Sample extraction efficiency estimation in nucleic acid extraction method development in molecular diagnostic workflows

Abstract

Methods of estimating nucleic acid extraction efficiency in a sequencing workflow for detection of at least one target are disclosed. The methods utilize a process calibrant added to a sample prior to nucleic acid extraction, and a process control added prior to performing a PCR reaction. Also disclosed are compositions, kits, and systems containing the process calibrant and process control.

Description

TITLESAMPLE EXTRACTION EFFICIENCY ESTIMATION IN NUCLEIC ACID EXTRACTION METHOD DEVELOPMENT IN MOLECULAR DIAGNOSTIC WORKFLOWSREFERENCE TO RELATED APPLICATIONS

[0001] The subject application claims benefit under 35 USC § 119(e) of US Provisional Application No. 63 / 675,961, filed July 26, 2024. The entire contents of the above-referenced patent application(s) are hereby expressly incorporated herein by reference.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.BACKGROUND

[0003] Metagenomic next-generation sequencing (mNGS) is a novel useful strategy that is increasingly used for universal pathogen detection in hospitals and diagnostic labs. Reliable nucleic acid (NA) extraction is considered critical for the detection sensitivity of such a sequencing-based workflow. Sample preparation and extraction steps in such methods are designed to be non-specific to capture all nucleic acids regardless of their source, including (but not limited to) intact bacteria, viruses, parasites, and fungi, as well as their free-floating nucleic acid content. Such sample extraction protocols often suffer from inadequacies such as incomplete cell lysis, incomplete DNA binding, inadequate washing of inhibitors which can interfere with the library preparation steps, and incomplete elution of nucleic acids. The losses in the sample extraction process have serious implications on the analytical sensitivity and also on the clinical sensitivity of the sequencing-based workflows.

[0004] Currently used methods of nucleic acid extraction efficiency estimation involve assessment of samples separately from downstream sequencing, and analysis by methods such as spectroscopic analysis of nucleic acid quantity and quality, or alternatively using quantitative real-time PCR. However, even independent nucleic acid quantification does not give an indication of nucleic acid yields relative to starting material (i.e., actual efficiency of the extraction) from an unknown sample, because the level of starting material prior to extraction is not known.

[0005] The other known molecular diagnostic solutions in the literature determine process success rate by using overall process controls which are generally one sample processing control (SPC in Cepheid or IC in Versant kPCR assays) that will serve as sample preparation control and amplification controls. A threshold Ct value is set for such controls, and a Ct greater than the threshold would indicate a overall workflow failure.

[0006] In sequencing-based workflows like Karius test, whole assay internal normalization control (WINC) molecules are used to monitor the yield and quality of the entire workflow. These are synthetic DNA molecules and are spiked in plasma at defined quantity. By counting the number of the unique WINC molecules in the sequencing data obtained, the whole assay yield was monitored. Samples that failed to achieve a minimum number of unique WINC molecule reads for any reason (for example, poor yield due to ANY workflow step) results in a workflow repeat. (Blauwkamp et al. (2019) Nat Microbiol, 4:663-674).

[0007] In many of these sequencing-based workflows, accuracy of the method needs to be validated. In other words, when the workflow reports a negative result, there is a need to validate whether the result is a true negative or a false negative. False negatives can arise if there was a processing error upstream to sequencing. In such scenarios, quantitative controls are added to the workflow and can help validate and discern between the false negatives and true negatives.

[0008] Thus, there is a need in the art for new and improved methods for determining target losses (i.e., a percentage of starting nucleic acid successfully extracted from a sample and utilized in sequencing workflows) and also for validating the workflow in order to discern a true positive from false positive.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0009] FIG. 1 illustrates a sample (or nucleic acid) extraction efficiency estimation method of one non-limiting embodiment of the present disclosure.

[0010] FIG. 2 graphically depicts experimental data for calibrating an output ratio of process calibrant to process control with process calibrants in a PCR at 100 cp, 1000 cp, and 10000 cp at a fixed process control input.

[0011] FIG. 3 contains a block diagram of one non-limiting embodiment of a system constructed in accordance with the present disclosure.DETAILED DESCRIPTION

[0012] Before explaining at least one embodiment of the present disclosure in detail, it is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of the components or steps or methodologies set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting in any way.

[0013] Independent of the grammatical term usage, individuals with male, female, or other gender identities are included within the term.

[0014] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0015] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0016] All of the non-transitory computer readable mediums, results interfaces, automated analyzers, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the non-transitory computer readable mediums, results interfaces, automated analyzers, and / or methods disclosed herein have been described in terms of particular embodiments, it will be apparent to those of skillin the art that variations may be applied to the non-transitory computer readable mediums, results interfaces, automated analyzers, and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the inventive concept(s) as defined by the present disclosure and / or appended claims.

[0017] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0018] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0019] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, includes ranges of 1-20, 10-50, 50-100, 100-500, and 500-1,000, for example. Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10 includes 9, 8, 7, etc. all the way down to the number one (1).

[0020] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc.The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0021] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0022] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0023] Throughout this application, the terms "about" and "approximately" are used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method being employed to determine the value, or the variation that exists among the study subjects. That is, the terms "about" and "approximately" and variations thereof are intended to include not only the exact value qualified by the term, but to also include some slight deviations therefrom, such as deviations caused by measuring error, manufacturing tolerances, wear and tear on components or structures, settling or precipitation of cells or particles out of suspension or solution, chemical or biological degradation of solutions over time, stress exerted on structures, and combinations thereof, for example. In particular, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent,or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0024] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, unless otherwise noted, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may also include other elements not expressly listed or inherently present therein.

[0025] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0026] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0027] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodimentis included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0028] The term "isolated" as used herein means that a biological material, such as but not limited to a nucleic acid or protein, has been removed from its original environment in which it is naturally present. For example, a polynucleotide present in a plant, mammal or animal is present in its natural state and is not considered to be isolated. The same polynucleotide separated from the adjacent nucleic acid sequences in which it is naturally inserted in the genome of the plant or animal is considered as being "isolated".

[0029] The term "isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with the biological activity and which may be present, for example, due to incomplete purification, addition of stabilizers or mixtures with pharmaceutically acceptable excipients, and the like.

[0030] The term "purified" as used herein means at least one order of magnitude of purification is achieved compared to the starting material or of the natural material, for example but not by way of limitation, two, three, four or five orders of magnitude of purification of the starting material or of the natural material. Thus, the term "purified" as utilized herein does not necessarily mean that the material is 100% purified, and therefore such term does not exclude the presence of other material(s) present in the purified composition.

[0031] The term "polynucleotide" or "oligonucleotide" as used herein will be understood to refer to a polymer of two or more nucleotides. Nucleotides, as used herein, will be understood to include deoxyribose nucleotides and / or ribose nucleotides, as well as artificial variants thereof. The term polynucleotide also includes single-stranded and double-stranded molecules.

[0032] As used herein, the terms "nucleic acid segment," "nucleic acid sequence," "nucleotide segment," "nucleotide sequence," "DNA sequence," and "DNA segment" are used interchangeably and refer to, for example, a synthetic DNA molecule or a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a "purified" or "isolated" nucleotide sequence as used herein refers to a DNA segment that is isolated away from, or purified free from, unrelated genomic DNA. Included within theseterms are DNA segments and smaller fragments of such segments, and also recombinant vectors including, for example (but not by way of limitation), plasmids, cosmids, phage, viruses, and the like.

[0033] As used herein, the phrases "associated with" and "coupled to" include both direct association / binding of two moieties to one another as well as indirect association / binding of two moieties to one another. Non-limiting examples of associations / couplings include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example.

[0034] Circuitry, as used herein, may be analog and / or digital components, or one or more suitably programmed processors (e.g., microprocessors) and associated hardware and software, or hardwired logic. Also, a "processing component" may perform one or more functions. The term "processing component," may include hardware, such as a processor (e.g., microprocessor), an application specific integrated circuit (ASIC), field programmable gate array (FPGA), a combination of hardware and software, and / or the like.

[0035] Software may include one or more computer readable instructions that when executed by one or more processing components cause the processing component to perform a specified function. It should be understood that the algorithms described herein may be stored on one or more non-transitory memory. Exemplary non-transitory memory may include random access memory, read only memory, flash memory, and / or the like. Such non-transitory memory may be electrically based, optically based, and / or the like.

[0036] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where arange of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value.

[0037] It is to be further understood that, as used herein, the term "user" includes but is not limited to a human being, and may comprise, a computer, a server, a website, a processor, a network interface, a human, a user terminal, a virtual computer, combinations thereof, and the like, for example.

[0038] The term "calibration parameters" as used herein refers to a collection of data points or one or more functions used to derive a collection of data points that correlates the signals from the sensor to known analyte concentrations. The calibration parameters can be derived by a calibration algorithm, such as a linear algorithm, a spline-based algorithm, exponential algorithm, a least squares algorithm, a logarithmic algorithm, or the like that is configured to fit a function to at least two calibration points.

[0039] The term "calibration logic" as used herein refers to the program logic used by a processing component to interpret data measured by one or more electrodes. In particular, the term "calibration logic" is the program logic used by a processing component to interpret data from an electrochemical sensor having at least a working electrode and a reference electrode.

[0040] The term "sample" as used herein will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluidic biological samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum (such as, but not limited to, bronchoalveolar lavage sputum), cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and the like, as well as any combinations thereof.

[0041] Certain non-limiting embodiments of the present disclosure are directed to a method of estimating nucleic acid extraction efficiency in a sequencing workflow for detection of at least one target. The method includes the following steps: (a) adding a specific amount of a process calibrant to a volume of biological sample that may contain at least one target to be detected, wherein the process calibrant comprises synthetic DNA; (b) performing a nucleic acid extraction process and eluting nucleic acid from the biological sample and the process calibrant in a specific eluate volume; (c) adding a specific amount of a process controlto a portion or all of the eluate volume to form an eluate mixture, wherein the process control comprises synthetic DNA, and wherein the process calibrant and process control have the same primer binding sites, the same length, and the same base compositions, but different sequences; (d) performing a PCR reaction on the eluate mixture to provide a PCR product pool; (e) performing a sequencing reaction on the PCR product pool, wherein an output of the sequencing reaction comprises process calibrant reads and process control reads, and wherein the output further comprises at least one target reads if the at least one target is present in the biological sample; (f) determining a positive or negative detection or concentration result for the at least one target in the biological sample based on the at least one target reads present in the output of the sequencing reaction; and (g) calculating an extraction efficiency for the nucleic acid extraction using Formula (I):Extraction EfficiencyFormula (I) wherein Cs represents an amount of process calibrant reads from the output of the sequencing reaction, Ps represents an amount of process control reads from the output of the sequencing reaction, M represents a maximum amount of process calibrant present in the PCR template or the eluate mixture if extraction was 100% efficient, and P represents a number of copies of process control DNA added in step (c); and ( h ) comparing the extraction efficiency calculated in step (f) to a threshold value. This method allows for discerning true negatives from false negatives based on the sample extraction efficiency calculation. False negatives result from process failure.

[0042] In certain non-limiting embodiments, M in Formula (I) is calculated using Formula (II):wherein C represents the specific amount (in copies / ml) of process calibrant added in step (a), B represents the volume of the biological sample to which the process calibrant is added in step (a), Vi represents the specific eluate volume from step (b), and V2 represents an amount of the eluate volume to which the process control is added in step (c).

[0043] This equation assumes that the amplification efficiency of the PCRs of process calibrant and process control are same, as the same primers are used for both the process calibrant and process control, and as the process calibrant and process control have identical base compositions and identical primer binding sites (although different internal base sequences for differentiation). However, in certain non-limiting embodiments, a linear response between both these amplifications may not be seen. In these instances, assuming an identical amplification efficiency of the process calibrant and process control would lead to overestimation of extraction efficiencies. Hence, the method may optionally include the step of calibrating the output ratio of process calibrant to process control. In these instances, the analysis software is equipped with the ratio calibrant function, and can be calculated, for example, from experimental data such as is shown in FIG. 2.

[0044] In certain non-limiting embodiments, the method further comprises the step(s) of: (ii) flagging a negative detection or concentration result for the at least one target in step (f) if the extraction efficiency calculated in (g) is below a threshold value; and / or (iz) repeating steps (a)-(h) if the extraction efficiency calculated in (g) is below the threshold value. In addition, the method may optionally further comprise the step of: (j) reporting the at least one target positive or negative detection or concentration result of step (f) if the extraction efficiency calculated in (g) is above the threshold value.

[0045] Any type of target or target analyte may be detected in the method. For example (but not by way of limitation), the target to be detected may be at least one pathogen (or the free-floating nucleic acid content thereof), such as (but not limited to), a bacteria, a virus, a parasite, a fungus, and / or any combination thereof. In another non-limiting example, the target to be detected may include mammalian cells or the free-floating nucleic acid content thereof. In a specific (but non-limiting) example, the target to be detected may include at least one human gene.

[0046] In certain particular (but non-limiting) embodiments, one or more of the steps of the methods are performed by an automated sequencing system. In addition, one or more steps may be performed by analysis software of the automated sequencing system. The automated sequencing system can be any next generation sequencing-based system based on Oxford Nanopore Technology or Illumina Sequencing Technology, which is based on sequencing by synthesis, or any other commercially known sequencing technology.

[0047] In certain particular (but non-limiting) embodiments, step (a) of the method may optionally further comprise adding at least one quantitation control to the biological sample and process calibrant. In a particular (but non-limiting) example, the at least one quantitation control is a synthetic DNA.

[0048] The nucleic acid may be eluted from the biological sample in step (b) in any specific eluate volume that is sufficient for performing the remaining steps of the methods. Certain non-limiting examples of specific eluate volumes that may be utilized in accordance with the present disclosure include about 1 pl, about 5 pl, about 10 pl, about 20 pl, about 30 pl, about 40 pl, about 50 pl, about 60 pl, about 70 pl, about 80 pl, about 90 pl, about 100 pl, about 125 pl, about 150 pl, about 175 pl, about 200 pl, about 225 pl, about 250 pl, about 275 pl, about 300 pl, about 325 pl, about 350 pl, about 375 pl, about 400 pl, about 425 pl, about 450 pl, about 475 pl, about 500 pl, about 525 pl, about 550 pl, about 575 pl, about 600 pl, and the like, as well as any ranges formed from two of the above values (e.g., a range of from about 1 pl to about 600 pl, a range of from about 10 pl to about 500 pl, a range of from about 20 pl to about 400 pl, etc.).

[0049] The process calibrant and the process control may be provided with any length that allows the process calibrant and process control to function as described herein in the estimation of nucleic acid extraction efficiency in a sequencing workflow. Non-limiting examples of lengths that may be utilized include about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 105 bp, about 110 bp, about 115 bp, about 120 bp, about 125 bp, about 130 bp, about 135 bp, about 140 bp, about 145 bp, about 150 bp, about 155 bp, about 160 bp, about 165 bp, about 170 bp, about 175 bp, about 180 bp, about 185 bp, about 190 bp, about 195 bp, about 200 bp, about 205 bp, about 210 bp, about 215 bp, about 220 bp, about 225 bp, about 230 bp, about 235 bp, about 240 bp, about 245 bp, about 250 bp, and the like, as well as any ranges formed from two of the above values (e.g., a range of from about 50 bp to about 250 bp, etc.).

[0050] The process calibrant and the process control may be provided with any G / C content that allows the process calibrant and process control to function as described herein in the estimation of nucleic acid extraction efficiency in a sequencing workflow. Non-limiting examples of G / C contents that may be utilized include about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%,about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 55%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 65%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 45%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, and the like, as well as any ranges formed from two of the above values (e.g., a range of from about 30% to about 80%, etc.).

[0051] The 5' and 3' ends of the process calibrant and the process control may be provided with primer binding sites of any length that allows the process calibrant and process control to function as described herein in the estimation of nucleic acid extraction efficiency in a sequencing workflow. Non-limiting examples of primer binding site lengths that may be utilized include about 15 bp, about 16 bp, about 17 bp, about 18 bp, about 19 bp, about 20 bp, about 21 bp, about 22 bp, about 23 bp, about 24 bp, about 25 bp, about 26 bp, about 27 bp, about 28 bp, about 29 bp, about 30 bp, about 31 bp, about 32 bp, about 33 bp, about 34 bp, about 35 bp, about 36 bp, about 37 bp, about 38 bp, about 39 bp, about 40 bp, about 41 bp, about 42 bp, about 43 bp, about 44 bp, about 45 bp, about 46 bp, about 47 bp, about 48 bp, about 49 bp, about 50 bp, and the like, as well as any ranges formed from two of the above values (e.g., a range of from about 15 bp to about 50 bp, etc.).

[0052] As stated herein above, while the process calibrant and process control have identical lengths, primer binding sites, and G / C contents, at least a portion of a sequence of the process calibrant is different from a sequence of the process control. The differences between the two sequences is sufficient to allow for distinction between the process calibrant reads and the process control reads in the output of the sequencing reaction. In certain nonlimiting embodiments, a sequence of the process calibrant is produced by randomly scrambling at least a portion of an interior of the amplified region of the process control between the primer binding sites.

[0053] In certain particular (but non-limiting) embodiments, the process control and process calibrant have sequence identities that are similar to the at least one target pathogen's genome. That is, the process control and process calibrant may have lengths and G / C contents similar to that of the target pathogen sequences being detected in the sequencing workflow. In addition, the primers utilized in amplifying the process control andthe process calibrant may be very similar in terms of base composition compared to the one or more target pathogens to be detected.

[0054] In addition, in certain particular (but non-limiting) embodiments, targeted sequencing may utilize universal primers.

[0055] While the methods described herein above reference the detection of "at least one target," it will be understood that many different targets may be detected within each sequencing workflow. For example, the sequencing workflow may contain a multiplex PCR with about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about48, about 49, about 50 (or more) primer sets for detection of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50 (or more) target sequences.

[0056] In certain particular (but non-limiting) embodiments, the sequencing workflow is a metagenomic next-generation sequencing workflow used for detection of at least one pathogen.

[0057] In certain particular (but non-limiting) embodiments, the sequencing workflow described herein is capable of detecting as low as about 10 CFU of pathogen in a biological sample.

[0058] Certain non-limiting embodiments of the present disclosure are directed to kits or systems for use in methods of estimating nucleic acid extraction efficiency in a sequencing workflow for detection of at least one target. The kit or system comprises: (i) at least one of any of the process calibrants disclosed or otherwise contemplated herein, for adding to a biological sample prior to nucleic acid extraction; and (ii) at least one of any of the process controls disclosed or otherwise contemplated herein, for adding prior to performing a PCR reaction. As disclosed herein above, the process calibrant and process control have the sameprimer binding sites, the same length, and the same base compositions, and wherein at least a portion of a sequence of the process calibrant is different from a sequence of the process control.

[0059] The compositions / reagents of the kits or systems may be provided in any form that allows them to function in accordance with the present disclosure. For example, but not by way of limitation, each of the reagents may be provided in liquid form and disposed in bulk and / or single aliquot form within the kit or system. Alternatively, in a particular (but nonlimiting) embodiment, one or more of the reagents may be disposed in the kit or system in the form of a single aliquot lyophilized reagent. The use of dried reagents in kits / microfluidics devices is described in detail in US Patent No. 9,244,085 (Samproni), the entire contents of which are hereby expressly incorporated herein by reference.

[0060] In addition to the compositions / reagents described in detail herein above, the kits or systems may further contain other reagent(s) for conducting any of the methods described or otherwise contemplated herein. For example, but not by way of limitation, the kits or systems may include at least one quantitation control, forward and reverse primers for PCR amplification of the process calibrant and process control, and / or forward and reverse primers for PCR amplification of the at least one target. The nature of these additional reagent(s) will depend upon the particular assay format, and identification thereof is well within the skill of one of ordinary skill in the art; therefore, no further description thereof is deemed necessary.

[0061] Also, the compositions / reagents present in the kits or systems may each be in separate containers / compartments, or various compositions / reagents can be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the compositions / reagents. For example (but not by way of limitation), the forward and reverse primers for PCR amplification of the process calibrant and process control (and / or optionally the forward and reverse primers for PCR amplification of the at least one target) may be present in the same container / compartment as the process control. In addition, the kit or system may include a device in which one or more of the compositions / reagents are disposed.

[0062] The relative amounts of the various compositions / reagents in the kits or systems can vary widely to provide for concentrations of the compositions / reagents that substantially optimize the reactions that need to occur during the methods and further to optimize substantially the sensitivity and selectivity of an assay. Under appropriate circumstances, oneor more of the compositions / reagents in the kit or system can be provided as a dry powder, such as a lyophilized powder, and the kit or system may further include excipient(s) for dissolution of the dried reagents; in this manner, a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present disclosure can be obtained from these compositions. Positive and / or negative target controls may also be included with the kit or system. In addition, the kit or system can further include a set of written instructions explaining how to use the kit or system. A kit or system of this nature can be used in any of the methods described or otherwise contemplated herein.

[0063] In certain particular (but non-limiting) embodiments, the system further comprises any of the automated sequencing systems disclosed or otherwise contemplated herein that are capable of performing at least a portion of any of the methods disclosed or otherwise contemplated herein. In a particular (but non-limiting) embodiment, the automated sequencing system comprises analysis software capable of performing one or more steps of any of the methods disclosed or otherwise contemplated herein. For example, but not by way of limitation, the extraction efficiency calculation may be embedded in the software of the automated sequencing system, and a threshold efficiency percentage is set for the workflow. The software indicates, based on the threshold, if the sample is a true negative or a false negative. In addition, the extraction efficiency may indicate the process failure of the workflow if the extraction efficiency is below a threshold. The threshold is set by the manufacturer based on the average extraction efficiency value calculated over many runs during development.

[0064] In certain particular (but non-limiting) embodiments, the analysis software is equipped with the extraction efficiency calculation of Formula (II), as well as a threshold for the same. The analysis software may also be equipped with the ratio calibrant function, to correct for any differences between the amplification efficiencies of process calibrant and process control.EXAMPLES

[0065] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

[0066] The present disclosure is directed to compositions, kits, systems, and devices for estimating nucleic acid extraction efficiency in a sequencing workflow, such as (but not limited to) a metagenomic next-generation sequencing (mNGS) workflow. Metagenomic nextgeneration sequencing (mNGS) is a novel useful strategy that is increasingly used for pathogen detection in hospitals and diagnostic labs. Reliable nucleic acid (NA) extraction is considered critical for the success of sequencing. Sample preparation and extraction steps in such methods are designed to be non-specific to capture all nucleic acids regardless of their source, which includes but not limited to bacteria, viruses, parasites, and fungi. Such sample extraction protocols often suffer from inadequacies including incomplete cell lysis, incomplete DNA binding, inadequate washing of inhibitors which can interfere with the library preparation steps, and incomplete elution of nucleic acids. The losses in the sample extraction step have serious implications on the analytical sensitivity and in turn also on the clinical sensitivity of the sequencing-based workflows.

[0067] Estimating sample extraction (or NA) efficiency and using this parameter as a thresholding feature in the sequencing analysis software improves the robustness of the workflow.

[0068] The present disclosure is the first embodiment of an integrated commercial solution to automated and integrated nucleic acid extraction efficiency estimation concomitant with a NGS or molecular assay. Generally, samples would be assessed separately from downstream sequencing and analysis by methods such as spectroscopic analysis of nucleic acid quantity and quality, or alternatively using quantitative real-time PCR. However, even independent nucleic acid quantification does not give an indication of nucleic acid yields relative to starting material (i.e., actual efficiency of the extraction) from an unknown sample, because the level of starting material prior to extraction is not known. The presently disclosed compositions, kits, systems, and methods provide an integrated solution to the problem of the percentage of starting nucleic acid successfully extracted by extrapolation from the measurement of added quantitative synthetic controls.

[0069] In certain non-limiting embodiments, the method involves including a process calibrant and a process control in the reagents included in the sequencing workflow. Process calibrant is added to the starting sample, i.e., before sample extraction, and a process control is added at the PCR stage. Both calibrant and process control have the same primer binding sites and the same length and same base compositions. The sequence of the process calibrantY1is produced by randomly scrambling the interior of the amplified region of process control so that the process calibrant and process control differ from one another by a sufficient, detectable amount. The quantity of the process control in the PCR is known, and the quantity of process calibrant to be added to the starting sample is known. Knowing these quantities in addition to the sample volume, elution volume, and eluate volume to be used per PCR tube, allows one to estimate the extraction efficiency by also considering output sequence reads from process control and process calibrant.

[0070] A method 10 of estimating extraction efficiency is represented in FIG. 1. The steps of the method 10 are as follows.

[0071] (1) A process calibrant DNA 12 is added into a sample 14 (for example, but not by way of limitation, blood shown in the illustration and used as an example to describe this workflow; in particular, this example utilized 3000 pl blood). However, blood is utilized solely for purposes of illustration; the extraction efficiency estimation can be applied in a similar format for any other sample type in a sequencing workflow. The amount of process calibrant 12 added is known beforehand. Other than process calibrant 12, there is at least one target 16 to be detected present in the sample 14, and at least one quantitation control 18 is also added.

[0072] (2) The sample 14 then goes through a NA extraction process 20, and the collectiveNA from all the components in the blood sample is eluted in a certain volume 22 (an eluate volume of 300 pl was utilized in this particular example, but the volume can vary from about 10 to about 500 pl). Depending on the extraction efficiency of the method, a percentage of process calibrant DNA 12 added in step (1) is eluted in the eluate volume 22, along with target DNA 16 and quantitation control DNA 18.

[0073] (3) A portion 22a of the eluate volume (19 pl was utilized in this example, but the portion of the eluate volume utilized can vary depending on the number of amplification reactions performed) is then carried forward to one PCR reaction 30. Before the PCR reaction 30 is initiated, a known amount of process control DNA 24 is added to the eluate volume portion 22a of the amplification reaction. After the amplification reaction 30 is performed, the entire reaction volume is now prepared into a library to be added to the sequencing reaction 40.

[0074] (4) An output of the sequencing reaction will contain target reads 42, process calibrant reads 44 (referred to in Formula (I) as "Cs"), process control reads 46 (referred to inFormula (I) as "Ps"), as well as quantitation control reads 48. Using these parameters, the extraction efficiency is calculated using Formulas (I) and (II). In Formulas (I) and (II): Cs = Number of sequence reads for Process Control Calibrant; Ps = Number of sequences for Process Control; M = Maximum process calibrant added as a template to the PCR reaction 30 (if extraction was 100% efficient); P = Copies of Process Control in PCR; C = Copies / mL of Process Calibrant added to blood sample; B = Blood sample volume (in pL); E = Extraction elution volume (in piL); R = Volume of eluate added to PCR (19 pL in this example).

[0075] (5) Exemplary data obtained from the method is as follows.• C=10, 000 copies / mL• In 3.0 mL blood volume, M = 1,900 = [((10,000 x 3) / (300)) x 19],• Cs = 1000 x 250 (Cs is the measured number of process calibrant sequences in the workflow).• Process Control P = 1000 copies• Ps = 1000 x 250 (Ps is the measured number of process control sequences in workflow)._. 1000 x 250 1000 >The extraction efficiency based on these values is = - 1900 x - 1000 x 250 = 0.526 or 52.6%.

[0076] This example assumes 100% PCR efficiency, and in 50 cycles, the template x will increase by x*250. Essentially, this amplification fold does not matter, as this factor will be cancelled out in the equation from the values of Cs and Ps.

[0077] (6) This equation assumes that the amplification efficiencies of the PCRs of ProcessCalibrant and Process Control are same, because the same primers are utilized for both the targets that have identical base compositions (although different base sequences for differentiation, exceptforthe primer binding sites, which are identical in the Process Calibrant and Process Control). However, a linear response between both these amplifications may not always be seen, and assuming an identical amplification efficiency of the Process Calibrant and Process Control would lead to overestimation of extraction efficiencies. Hence, the output ratio of process calibrant to process control should be calibrated. The analysis software is equipped with the ratio calibrant function, calculated from the experimental data in FIG. 2.

[0078] (7) A threshold is set for the estimated sample extraction efficiency after the completion of the sequencing run. If the efficiency is lower than the threshold, and the sampleis negative for pathogen detection, the run is then declared invalid, and the sample should be re-run with the workflow.

[0079] FIG. 3 contains a block diagram of one non-limiting embodiment of a system 100 which can be configured to perform any of the processes and methods as described therein. The system 100 includes a processing unit 101, a memory 102, a storage unit 103, an input unit 104, a bus 106, an output unit 105, and a network interface 107.

[0080] The processing unit 101, as used herein, means any type of computational circuit, such as, but not limited to, a microprocessor, microcontroller, complex instruction set computing microprocessor, reduced instruction set computing microprocessor, very long instruction word microprocessor, explicitly parallel instruction computing microprocessor, graphics processor, digital signal processor, or any other type of processing circuit. The processing unit 101 may also include embedded controllers, such as generic or programmable logic devices or arrays, application specific integrated circuits, single-chip computers, and the like.

[0081] The memory 102 may include volatile memory and / or non-volatile memory. The memory 102 may be coupled, for example, for communication with said processing unit 101. The processing unit 101 may execute instructions and / or code stored in the memory 102. A variety of computer-readable storage media may be stored in and accessed from said memory 102. The memory 102 may include any suitable elements for storing data and machine- readable instructions, such as read only memory, random access memory, erasable programmable read only memory, electrically erasable programmable read only memory, a hard drive, a removable media drive for handling compact disks, digital video disks, diskettes, magnetic tape cartridges, memory cards, and the like. In the present non-limiting embodiment of FIG. 3, the memory 102 includes a susceptibility module 110 stored in the form of machine-readable instructions on any of said above-mentioned storage media and may be in communication to and executed by processor 101. In certain non-limiting embodiments, method steps executed by the processor 101 to achieve the abovementioned functionality are elaborated upon in detail in FIG. 1 as well as the various other descriptions provided herein.

[0082] The storage unit 103 may be a non-transitory storage medium which stores a database 112 or other information. The database 112 is a repository of data that is maintained by, for example, a healthcare service provider. The input unit 104 may include input meanssuch as keypad, touch-sensitive display, camera (such as a camera receiving gesture-based inputs), etc. capable of receiving input signal. The bus 106 acts as interconnect between the processing unit 101, the memory 102, the storage unit 103, the input unit 104, the output unit 105, and the network interface 107.

[0083] Those of ordinary skilled in the art will appreciate that said hardware depicted in FIG. 3 may vary for particular implementations. For example, other peripheral devices such as an optical disk drive and the like, Local Area Network (LAN) / Wide Area Network (WAN) / Wireless (e.g., Wi-Fi) adapter, graphics adapter, disk controller, input / output (I / O) adapter also may be used in addition or in place of the hardware depicted. Said depicted example is provided for the purpose of explanation only and is not meant to imply architectural limitations with respect to the present disclosure.

[0084] The system 100 in accordance with an embodiment of the present disclosure includes an operating system employing a graphical user interface. Said operating system permits multiple display windows to be presented in the graphical user interface simultaneously with each display window providing an interface to a different application or to a different instance of the same application. A cursor in said graphical user interface may be manipulated by a user through a pointing device. The position of the cursor may be changed and / or an event such as clicking a mouse button, generated to actuate a desired response. One of various commercial operating systems, such as a version of Microsoft Windows™, a product of Microsoft Corporation located in Redmond, Washington, may be employed if suitably modified. Said operating system is modified or created in accordance with the present disclosure as described.

[0085] Additionally, non-transitory computer readable media containing executable instructions that when executed cause a processor to perform operations including a method as provided herein are provided. In certain non-limiting embodiments, the present disclosure includes an article of manufacture, such as a system or component thereof including a non- transitory computer-readable medium with instructions encoded thereon, the instructions configured to cause one or more processors to perform any of the methods disclosed or otherwise contemplated herein (such as, but not limited to, the method 10 of FIG. 1 and / or any of the methods disclosed in the non-limiting illustrative embodiments section below).

[0086] It will be understood that the method 10 and / or system 100 of FIGS. 1 and 3, respectively, may be integrated to be performed on a single machine. Alternatively, the method 10 and / or system 100 may be integrated across multiple platforms.

[0087] In summary, a method for estimating the nucleic acid extraction efficiency in sequencing workflows has been described in this Example. The method includes introducing synthetic DNA calibrants at the sample stage and at the amplification steps. The method includes an estimation equation that is integrated in the analysis software. A correction factor is introduced to account for the differences in amplification efficiencies of the Process control and Process calibrant to improve the equation robustness. A threshold for sample extraction efficiency is included in the software, to determine the validity of the run.

[0088] The methods of the present disclosure combine the unique steps of introducing Process Calibrant and Process Control (amplified by same primers) at known quantities during the sample preparation and amplification stages, respectively, and using these quantities and the sequence outputs thereof to determine sample extraction efficiency. This efficiency estimation indicates if the sample extraction was carried out successfully and if the yield is optimum. In addition, this estimation identifies true negatives: when a sample detection result is negative, the software looks for the sample extraction efficiency parameter, and if the efficiency is more than the set threshold, the result is flagged as a "true negative." If the efficiency is less than the set threshold, the result is flagged as a "false negative," and also indicates that a workflow repeat should be performed for that sample.

[0089] The present disclosure possesses multiple advantages over the prior art including (but not limited to) the following. First, the method can be integrated in any sequencingbased target detection workflows. Second, the method can improve the robustness of the analysis outcome. If the analysis outcome is negative, i.e., no causative pathogen has been determined, the software then looks for the sample extraction efficiency parameter to check if the result is negative due to lack of target or due to failure of the sample extraction. In other words, this feature enables the method to distinguish "true negatives" from "false negatives" in cases where the extraction efficiency was compromised. Third, this feature can provide feedback during development of sequencing-based detection workflows, where the developer can optimize the sample preparation method for better efficiency and integrate the optimized method in the full assay.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0090] The following is a list of non-limiting illustrative embodiments disclosed herein:

[0091] Illustrative embodiment 1. A method of estimating nucleic acid extraction efficiency in a sequencing workflow for detection of at least one target, the method comprising the steps of: (a) adding a specific amount of a process calibrant to a volume of biological sample that may contain the at least one target to be detected, wherein the process calibrant comprises synthetic DNA; (b) performing a nucleic acid extraction process and eluting nucleic acid from the biological sample and the process calibrant in a specific eluate volume; (c) adding a specific amount of a process control to a portion or all of the eluate volume to form an eluate mixture, wherein the process control comprises synthetic DNA, and wherein the process calibrant and process control have the same primer binding sites, the same length, and the same base compositions, and wherein at least a portion of a sequence of the process calibrant is different from a sequence of the process control; (d) performing a PCR reaction on the eluate mixture to provide a PCR product pool; (e) performing a sequencing reaction on the PCR product pool, wherein an output of the sequencing reaction comprises process calibrant reads and process control reads, and wherein the output further comprises at least one target reads if the target is present in the biological sample; (f) determining a positive or negative detection result and / or concentration result for the at least one target in the biological sample based on the at least one target reads present in the output of the sequencing reaction; and (g) calculating an extraction efficiency for the nucleic acid extraction using Formula (I):Extraction EfficiencyFormula (I) wherein C5represents an amount of process calibrant reads, Ps represents an amount of process control reads, M represents a maximum amount of process calibrant present in the eluate mixture (i.e., template present in the PCR reaction) if extraction was 100% efficient, and P represents a number of copies of process control DNA added in step (c); and (h) comparing the extraction efficiency calculated in step (f) to a threshold value.

[0092] Illustrative embodiment 2. The method of Illustrative embodiment 1, wherein M in Formula (I) is calculated using Formula (II):Formula (II) wherein C represents the specific amount (in copies / ml) of process calibrant added in step (a), B represents the volume of the biological sample to which the process calibrant is added in step (a), Vi represents the specific eluate volume from step (b), and V2 represents an amount of the eluate volume to which the process control is added in step (c).

[0093] Illustrative embodiment 3. The method of Illustrative embodiment 1 or 2, further comprising the step of: (ii) flagging a negative detection or concentration result for the at least one target in step (f) if the extraction efficiency calculated in (g) is below a threshold value.

[0094] Illustrative embodiment 4. The method of Illustrative embodiment 1 or 2, further comprisingthe step of: (iz) repeating steps (a)-(h) if the extraction efficiency calculated in step (g) is below the threshold value.

[0095] Illustrative embodiment 5. The method of any one of Illustrative embodiments 1-4, further comprising the step of: (j) reporting the at least one target positive or negative detection or concentration result of step (f) if the extraction efficiency calculated in (g) is above the threshold value.

[0096] Illustrative embodiment 6. The method of any one of Illustrative embodiments 1-5, wherein the at least one target to be detected is at least one pathogen and / or at least one human gene.

[0097] Illustrative embodiment 7. The method of Illustrative embodiment 6, wherein the at least one pathogen comprises a bacteria, a virus, a parasite, a fungus, and combinations thereof.

[0098] Illustrative embodiment 7A. The method of Illustrative embodiment 6, wherein the at least one human gene comprises DNA or RNA.

[0099] Illustrative embodiment 8. The method of any one of Illustrative embodiments 1- 7A, wherein the steps are performed by an automated sequencing system.

[0100] Illustrative embodiment 9. The method of Illustrative embodiment 8, wherein one or more steps is performed by analysis software of the automated sequencing system.

[0101] Illustrative embodiment 9A. The method of Illustrative embodiment 9, wherein the extraction efficiency calculation is embedded in the software of the automated sequencing system, and a threshold efficiency percentage is set for the workflow. The software indicates, based on the threshold, if the sample is a true negative or a false negative.

[0102] Illustrative embodiment 9B. The method of Illustrative embodiment 9 or 9A, wherein the extraction efficiency indicates the process failure of the workflow if the extraction efficiency is below a threshold.

[0103] Illustrative embodiment 10. The method of any one of Illustrative embodiments 1-9, wherein step (a) further comprises adding at least one quantitation control to the biological sample and process calibrant.

[0104] Illustrative embodiment 11. The method of any one of Illustrative embodiments 1-10, wherein the specific eluate volume in step (b) is in a range of from about 10 pl to about 500 pl.

[0105] Illustrative embodiment 12. The method of any one of Illustrative embodiments 1-11, wherein the biological sample is selected from the group consisting of whole blood or any portion thereof, urine, saliva, sputum (such as, but not limited to, bronchoalveolar lavage sputum), cerebrospinal fluid, skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0106] Illustrative embodiment 13. The method of any one of Illustrative embodiments 1-12, wherein each of the process calibrant and process control has a length in a range of from about 50bp to about 150bp and a G / C content in a range of from about 30% to about 80%.

[0107] Illustrative embodiment 14. The method of Illustrative embodiment 13, wherein a sequence of the process calibrant is produced by randomly scrambling at least a portion of an interior of the amplified region of the process control between the primer binding sites.

[0108] Illustrative embodiment 15. The method of any one of Illustrative embodiments 1-14, wherein the sequencing workflow is a metagenomic next-generation sequencing workflow used for detection of at least one pathogen.

[0109] Illustrative embodiment 16. A method of performing a sequencing-based detection workflow, comprising: performing the steps of any of Illustrative embodiments 1-15; and optimizing a sample preparation method for better efficiency in detecting the at least one target.

[0110] Illustrative embodiment 17. The method of claim 16, wherein the step of optimizing the sample preparation method comprises optimizing step (b) of Illustrative embodiment 1.

[0111] Illustrative embodiment 18. A kit for use in estimating nucleic acid extraction efficiency in a sequencing workflow for detection of at least one target, the kit comprising: a process calibrant for adding to a biological sample prior to nucleic acid extraction; and a process control for adding prior to performing a PCR reaction; and wherein the process calibrant and process control have the same primer binding sites, the same length, and the same base compositions, and wherein at least a portion of a sequence of the process calibrant is different from a sequence of the process control.

[0112] Illustrative embodiment 19. The kit of Illustrative embodiment 18, wherein each of the process calibrant and process control has a length in a range of from about 50bp to about 150bp and a G / C content in a range of from about 30% to about 80%.

[0113] Illustrative embodiment 20. The method of Illustrative embodiment 18 or 19, wherein a sequence of the process calibrant is produced by randomly scrambling at least a portion of an interior of the amplified region of the process control between the primer binding sites.

[0114] Illustrative embodiment 21. The kit of any one of Illustrative embodiments 18-20, further comprising at least one quantitation control.

[0115] Illustrative embodiment 22. The kit of any one of Illustrative embodiments 18-21, further comprising at least one of: forward and reverse primers for PCR amplification of the process calibrant and process control; and / or forward and reverse primers for PCR amplification of the at least one target.

[0116] Thus, in accordance with the present disclosure, there have been provided methods, devices, systems, and / or apparatus which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparentto those skilled in the art. Accordingly, it is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

CLAIMS1. A method of estimating nucleic acid extraction efficiency in a sequencing workflow for detection of at least one target, the method comprising the steps of:(a) adding a specific amount of a process calibrant to a volume of biological sample that may contain the at least one target to be detected, wherein the process calibrant comprises synthetic DNA;(b) performing a nucleic acid extraction process and eluting nucleic acid from the biological sample and the process calibrant in a specific eluate volume;(c) adding a specific amount of a process control to a portion or all of the eluate volume to form an eluate mixture, wherein the process control comprises synthetic DNA, and wherein the process calibrant and process control have the same primer binding sites, the same length, and the same base compositions, and wherein at least a portion of a sequence of the process calibrant is different from a sequence of the process control;(d) performing a PCR reaction on the eluate mixture to provide a PCR product pool;(e) performing a sequencing reaction on the PCR product pool, wherein an output of the sequencing reaction comprises process calibrant reads and process control reads, and wherein the output further comprises at least one target reads if the target is present in the biological sample;(f) determining a positive or negative detection or concentration result for the at least one target in the biological sample based on the at least one target reads present in the output of the sequencing reaction; and(g) calculating an extraction efficiency for the nucleic acid extraction using Formula (I):Extraction EfficiencyFormula (I)wherein Cs represents an amount of process calibrant reads, Ps represents an amount of process control reads, M represents a maximum amount of process calibrant present in the eluate mixture if extraction was 100% efficient, and P represents a number of copies of process control DNA added in step (c); and (h) comparing the extraction efficiency calculated in step (f) to a threshold value.

2. The method of claim 1, wherein M in Formula (I) is calculated using Formula (II):Formula (II) wherein C represents the specific amount (in copies / ml) of process calibrant added in step (a), B represents the volume of the biological sample to which the process calibrant is added in step (a), Vi represents the specific eluate volume from step (b), and V2 represents an amount of the eluate volume to which the process control is added in step (c).

3. The method of claim 1, further comprising the step of:(ii) flagging a negative detection or concentration result forthe at least one target in step (f) if the extraction efficiency calculated in (g) is below a threshold value.

4. The method of claim 1, further comprising the step of:(is) repeating steps (a)-(h) if the extraction efficiency calculated in step (g) is below the threshold value.

5. The method of claim 1, further comprising the step of:(j) reporting the at least one target detection or concentration result of step (f) if the extraction efficiency calculated in (g) is above the threshold value.

6. The method of claim 1, wherein the at least one target to be detected is at least one pathogen and / or at least one human gene.

7. The method of claim 6, wherein the at least one pathogen comprises a bacteria, a virus, a parasite, a fungus, and combinations thereof.

8. The method of claim 1, wherein the steps are performed by an automated sequencing system.

9. The method of claim 8, wherein one or more steps is performed by analysis software of the automated sequencing system.

10. The method of claim 1, wherein step (a) further comprises adding at least one quantitation control to the biological sample and process calibrant.

11. The method of claim 1, wherein the specific eluate volume in step (b) is in a range of from about 10 pl to about 500 pl.

12. The method of claim 1, wherein the biological sample is selected from the group consisting of whole blood or any portion thereof, urine, saliva, sputum, cerebrospinal fluid, skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.

13. The method of claim 1, wherein each of the process calibrant and process control has a length in a range of from about 50bp to about 150bp and a G / C content in a range of from about 30% to about 80%.

14. The method of claim 13, wherein a sequence of the process calibrant is produced by randomly scrambling at least a portion of an interior of the amplified region of the process control between the primer binding sites.

15. The method of claim 1, wherein the sequencing workflow is a metagenomic nextgeneration sequencing workflow used for detection of at least one pathogen.

16. A method of performing a sequencing-based detection workflow for detection of at least one target, comprising the steps of: performing the method of claim 1; and optimizing a sample preparation method for better efficiency in detecting the at least one target.

17. A kit for use in estimating nucleic acid extraction efficiency in a sequencing workflow for detection of at least one target, the kit comprising: a process calibrant for adding to a biological sample prior to nucleic acid extraction; and a process control for adding prior to performing a PCR reaction; and wherein the process calibrant and process control have the same primer binding sites, the same length, and the same base compositions, and wherein at least a portion of a sequence of the process calibrant is different from a sequence of the process control.

18. The kit of claim 17, wherein each of the process calibrant and process control has a length in a range of from about 50bp to about 150bp and a G / C content in a range of from about 30% to about 80%.

19. The method of claim 17, wherein a sequence of the process calibrant is produced by randomly scrambling at least a portion of an interior of the amplified region of the process control between the primer binding sites.

20. The kit of claim 17, further comprising at least one of: at least one quantitation control; forward and reverse primers for PCR amplification of the process calibrant and process control; and / or forward and reverse primers for PCR amplification of the at least one target.

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