIM_Y18050_3464_3614
blaVIM, beta-lactamase VIM-1

VIM_AY635904_58_203
blaVIM-11, metallo-beta-lactamase

VIM_HM750249_275_454
bla, metallo-beta-lactamase VIM-25

VIM_AJ536835_313_481
blaVIM-7, metallo-b-lactamase

VIM_EU118148_131_300
near intI1, DNA integrase INTI1

VIM_DQ143913_921_1063
near intI1, IntI1

VIM_EU118148_1060_1229
blaVIM-17, metallo-beta-lactamase VIM-17

van_NC_008821.1_11898_12045
vanB, pVEF236, D-alanine--D-lactate ligase

mecA_AY820253.1_1431_1608
mecA, PBP2a-like protein

mecA_AY952298.1_130_302
Pbp2′

erm_NC_002745.2_871803_871973

erm_NC_002745.2_871666_871841
ermA, SA1951, rRNA methylase Erm(A)

TABLE 10

Resistance probes

Probe Coordinates
Binding region 1
Binding region 2

plasmids_NC_013950_90185_90338
GAGGACCGAAGGAGCTAACCG
CGCCGCATACACTATTCTC

plasmids_NC_013452_4052_4209
CTCATTCCAGAAGCAACTTCTTCTT
GGATAGCCATGGCTACAAGAATA

plasmids_NC_014208_52313_52469
GGTTCTGGACCAGTTGCGTGAGCGC
CGTAACATCGTTGCTGCTCCAT

betalactamase_AB372224_738_905
CGCTGGATTTCACGCCATAGGC
TGTCGCTACCGTTGATGATT

betalactamase_EF685371_398_548
CGTATAGGTGGCTAAGTGCAGC
GTAACTCATTCCTGAGGGTTTC

betalactamase_DQ149247_231_371
GTACATACTCGATCGAAGCACGA
CCGGAATAGCGGAAGCTTTC

betalactamase_AY750911_244_414
AAGGTCGAAGCAGGTACATACTCG
AGACATGAGCTCAAGTCCAAT

betalactamase_DQ519087_417_575
GAAGCTTTCATAGCGTCGCCTAG
TTAGCTAGCTTGTAAGCAAATTG

betalactamase_AM231719_379_537
GAAGCTTTCATGGCATCGCCTAG
AGCTAGCTTGTAAGCAAACTG

betalactamase_Y14156_663_819
CGCTACCGGTAGTATTGCCCTT
AGAATATCCCGACGGCTTTC

betalactamase_JN227085_763_931
ATCGCCACGTTATCGCTGTACT
TTTACCCAGCGTCAGATTCC

betalactamase_EU259884_1030_1170
CAAGTACTGTTCCTGTACGTCAGC
TCGCCAGTAACTGGTCTATTC

betalactamase_HQ913565_578_730
CAACGTCTGCGCCATCGCC
CGCAATATCATTGGTGGTGC

betalactamase_AY524988_385_552
GCCGCCCGAAGGACATCAAC
CAGACGGGACGTACACAAC

CARB_AF030945_646_795
CGTGCTGGCTATTGCCTTAGG
GTAATACTCCTAGCACCAAATC

CARB_U14749_1227_1390
CATTAGGAGTTGTCGTATCCCTCA
AATACTCCGAGCACCAAATC

CARB_AF313471_2731_2906
AAATTGCAGTTCGCGCTTAGC
GTTCCATAGCGTTAAGGTTTC

CMY_DQ463751_613_790
GCGCCAAACAGACCAATGCT
GATTTCACGCCATAGGCTC

CMY_EF685371_397_552
GTATAGGTGGCTAAGTGCAGCA
TCGTAACTCATTCCTGAGGG

CMY_EU515251_583_733
GTCATCGCCTCTTCGTAGCTC
GCCATATCGATAACGCTGG

CMY_JN714478_1882_2055
ACCAATACGCCAGTAGCGAGA
GCAACGTAGCTGCCAAATC

CMY_X91840_1872_2046
CAATCAGTGTGTTTGATTTGCACC
TACCCGGAATAGCCTGCTC

CTXM_EF219134_13713_13858
CGGATAACGCCACGGGATGA
ACCGGGTCAAAGAATTCCTC

CTXM_HQ398215_802_947
GCGGCGTGGTGGTGTCTC
CGCTGCCGGTCTTATCAC

CTXM_AM982522_639_788
GCCACGTCACCAGCTGCG
CGGCTGGGTGAAGTAAGTC

GES_HM173356_1163_1321
GCTCGTAGCGTCGCGTCTC
TTGACCGACAGAGGCAAC

GES_AF156486_1754_1905
CAGCAGGTCCGCCAATTTCTC
AGTGGACGTCAGTGCGC

GES_HQ874631_571_748
CCATAGAGGACTTTAGCCACAGT
TACACCGCTACAGCGTAAT

GES_FJ820124_1174_1338
CATATGCAGAGTGAGCGGTCC
TCAATTCTTTCAAAGACCAGC

IMG_DQ361087_489_645
CCATTAACTTCTTCAAACGATGTATG
ACCCGTGCTGTCGCTAT

IMG_JN848782_301_475
GTGCTGTCGCTATGGAAATGTG
AACCAAACCACTAGGTTATCTT

IMG_EF192154_182_328
GTCAGTGTTTACAAGAACCACCA
ATGCATACGTGGGAATAGATT

IMG_AF318077_871_1047
CGAACCAGCTTGGTTCCCAAG
TCACTGCGTGTTCGCTC

IMG_AF318077_515_657
GATGCTGTACTTTGTGATGCCTA
CGCTTGGCAAGTACTGTTC

KPC_HM066995_226_375
GCAAGAAAGCCCTTGAATGAGC
GCGTTATCACTGTATTGCAC

KPC_GQ140348_624_799
AATCAACAAACTGCTGCCGCT
GCTGTACTTGTCATCCTTGT

KPC_EU729727_683_840
CCAGTCTGCCGGCACCGC
TCGAGCGCGAGTCTAGC

KPC_FJ234412_691_839
CCGACTGCCCAGTCTGCCG
CGAGCGCGAGTCTAGCC

NDM_JN104597_64_211
GTAAATAGATGATCTTAATTTGGTTCAC
TTGCTGGCCAATCGTCG

NDM_FN396876_2744_2885
CACAGCCTGACTTTCGCCGC
CAAGCAGGAGATCAACCTGC

NDM_FN396876_2958_3117
GGTGGTCGATACCGCCTGG
GTGAAATCCGCCCGACG

NDM_JN104597_314_465
CATGTCGAGATAGGAAGTGTGC
TGATGCGCGTGAGTCAC

NDM_FN396876_2382_2548
CAATCTGCCATCGCGCGATT
CGGCAATCTCGGTGATGC

OXA_EF650035_239_388
CGAAGCAGGTACATACTCGGTC
ACGAGCTAAATCTTGATAAACTT

OXA_EU019535_389_537
TAGAATAGCGGAAGCTTTCATGG
AGCTAGCTTGTAAGCAAACTG

OXA_EF650035_423_594
CAAGTCCAATACGACGAGCTAAA
GAATAGCATGGATTGCACTTC

OXA_DQ309276_232_380
GGTACATACTCGGTCGAAGCAC
AATCTTGATAAACTGAAATAGCG

OXA_DQ445683_232_380
GGTACATACTCGGTCGATGCAC
TCTTGATAAACCGGAATAGCG

OXA_X75562_201_366
GTAATTGAACTAGCTAATGCCGTAC
TTATGACACCAGTTTCTAGGC

OXA_M55547_995_1154
CAAGTACTGTTCCTGTACGTCAG
GCCCAGTTGTGATGCATTC

OXA_AY445080_313_469
TCTCTTTCCCATTGTTTCATGGC
TGCGGAAATTCTAAGCTGAC

PER_Z21957_217_371
GTAGGTTATGCAGTTATTAGGTTCAG
GACTCAGCCGAGTCAAGC

PER_HQ713678_6002_6167
GCAGTACCAACATAGCTAAATGC
AAATAACAAATCACAGGCCAC

PER_GQ396303_667_844
GGTCCTGTGGTGGTTTCCACC
CGCGATAATGGCTTCATTGG

PER_X93314_954_1122
TAACCGCTGTGGTCCTGTGG
TGCGCAATAATAGCTTCATTG

PER_HQ713678_4517_4674
GGAAGCGTTGCTTGCCATAGT
AACCGAAGCACCATGTAATT

PER_HQ713678_5074_5219
GTTCGGTGCAAAGACGCCG
TCGCAGACTTCAATATCAATATT

PER_GQ396303_254_399
CACCTGATGCAGAACCAGCAT
AGGCCACGTTATCACTGTG

SHV_AY661885_656_806
CAGCTGCCGTTGCGAACG
CGCAGATAAATCACCACAATC

SHV_AF535128_587_761
GCTCAGACGCTGGCTGGTC
CCGCAGATAAATCACCACG

SHV_U92041_406_579
GCCAGTAGCAGATTGGCGGC
GAACGGGCGCTCAGACG

SHV_AY288915_617_764
CCACTGCAGCAGATGCCGT
GTATCCCGCAGATAAATCACC

SHV_HQ637576_88_245
TTAATTTGCTTAAGCGGCTGCG
CCAGCTGTTCGTCACCG

SHV_AF535128_188_362
GGGAAAGCGTTCATCGGCG
TCGCTCATGGTAATGGCG

SHV_X98102_763_913
TCTTATCGGCGATAAACCAGCC
CGTTGCCAGTGCTCGAT

TEM_GQ149347_3605_3747
GTCGGAAAGTTGACCAGACATTA
ATACTAGGAGAAGTTAATAAATACG

TEM_GU371926_11801_11944
GTGAAGTGAATGGTCAGTATGTTG
AGTGCGCAGGAGATTAGC

TEM_J01749_766_908
CCTGTCCTACGAGTTGCATGAT
ATAATGGCCTGCTTCTCGC

VEB_EU259884_6947_7094
CAAATACTAAATTATACAGTATCAGAG
ATGCAAAGCGTTATGAAATTTC

AG

VEB_EF136375_596_738
GTTCTTATTATTATAAGTATCTATTAA
CATTAGTGGCTGCTGCAAT

CAGTT

VEB_EF420108_234_380
CATCGGGAAATGGAAGTCGTTAT
GTTCAATCGTCAAAGTTGTTC

VEB_AF010416_89_230
CGTGGTTTGTGCTGAGCAAAG
CAAAGTTAAGTTGTCAGTTTGAG

VIM_AY524988_385_552
GCCGCCCGAAGGACATCAA
AGACGGGACGTACACAAC

VIM_Y18050_3464_3614
GCAACTCATCACCATCACGGA
TGATGCGTACGTTGCCAC

VIM_AY635904_58_203
GCGACAGCCATGACAGACGC
GGACAATGAGACCATTGGAC

VIM_HM750249_275_454
AAACGACTGCGTTGCGATATG
TTCCGAAGGACATCAACGC

VIM_AJ536835_313_481
ATGCGACCAAACGCCATCGC
ATCGTCATGGAAGTGCGTA

VIM_EU118148_131_300
GAACAGGCTTATGTCAACTGGG
CATAACATCAAACATCGACCC

VIM_DQ143913_921_1063
ACGAACCGAACAGGCTTATGTC
TAACGCGCTTGCTGCTT

VIM_EU118148_1060_1229
CATCATAGACGCGGTCAAATAGA
ACTCATCACCATCACGGAC

van_NC_008821.1_11898_12045
CAGGCTGTTTCGGGCTGTGA
GGGTTATTAATAAAGATGATAGGC

mecA_AY820253.1_1431_1608
TAATTCAAGTGCAACTCTCGCAA
TTTATTCTCTAATGCGCTATATATT

mecA_AY952298.1_130_302
GGATAGTTACGACTTTCTGCTTCA
TGTATTGCTATTATCGTCAACG

erm_NC_002745.2_871803_871973
GTCAGGCTAAATATAGCTATCTTATCG
TCAGTTACTGCTATAGAAATTGAT

erm_NC_002745.2_871666_871841
CATCCTAAGCCAAGTGTAGACTC
AAGATATATGGTAATATTCCTTATA

AC

TABLE 11

Additional regions may be used for additional discrimination and

characterization of organisms.

Probe Coordinates
Gene

peGFP_N1_730_925

CMY_X92508_126_301

TEM_X64523_2037_21
near tnpR, resolvase

91

TEM_J01749_2068_22
near ROP protein

39

TEM_AF091113_1529_

1699

TEM_J01749_1634_17

83

TEM_U36911_6901_70

69

TEM_GU371926_33909_
klcA, KlcA

34082

VIM_EU118148_2821_
qacEdeltal, quarternary ammomium compound-resistance

2961
protein QacEdeltal sull, dihydropteroate synthase SUL1

van_DQ018710.1_648

1_6652

van_DQ018710.1_676

4_6926

van_AY926880.1_364

0_3785

van_FJ545640.1_517_

690

van_AE017171.1_347

15_34859

van_FJ349556.1_560

1_5765

mecA_AM048806.2_15

74_1720

mecA_EF692630.1_23

9_405

mex_AF092566.1_371_

520

mex_AF092566.1_50_

193

mex_CP000438.1_487

178_487357

mex_NZ_AAQW0100000

1.1_461304_461466

erm_EU047809.1_79_

229

gyrB_NC_015663_145
EAE_24795, hemagluttinin domain-containing

5472_1455621
protein, gyrB, EAE_07020, DNA gyrase subunit B

gyrB_NC_010410_421
gyrB, ABAYE0004, DNA gyrase, subunit B

5_4366

gyrB_NC_005773_490
gyrB, PSPPH_0004, DNA gyrase subunit B

4_5052

gyrB_NC_016514_534
gyrB, EcWSU1_00004, DNA gyrase subunit B

3_5487

gyrB_NC_016603_263
gyrB, BDGL_002434, DNA gyrase, subunit B

1439_2631616

gyrB_NC_009436_436
gyrB, Ent638_0004, DNA gyrase subunit B

6_4524

gyrB_NC_009512_420
gyrB, Pput_0004, DNA gyrase subunit B

3_4373

TABLE 12

Additional arms

Probe Coordinates
Binding region 1
Binding region 2

peGFP_N1_730_925
GTGGTATGGCTGATTATGATCTAGAGT
GAGTTTGGACAAACCACAACTAGAA

CMY_X92508_126_301
AGTATCTTACCTGAAATTCCCTCAC
CCTCTCGTCATAAGTCGAATG

TEM_X64523_2037_2191
CAGTCCCTCGATATTCAGATCAGA
TTAACAATTTCGCAACCGTC

TEM_J01749_2068_2239
CAGCTGCGGTAAAGCTCATCA
CATAGTTAAGCCAGTATACACTC

TEM_AF091113_1529_1699
GTAACAACTTTCATGCTCTCCTAAA
CGGTAACTGATGCCGTATTT

TEM_J01749_1634_1783
CGTTTCCAGACTTTACGAAACAC
ACGTTGTGAGGGTAAACAAC

TEM_U36911_6901_7069
CATCATGTTCATATTTATCAGAGCTC
TAGATTTCATAAAGTCTAACACAC

TEM_GU371926_33909_34082
GTTTCCACATGGTGAACGGTG
AAACCTGTCACTCTGAATGTT

VIM_EU118148_2821_2961
GCTGTAATTATGACGACGCCG
CTCGGTGAGATTCAGAATGC

van_DQ018710.1_6481_6652
GTGTATGTCAGCGATTTGTCCAT
TGTCATATTGTCTTGCCGATT

van_DQ018710.1_6764_6926
GTCCACCTCGCCAACAATCAA
ATATCAACACGGGAAAGACCT

van_AY926880.1_3640_3785
GCGTGATTATCACGTTCGGCA
CTTGCAGATTTAACCGACAC

van_FJ545640.1_517_690
GGCTCGACTTCCTGATGAATACG
TGAAACCGGGCAGAGTATT

van_AE017171.1_34715_34859
CAACGATGTATGTCAACGATTTGT
ATTGCGTAGTCCAATTCGTC

van_FJ349556.1_5601_5765
GGCTCGGCTTCCTGATGAATAC
AGGCATGGTATTGACTTCATT

mecA_AM048806.2_1574_1720
CAGTATTTCACCTTGTCCGTAACC
GTTTACGACTTGTTGCATGC

mecA_EF692630.1_239_405
AATGTTTATATCTTTAACGCCTAAACT
ATGCTTTGGTCTTTCTGCAT

mex_AF092566.1_371_520
CTGGCCCTTGAGGTCGCGG
CGGTCTTCACCTCGACAC

mex_AF092566.1_50_193
GACGTAGATCGGGTCGAGCT
ACGGAAACCTCGGAGAATT

mex_CP000438.1_487178_487357
GGCGTACTGCTGCTTGCTCA
TGACGTCGACGTAGATCG

mex_NZ_AAQW01000001.1_461304_461466
CCTGTTCCTGGGTCGAAGCC
CTTCGGTCACCGCGGA

erm_EU047809.1_79_229
GTTTATAAGTGGGTAAACCGTGAAT
GAAACGAGCTTTAGGTTTGC

gyrB_NC_015663_1455472_1455621
GCCCTTTCAGGACTTTGATACTGG
TGTACGGAGACGGAGTTATCG

gyrB_NC_010410_4215_4366
ACACTGACCGATTCATCCTCGTG
CTTGAAAGTGCGTTAACAACC

gyrB_NC_005773_4904_5052
CGGAAGCCCACCAAGTGAGTAC
CGAAACCAGTTTGTCCTTAGTC

gyrB_NC_016514_5343_5487
ACCAGCTTGTCTTTAGTCTGAGAG
CTTTACGACGGGTCATTTCAC

gyrB_NC_016603_2631439_2631616
CATTGGTTTGTTCTGTTTGAGAGGC
GATTCATCTTCGTGAATTGTGAC

gyrB_NC_009436_4366_4524
GGACTTTGATACTGGAGGAGTCATA
TGTACGGAAACGGAGTTATCG

gyrB_NC_009512_4203_4373
ATGCTGGAGGAGTCGTACGTTT
GTCGCGCACACTAATAGATTC

TABLE 13

Plasmid regions can be used for identification purposes and can evidence

horizontal gene transfer.

Probe Coordinates
Gene

plasmids_NC_010660_187

035_187205

plasmids_NC_014232_550
parB1, ETEC1392/75_p1018_014, putative ParB plasmid

1_5677
stabilisation protein

plasmids_NC_011838_178
pCAR12_p001, putative ABC transporter

818_178996
subunit, tnpAb, pCAR12_p172, transposase

plasmids_FN554767_1301
EC042_pAA016, site-specific recombinase

7_13190

plasmids_NC_013655_115
ECSF_P1-0138, hypothetical protein

365_115542

plasmids_NC_013951_698

99_70067

plasmids_NC_007635_383
pCoo017, resD, pCoo052, putative resolvase

95_38566

plasmids_NC_009787_179
EcE24377A_C0013, putative methylase

46_18116

plasmids_NC_006671_562
near yfcB, O2R_81, YfcB

59_56438

plasmids_NC_014385_531

51_53310

plasmids_FN649418_5716
ETEC_p948_0010, IS66-family transposase

9_57339

plasmids_NC_005011_862
blaR1, MWP012, bla regulator protein blaRl

0_8785

plasmids_NC_014843_984
yfhA, p3521_p111, YfhA

13_98578

plasmids_NC_008490_516

5_5334

plasmids_NC_015963_147
Entas_4593, integrase catalytic subunit

516_147686

plasmids_NC_007365_100
resD, LH0122, site-specific recombinase

545_100708

plasmids_NC_009838_104
qacEdelta1, APECO1_O1R94, quaternary ammonium

163_104332
compound resistance protein

plasmids_NC_010409_397
pVM01_p034, insertion sequence 2 OrfA protein

68_39935

plasmids_NC_014233_503
ETEC1392/75_p557_00068

37_50492

plasmids_NC_013362_566
ECO26_p2-76, conjugal transfer nickase/helicase TraI

51_56805

TABLE 14

Plasmid arms

Probe Coordinates
Binding region 1
Binding region 2

plasmids_NC_010660_187035_187205
GCTGTCACCGTCCAGACGCTGTTGGC
TCCGTGCCTTCAAGCGCG

plasmids_NC_014232_5501_5677
GACTCCGCAGAATACGGCACCGTGCGCA
GCGTACAGGCCAGTCAGC

plasmids_NC_011838_178818_178996
GCTGTCCTGGCTGCAAGCCTGG
CCGAACTGCTGATGGACGT

plasmids_FN554767_13017_13190
GACAGCAGACTCACCGGCTGGTTCCGCT
GCAAGATGCTGCTGGCCACACTG

plasmids_NC_013655_115365_115542
GACAGAACAAGTTCCGCTCCGG
CACGGATACGCCGCGCAT

plasmids_NC_013951_69899_70067
GAACGTCTGGCGCTGGTCGCCTGCC
GCACAGGTGCTGACGTGGT

plasmids_NC_007635_38395_38566
AATCCAGGTCCTGACCGTTCTGTCCGT
ACCTCCGTTGAGCTGATGGA

plasmids_NC_009787_17946_18116
GAGGTGGCCAACACCATGTGTGACC
GACGCCGGTATATCGGTATCGAGCT

GCT

plasmids_NC_006671_56259_56438
GAAGTGCCGGACTTCTGCAGA
GCACGGCCTGATGGAGGCCGC

plasmids_NC_014385_53151_53310
GCTAATCGCATAACAGCTAC
CATCACGTAACTTATTGATGATATT

plasmids_FN649418_57169_57339
GCTGCGGTATTCCACGGTCGGCC
GCAGGAACGCTGCCTGTGGTC

plasmids_NC_005011_8620_8785
GAATCAATTATCTTCTTCATTATTGAT
CTGCGGCTCAACTCAAGCA

plasmids_NC_014843_98413_98578
GTCACACGTCACGCAGTCC
GCATTCATGGCGCTGATGGC

plasmids_NC_008490_5165_5334
GTGTTACTCGGTAGAATGCTCGCAAGG
ACTAGATGACATATCATGTAAGTT

plasmids_NC_015963_147516_147686
CGGAACTGCCTGCTCGTAT
AACGATATAGTCCGTTAT

plasmids_NC_007365_100545_100708
GCTCTCCGACTCCTGGTACGTCAG
GCGCGCATTAATGAAGCAC

plasmids_NC_009838_104163_104332
GATGTTGCGATTACTTCGCCAACTATTG
GCTGTAATTATGACGACGCCG

plasmids_NC_010409_39768_39935
GCAATACCAGGAAGGAAGTCTTACTG
GTCATTGGAGAACAGATGATTGATGT

plasmids_NC_014233_50337_50492
GTATCGCCACAATAACTGCCGGAA
AACGATATAGTCCGTTATG

plasmids_NC_013362_56651_56805
GTGAAGCGCATCCGGTCACC
ATGGCATAGGCCAGGTCAATAT

TABLE 15

A list of antibiotic resistance genes for which probes can be used to identify,

distinguish and/or sequence

Source Sample ID

CARB

CMY

CTX-M

GES

IMP

KPC

NDM

Other ampC

OXA

PER

SHV

VEB

VIM

ermA

vanA

vanB

mecA

mexA

In some embodiments, the oligonucleic acid probes provided by the invention are molecular inversion probes (MIP). Advantages that the MIP probes described herein offer over PCR include:

1) Multiplexing: there are published studies using 10k+ inversion probes to genotype humans including: http://www.ncbi.nlm.nih.gov/pubmed/17934468 (Porreca et. al.), 55k probes http://www.ncbi.nlm nih gov/pmc/articles/PMC2715272/?tool=pubmed 30k probes http://www.ncbi.nlm.nih.gov/pubmed/19329998 10k probes.

This offers a huge capability to expand panels. First uses might be to capture more rare strains/variants that work poorly with current PCR primers. Later uses might involve genotyping HIV and human loci as well as testing for diseases common in HIV patients—such a test can still be performed in a single tube with minimal per-test increase in reagents cost.

2) Specificity: the probes described herein are less likely to produce off-target products because the two probe arms must bind together. This provides a thermodynamic advantage for on-target binding compared to mis-priming. Furthermore, the exonuclease step will eliminate extension products that occur when only a single probe arm binds.

PCR primers can create long extension products that serve as templates for mis-priming in later rounds. This is particularly a problem when there's lots of background (e.g. human) DNA compared to the target sequence; such as when the exonuclease step didn't remove all of the template and the amplification/barcoding primers misprimed against human DNA. This ends up wasting reads and would have been worse had enrichment for the circularized probes was not being performed. Preventing such reads in a PCR-only system is difficult.

3) Design optimization: the large published datasets provide good training data for a probe picking algorithm. These large datasets can be useful for picking probe sets that will work reliably and with uniform efficiency. Furthermore, we can generate a set of 10k+ probes on a microarray to generate datasets using preferred enzymes. Currently being tested is the entire set of 10k+ probes in a single reaction and then analyzing the read counts to see what made a good probe and what didn't.

Understanding the probe behavior is important for pathogens as it helps to understand the sensitivity and specificity, particularly when considering rare strains or the possibility of previously unknown strains. Pathogenica has thermodynamic models of probe behavior that provide quantitative predictions of how well a probe will work against a target.

4) Simplicity: the probe protocol can be one-tube all the way through, adding reagents until all of the samples are pooled. PCR protocols often require multiple tubes to purify intermediate or final product from the template (e.g., Ampliseq requires 7, PCR+ Nextera likely requires 3+). Also being used are standard reagents (enzymes+oligos) and equipment (thermal cycler).

The following references are incorporated by reference in their entirety: Roberts R R, et al., “Costs attributable to healthcare-acquired infection in hospitalized adults and a comparison of economic methods,” Medical Care, 48(11):1026-1035, November 2010; Scott, R. D., II., “The Direct Medical Costs of Healthcare-Associated Infections in U.S. Hospitals and the Benefits of Prevention,” U.S. Centers for Disease Control and Prevention, March 2009; and Edwards, J. R., et al., National Healthcare Safety Network (NHSN) report: data summary for 2006 through 2008, issued December 2009, American Journal of Infection Control. 37:783-805, December 2009.

It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description at least 1, 2, 3, 4, or 5 also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.

For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited. Where any conflict exits between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GeneIDs, Unigene IDs, or HomoloGene ID, or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures) are hereby incorporated by reference in their entirety.

Headings used in this application are for convenience only and do not affect the interpretation of this application.

Preferred features of each of the aspects provided by the invention are applicable to all of the other aspects of the invention mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features (e.g., elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the invention including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed invention piecemeal without departing from the invention. For example, for material is that are disclosed, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, elements of a composition of matter and steps of method of making or using the compositions.

The forgoing aspects of the invention, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art—thus to the extent an element is described in one or more references known to the person having ordinary skill in the art, they may be excluded from the claimed invention by, inter alia, a negative proviso or disclaimer of the feature or combination of features.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Examples

Procedure:

- 1) Remove the DxSeq Kit from the −20° C. freezer.
- 2) Remove one Reagent Set Pack from the DxSeq Kit, and place the tubes on ice.
- 3) Remove two blue FrameStrips and matching strip caps from the kit. [The Break-A-Way Plate with primers is not needed at this point in the protocol.]
- 4) Label the FrameStrips and the strip caps #1 and #2 with a permanent marker. [Both the FrameStrips and strip caps should be labeled to avoid cross-contamination during subsequent handling steps.]
- 5) Return the kit to the −20° C. freezer for later use.
- 6) After the components have thawed, pulse-spin any droplets from the cap or sidewalls to the bottom of the tubes using a microcentrifuge.
- 7) Using barrier pipette tips, prepare 75 μL Hybridization Master Mix for 12 samples and 2 controls, as follows:
  - a. 22.5 μL 10× Buffer A
  - b. 15 μL MIP Probe mixture
  - c. 37.5 μl of nuclease-free water
- 8) Using barrier pipette tips, pipette 5 μL of Hybridization Master Mix into wells A-G of two blue FrameStrip PCR 8-strips (n=14 wells). [Do not pipette Hybridization Master Mix into wells H: these are reserved for negative controls.]
- 9) Being very careful not to cross-contaminate the wells, add 10 μL of each DNA sample to the A-F wells of the two FrameStrips (n=12 wells). [Do not pipette your DNA samples into the G & H wells: these four wells are reserved for control reactions.]
- 10) Add 10 μL of nuclease-free water to the G wells (n=2 wells). These will serve as the “no target DNA” negative controls.
- 11) Add 13.5 of nuclease-free water and 1.5 of 10× Buffer A to the H wells (n=2 wells). These will serve as the “no probe” negative controls.
- 12) Seal the two FrameStrips with the flat strip caps.
- 13) Vortex the sealed FrameStrips briefly to mix the contents; and then pulse-spin down the contents in a microcentrifuge with a rotor that accommodates 8-well strip PCR tubes.
- 14) Enter the following program into a thermocycler, using the heated lid option.

a.
94° C., 10 min
Hybridization

b.
Ramp to 60° C., 0.1° C./sec

c.
60° C., 10 min

d.
60° C. hold

e.
60° C., 10 min
Extension

f.
15° C. hold

g.
94° C., 2 min
Exonuclease cleanup

h.
37° C. hold

i.
37° C., 30 min

j.
94° C., 15 min

k.
4° C. hold

- 15) Place the sealed FrameStrips in the thermocycler; and begin the hybridization portion of the MIP Program.
- 16) While the hybridization is underway, prepare the Polymerase/Ligase Master Mix on ice:
  - a. 5 μL Polymerase
  - b. 5 μL 10× Buffer A
  - c. 1 μL Ligase
  - d. 1.25 μL dNTPs
  - e. 37.75 μL nuclease-free water
- 17) When the hybridization reaction reaches the 60° C. hold step (approximately 26 minutes into the program), add 2 μL of the Polymerase/Ligase Master Mix to every well (n=16 wells).
- 18) Reseal the FrameStrips with the same strip caps as before and mix. [Special care needs to be taken not to cross-contaminate the samples.]
- 19) Advance the thermocycler to the next step in the MIP Program (60° C. for 10 min).
- 20) When the thermocycler reaches the 15° C. hold step, advance the thermocycler to the next step (94° C. for 2 min) in the MIP Program.
- 21) When the thermocycler reaches the 37° C. hold step, immediately add 1 μL of Exonuclease to each sample.
- 22) Reseal the FrameStrips with the same strip caps as before and mix. [Special care needs to be taken not to cross-contaminate the samples.]
- 23) Advance the thermocycler to the next step (37° C. for 30 min) in the MIP Program.
- 24) While the reactions are incubating at 37° C., prepare the amplification mix:
  - a. (components of the PCR reaction)
- 25) Remove the Purple Break-A-Way 96 Well Plate containing PCR primers from the −20° C. freezer. Break off three columns from the left side of the plate.
- 26) Return the unused portion of the Break-A-Way 96 Well Plate to the freezer (before the primers thaw).
- 27) When the thermocycler reaches the 4° C. hold, add 2.5 μL of tube-specific barcoding primer and 29.5 μL of amplification mix.
- 28) Begin the PCR Amplification Program on the thermocycler:
  - a. 94° C., 3 min
  - b. 30 cycles of:
    - i) 94° C., 15 sec
    - ii) 60° C., 15 sec
    - iii) 72° C., 30 sec
  - c. 72° C., 4 min
  - d. 4° C. hold
- 29) Purify the PCR amplicons using AMPure beads (Beckman Coulter).
- 30) Proceed to the IonTorrent Template preparation workflow.

Pathogenica Software installed on the Ion Torrent PGM reports the results.

Method and System for Detection of an Organism

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