Optimized ultra-low volume liquid biopsy methods, systems, and devices

Abstract

To provide a method for obtaining genetic information from an ultra-low volume of a biological sample.SOLUTION: Provided is a method comprising: generating a blunt end of a cell-free deoxyribonucleic acid (cfDNA) by contacting the tagged cfDNA with one or more polymerases and one or more exonucleases in the presence of a crowding reagent, thereby removing a 5' overhang or a 3' recessed end, and dephosphorylating the blunt end of the cfDNA, and ligating the cfDNA to an adaptor oligonucleotide by contacting the cfDNA with the adaptor oligonucleotide in the presence of a ligase and one or more of the crowding reagent and a small molecule enhancer, thereby producing the tagged cfDNA; and sequencing at least a portion of the tagged cfDNA or an amplicon thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross-reference This international application claims the benefit of U.S. Provisional Patent Application No. 62 / 824,757, filed Mar. 27, 2019, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Genetic testing is a means for obtaining information regarding a subject's DNA and / or the expression of that DNA. Genetic testing has been continuously developed to obtain biological information regarding a subject. This biological information has many uses, including determination of an individual's health status, diagnosis of an individual carrying an infectious disease or disorder, determination of an appropriate treatment for an individual, solving of crimes, and identification of paternity. Currently, genetic testing is mainly performed in clinics and laboratories by trained personnel using expensive and bulky instruments that require technical training and specialized knowledge for use. Typically, it takes several days to several weeks from obtaining a biological sample from a patient to communicating the genetic test results to the patient.

[0003]

[0004] As an example, many people who learn they are pregnant wish to know the sex of the fetus (referred to as gender in this application) as soon as possible. There are several tests that can obtain gender information from DNA in a mother's blood. Blood collected from the mother must be analyzed using sophisticated machinery by a highly trained technician. If the blood is collected at a location remote from the laboratory performing the DNA analysis, the sample must be stored, transported, and analyzed in a timely manner, or else there is a risk of sample degradation.Cell-free nucleic acids are derived from various tissue types and are released into an individual's circulation. The pool of cell-free nucleic acids in circulation often represents the genetic constitution contributing to the tissue type. In the case of healthy individuals, it can be a very homogeneous pool with little variation. However, when tissues contain significantly different genomes, a more heterogeneous cell-free nucleic acid pool can be observed. Common examples of subjects having tissues with significantly different genomes include, but are not limited to: (a) cancer patients in whom tumor DNA contains mutated sites, (b) organ transplant patients in whom the transplanted organ releases donor DNA into the pool of cell-free DNA, and (c) pregnant women in whom the placenta contributes to cell-free DNA that mainly represents fetal DNA. In some examples, the genomes can differ significantly due to epigenetic modifications. DNA from various tissues, organs, and cell types has been shown to have characteristic epigenetic patterns. Thus, it may be possible to detect cell-free nucleic acids from tissues, organs, and cells including, but not limited to, the brain, liver, fat, pancreas, endothelium, and immune cells. In addition, when an individual's tissue or cell type is affected by a disease or infection, there may be more cell-free DNA from that tissue or cell type circulating in that individual. Summary of the Invention

[0005] This specification discloses devices, systems, kits, and methods for analyzing components (e.g., nucleic acids, proteins) of a biological sample that includes a sample from an animal (human or non-human). Generally, the devices, systems, kits, and methods disclosed herein can provide genetic information from ultra-trace samples by utilizing cell-free DNA fragmentation. For the sake of brevity, this may be referred to as "ultra-trace liquid biopsy". Prior to this disclosure, it was not expected that reliable and useful genetic information could be obtained from ultra-trace samples. This is because it was not believed that sufficient amounts of cell-free nucleic acids could be obtained from a particular desired tissue (e.g., brain, liver, placenta, tumor) that was detectable or beneficial at ultra-trace levels. Additionally, the background signal from other cell-free nucleic acids, particularly from blood cells, is abundant and varies from subject to subject, making reproducible and reliable comparisons between test and control subjects seem nearly impossible. Furthermore, it was not previously anticipated that the profiles of the relative amounts and size distributions of DNA extracted from ultra-trace samples could be significantly different from what has been described heretofore.

[0006] In contrast to intracellular DNA, cell-free DNA is fragmented. To analyze cell-free DNA from ultra-trace samples, the methods, devices, systems, and kits disclosed herein utilize cell-free DNA fragments from repetitive regions (e.g., regions having common sequences) and / or multiple regions as statistically independent markers. The methods, devices, systems, and kits disclosed herein are possible because cell-free DNA fragments from repetitive regions (e.g., genomic regions containing multiple copies of the same or similar sequences) or many regions are grouped together and present at a higher effective concentration in the sample than non-fragmented DNA sequences. Thus, the sample amount containing enough analytes to obtain useful genetic information is less than previously thought. Advantageously, fragments from repetitive regions can be amplified with a pair of primers or detected using a single probe. Alternatively, or in addition, multiple detection regions that do not share similar sequences can be detected in small amounts, for example, by tagging or amplifying them with universal primers or amplifying them with multiple primer pairs (e.g., in a multiplex format).

[0007] Since useful genetic information can be obtained from ultra-trace biological samples, the devices, systems, kits, and methods described above have the advantages that (1) they are minimally invasive, (2) can be used at home with little or no technical training (e.g., do not require complex equipment), and (3) information can be obtained at the early stages of disease (e.g., pregnancy, infectious diseases). These advantages reduce or eliminate the requirements for laboratories or experts, thereby improving patient convenience, compliance, and monitoring. This ultimately leads to improving health outcomes at low cost to the healthcare system.

[0008] The analysis of cell-free circulating nucleic acids encounters many technical difficulties. For example, the amplification of circulating nucleic acids in blood can be inhibited by some of the components in whole blood (e.g., hemoglobin). One way in which the methods, systems, and devices of the present application solve this technical problem is to obtain plasma (containing cell-free nucleic acids) from the blood in capillaries in a way that avoids sample damage or contamination of the sample from components in whole blood or surrounding tissues (e.g., DNA in the skin contaminates the whole blood sample due to percutaneous puncture of the skin).

[0009] The analysis of cell-free circulating nucleic acids in a small amount of sample is particularly difficult. Despite past attempts to achieve this goal and for the reasons described herein, the art has remained skeptical as to whether useful and accurate genetic information can be obtained from cell-free DNA in a small amount of sample (e.g., capillary blood obtained by finger prick) that can be taken when needed. The methods, systems, and devices disclosed herein not only overcome these technical difficulties but can be used when needed, which was actually unthinkable considering the state of the art.

[0010] For example, past attempts to analyze circulating cell-free tumor DNA in more than 5 milliliters of blood have provided information only when the sample has a relatively high tumor load (e.g., 15 - 20%) and a fraction of the altered genome (FGA) of 15% or more, which makes it easier to detect alterations. However, in most patients, the tumor load is much lower. Thus, past attempts have excluded most of the patient population. The methods, systems, devices, and kits disclosed herein enable the detection of cell-free nucleic acids from tumors in samples with a lower tumor load (e.g., less than 15%).

[0011] In further attempts, the analysis of small amounts of biological samples was unsuccessful due to white blood cell contamination or nucleic acid damage. In some embodiments, examples indicative of DNA damage or contamination are disclosed herein in the context of measuring fetal components of cell-free nucleic acids in samples obtained from a mother. As shown herein (see Example 19), DNA damage and / or contamination at the percutaneous puncture site (e.g., finger puncture) results in the presence of nucleic acids of fragment lengths in the sample, which may in some cases be misidentified as cell-free nucleic acid fragments. In fact, as shown herein, the overabundance of short fragment lengths was due to DNA from the surrounding skin and DNA damage by the lancet caused by percutaneous puncture to obtain the sample. The DNA damage and contamination described herein for the first time pose a major challenge when collecting small amounts of sample. For example, in a 20 microliter blood draw, based on these findings, the fetal fraction would decrease from 10% to about 5%. The methods, systems, devices, and kits disclosed herein provide solutions to contamination and DNA damage introduced by percutaneous puncture, which include: (1) discarding the first blood and obtaining subsequent blood for analysis; (2) a capture method that selects longer DNA fragments; (3) electrophoresis; (4) selection of library products by size; (5) or using bioinformatics methods for annotation / removal or differential analysis based on size information

[0012] In addition, obtaining more than 5 milliliters of blood from an individual requires a technician, which increases the cost of genetic analysis and the inconvenience to the patient (e.g., the inconvenience caused by the time, discomfort, and cost of genetic analysis). The present method, device, and system are configured to provide useful and accurate genetic information by analyzing biological samples, such as capillary blood in an amount less than 5 milliliters, that can be collected when needed (e.g., capillary blood by finger puncture).

[0013] Even if past attempts analyzed small amounts of sample, they resulted in artificial results. For example, some past attempts to analyze small amounts of sample involved diluting genomic DNA from cell lines, cutting the genomic DNA to generate and detect a cell-free (cfDNA) surrogate. Downsampling or dilution of cell line DNA / cut DNA, and in silico methods, result in artificial results because they do not reflect the size and length distribution and bin information of individual samples with a low number of molecular inputs. In another example, past attempts to analyze small amounts of sample result in artificial results because they rely on the detection of predefined mutations, which can also be referred to as "known events". The present disclosure shows methods, systems, and devices for obtaining plasma (including cell-free nucleic acids) from a small amount of capillary blood (e.g., finger prick) in a way that provides accurate and undefined genetic information from non-surrogate cfDNA.

[0014] The past attempts described herein used a combination of low-pass / low-coverage whole genome sequencing in the initial detection step and then had to perform additional analysis in more detail to accurately perform genetic analysis. Low-pass / low-coverage whole genome sequences are not optimal for detecting unknown events with high sensitivity and require more detailed follow-up assays. Using multiple assays to provide genetic analysis is costly, inefficient, and not an alternative solution when needed. In contrast, the present methods, devices, and systems solve the above problems by providing a method for obtaining accurate genetic analysis from ultra-trace sample amounts by using multiple fragments of cell-free DNA that are present in high concentrations and can be detected even in small amounts of sample.

[0015] The devices, systems, kits, and methods disclosed herein are summarized as follows.

[0016] Aspects disclosed herein include: (a) obtaining a biological sample from a subject, wherein the biological sample contains cell-free deoxyribonucleic acid (cfDNA), and when the biological sample is obtained from the subject, it has a volume of at most 120 microliters (μl); (b) (i) generating blunt ends of cfDNA, where one or more polymerases and one or more exonucleases are used to remove 5' overhangs or 3' recessed ends; (2) dephosphorylating the blunt ends of cfDNA; (3) contacting cfDNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and cfDNA; or (4) repairing or removing DNA damage in cfDNA using a ligase, to generate ligation-capable cfDNA by one or more of these steps; and (ii) ligating ligation-capable cfDNA to an adapter oligonucleotide by contacting ligation-capable cfDNA with the adapter oligonucleotide in the presence of a ligase and one or more of a crowding reagent and a small molecule enhancer, to tag at least a portion of the cfDNA to generate tagged cfDNA; (c) optionally, amplifying the tagged cfDNA; (d) sequencing at least a portion of the tagged cfDNA. In some embodiments, when obtained from the subject, the volume is at most 100 microliters. In some embodiments, when obtained from the subject, the volume is at most 55 microliters. In some embodiments, when obtained from the subject, the volume is at most 50 microliters. In some embodiments, when obtained from the subject, the volume is at most 40 microliters. In some embodiments, when obtained from the subject, the volume is between about 10 microliters and about 40 microliters. In some embodiments, the biological sample obtained from the subject is capillary blood.In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains from about 25 picograms (pg) to about 250 pg of total circulating cfDNA molecules. In some embodiments, the biological sample is about 10. 4 ~ about 10 9 of cfDNA molecules. In some embodiments, the biological sample is about 10 4 ~ about 10 7It contains cfDNA molecules. In some embodiments, the cfDNA in the biological sample is about 10 genome equivalents. In some embodiments, the cfDNA in the biological sample is at most 10 genome equivalents. In some embodiments, the cfDNA in the biological sample is between 5 - 6, 6 - 7, 7 - 8, 8 - 9, 9 - 10, 10 - 11, 11 - 12, 12 - 13, 13 - 14, or 14 - 15 genome equivalents. In some embodiments, the biological sample is obtained from a subject by a process that includes: (a) a process of inducing a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process of discarding the first fraction of the biological sample; and (c) a process of collecting a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by lysis of white blood cells. In some embodiments, the method further includes detecting normal expression, overexpression, or underexpression of at least one target sequence in at least a portion of the tagged cfDNA. In some embodiments, the subject is pregnant with a fetus. In some embodiments, the component of the cfDNA is a fetal cfDNA component from the fetus. In some embodiments, the method includes analyzing genotype information from an individual and a fetal cfDNA component to determine whether the individual has paternally contributed to the fetus by identifying a genotype match between the fetal cfDNA component and the genotype information. In some embodiments, the above method further includes a step of amplifying in (c), wherein the step of generating ligatable cfDNA includes: (a) generating blunt ends of the cfDNA, wherein one or more polymerases and one or more exonucleases are used to remove 5' overhangs or 3' recessed ends; (b) dephosphorylating the blunt ends of the cfDNA; (c) contacting the cfDNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cfDNA; and (d) using a ligase to repair or remove DNA damage in the cfDNA. In some embodiments, the cfDNA is selected from a tumor of the subject, a transplanted tissue or organ, or one or more pathogens.In some embodiments, the one or more pathogens include bacteria or components thereof. In some embodiments, the one or more pathogens include viruses or components thereof. In some embodiments, the one or more pathogens include fungi or components thereof. In some embodiments, the method includes amplifying by large-scale multiplex amplification. In some embodiments, the large-scale multiplex amplification assay is isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is large-scale multiplex polymerase chain reaction (mmPCR). In some embodiments, the method further includes pooling two or more biological samples, each sample being obtained from a different subject. In some embodiments, the method further includes contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample obtained from the subject is collected using a device configured to lyse the intercellular junctions of the epidermis of the subject.In some embodiments, the tagging step comprises the library performing: (a) end repair, 5′ phosphorylation, and A-tailing with an incubation at 20° C. for 30 minutes followed by an incubation at 65° C. for 30 minutes; (b) ligating cfDNA to adapter oligonucleotides with an incubation at 20° C. for 15 minutes; (c) cleaving the ligated adapter loop from the adapter oligonucleotides with an incubation at 37° C. for 15 minutes to produce ligatable cfDNA; (d) (i) denaturing the ligatable cfDNA at 98° C. for 1 minute followed by 13 cycles of denaturing at 98° C. for 10 seconds; (ii) annealing the denatured ligatable cfDNA to one or more complementary primers from (i) at 65° C. for 75 seconds; and (iii) extending the ligatable cfDNA at 65° C. for 5 minutes to produce an amplified library of ligatable cfDNA; and (iv) purifying the amplified library of ligatable cfDNA using SPRI beads to amplify the ligatable cfDNA, thereby producing a library of cfDNA tagged with an efficiency of at least 0.5. In some embodiments, tagging produces a library of cfDNA tagged with an efficiency of at least 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.00.

[0017] Aspects disclosed herein include: (a) obtaining a biological sample from a subject during pregnancy who is carrying a fetus, wherein the biological sample contains cell-free deoxyribonucleic acid (cfDNA), and wherein the biological sample, when obtained from the subject, has a volume of about 120 microliters or less; (b) contacting at least one cfDNA in the biological sample with a polynucleotide primer that anneals to a sequence corresponding to a desired sequence and an amplification reagent to generate an amplification product; and (c) detecting the presence or absence of the amplification product. In some embodiments, the method further includes annealing an oligonucleotide probe having a detectable label to at least one cfDNA. In some embodiments, the method further includes detecting an epigenetic modification of the cfDNA. In some embodiments, the epigenetic modification includes methylation at a locus of the cfDNA. In some embodiments, detecting the presence of the amplification product indicates the gender of the fetus. In some embodiments, the component of the cfDNA is of fetal origin. In some embodiments, the method includes contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, when obtained from the subject, the volume is at most 100 microliters. In some embodiments, when obtained from the subject, the volume is at most 55 microliters. In some embodiments, when obtained from the subject, the volume is at most 50 microliters. In some embodiments, when obtained from the subject, the volume is at most 40 microliters. In some embodiments, when obtained from the subject, the volume is between about 10 microliters and about 40 microliters. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains about 25 picograms (pg) to about 250 pg of total circulating cfDNA molecules. In some embodiments, the biological sample contains about 10 4 to about 10 9contains cfDNA molecules. In some embodiments, the biological sample contains about 10 4 to about 10 7 cfDNA molecules. In some embodiments, the cfDNA in the biological sample is about 10 genome equivalents. In some embodiments, the cfDNA in the biological sample is at most 10 genome equivalents. In some embodiments, the cfDNA in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the biological sample is collected from a subject by: (a) a process that induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by lysis of white blood cells.

[0018] Aspects disclosed herein provide a method for increasing the relative amount of a target nucleic acid in a biological sample obtained from a subject, the method comprising: (a) inducing a percutaneous puncture at a site of the subject to generate a first fraction and a second fraction of the biological sample; (b) discarding the first fraction of the biological sample; and (c) collecting the second fraction of the biological sample, thereby reducing or removing contamination or nucleic acid damage of the biological sample, wherein the first fraction contains a lower fraction of the target nucleic acid compared to the fraction of the target nucleic acid in the second fraction. In some embodiments, the method further comprises washing the site prior to inducing the percutaneous puncture, thereby removing or reducing unwanted contaminants. In some embodiments, the unwanted contaminants include DNA from the percutaneous puncture site. In some embodiments, the nucleic acid damage includes damage to non-apoptotic DNA in the biological sample. In some embodiments, the biological sample is capillary blood. In some embodiments, the method further comprises detecting the target nucleic acid in the second fraction of the biological sample using an assay selected from a large-scale multiplex polymerase chain reaction (mmPCR) or nucleic acid sequencing. In some embodiments, the first fraction, the second fraction, or a combination thereof, when obtained from the subject, has a volume of at most 300 microliters. In some embodiments, when obtained from the subject, the volume is at most 100 microliters. In some embodiments, when obtained from the subject, the volume is at most 55 microliters. In some embodiments, when obtained from the subject, the volume is at most 50 microliters. In some embodiments, when obtained from the subject, the volume is at most 40 microliters. In some embodiments, when obtained from the subject, the volume is between about 10 microliters and about 40 microliters. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the target nucleic acid is a circulating cell-free nucleic acid molecule.In some embodiments, the biological sample contains from about 25 picograms (pg) to about 250 pg of total circulating cell-free nucleic acid molecules. In some embodiments, the biological sample contains up to about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample contains up to about 10 4 ~ about 10 7 of cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is up to 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the cell-free nucleic acid molecules are cell-free DNA molecules.

[0019] Aspects disclosed herein provide a device, the device comprising: (a) a sample collector for obtaining a biological sample having a volume of up to 120 microliters from a subject, wherein the biological sample contains target cell-free DNA (cfDNA); (b) a sample purification device for removing cells from the biological sample to generate a cell-depleted sample; and (c) a nucleic acid detection device configured to detect target cfDNA in the cell-depleted sample. In some embodiments, the device further comprises a nucleic acid ligator, the nucleic acid ligator comprising: (a) a ligation formulation for creating ligatable target cfDNA, the ligation formulation comprising one or more of: (i) one or more exonucleases adapted to generate blunt ends of the target cfDNA and to remove 5' overhangs or 3' recessed ends of the blunt ends of the target cfDNA; (ii) a blunt-end cfDNA dephosphorylating agent; (iii) a crowding reagent; (iv) a DNA damage repair agent; or (v) a DNA ligase; and (b) one or more adapter oligonucleotides ligated to the ligatable target cfDNA. In some embodiments, the device further comprises a white blood cell stabilizer. In some embodiments, the nucleic acid detection device is a massively multiplexed PCR device (mmPCR). In some embodiments, the ligation formulation comprises: (a) one or more exonucleases adapted to generate blunt ends of the target cfDNA and to remove 5' overhangs or 3' recessed ends of the blunt ends of the target cfDNA; (i) a blunt-end cfDNA dephosphorylating agent; (ii) a DNA damage repair agent; and (iii) a DNA ligase. In some embodiments, the nucleic acid detection device comprises a nucleic acid sequencer or a lateral flow strip. In some embodiments, the nucleic acid sequencer comprises a signal detector. In some embodiments, the sample purification device comprises a filter having a pore size of from about 0.05 microns to about 2 microns. In some embodiments, the filter is a vertical filter.In some embodiments, the sample purification device comprises a binding moiety selected from antibodies, antigen-binding antibody fragments, ligands, receptors, peptides, small molecules, and combinations thereof. In some embodiments, the sample collection device is configured to lyse the intercellular junctions of the epidermis of a subject to obtain a biological sample. In some embodiments, when obtained from a subject, the volume is at most 100 microliters. In some embodiments, when obtained from a subject, the volume is at most 55 microliters. In some embodiments, when obtained from a subject, the volume is at most 50 microliters. In some embodiments, when obtained from a subject, the volume is at most 40 microliters. In some embodiments, when obtained from a subject, the volume is between about 10 microliters and about 40 microliters. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the target nucleic acid is a circulating cell-free nucleic acid molecule. In some embodiments, the biological sample contains about 25 picograms (pg) to about 250 pg of total circulating cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10 4 ~ about 10 7 of cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the cell-free nucleic acid molecule is a cell-free DNA molecule.

[0020] In some embodiments, the aspects disclosed herein include: (a) obtaining a biological sample from a subject; (b) optionally tagging at least a portion of the cell-free nucleic acids to generate a library of optionally tagged cell-free nucleic acids; (c) optionally amplifying the optionally tagged cell-free nucleic acids; (d) sequencing at least a portion of the optionally tagged cell-free nucleic acids; and (e) detecting normal expression, overexpression, or underexpression of at least one target sequence in at least a portion of the optionally tagged cell-free nucleic acids. In some embodiments, the biological sample includes blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the blood includes capillary blood. In some embodiments, the method further includes pooling two or more biological samples, each sample obtained from a different subject. In some embodiments, the method further includes contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample obtained from the subject is collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is not collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is collected using a device configured to lyse the intercellular junctions of the epidermis of the subject. In some embodiments, the biological sample obtained from the subject is collected from the subject by a process that includes: (a) a process of inducing a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process of discarding the first fraction of the biological sample; and (c) a process of collecting a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by lysis of leukocytes.In some embodiments, the tagging of (c) comprises one or more steps of: (i) generating blunt ends of cell-free DNA, wherein in some embodiments, 5' overhangs or 3' recessed ends are removed using one or more polymerases and one or more exonucleases; (ii) dephosphorylating the blunt ends of cell-free DNA; (iii) contacting the cell-free DNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cell-free DNA; or (iv) repairing or removing DNA damage in the cell-free DNA using a ligase, to generate ligation-capable cell-free DNA; and (b) ligating the ligation-capable cell-free DNA to an adapter oligonucleotide by contacting the ligation-capable cell-free DNA with the adapter oligonucleotide in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer. In some embodiments, the one or more polymerases include T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases include T4 polynucleotide kinase or exonuclease III. In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein. In some embodiments, the ligase includes T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7 ligase fusion protein. In some embodiments, the crowding reagent includes polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof. In some embodiments, the small molecule enhancer includes dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof.In some embodiments, the ligation of (b) includes blunt-end ligation or single-base overhang ligation. In some embodiments, the adapter oligonucleotide includes a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a degradable adapter, blocked self-ligating adaptors, or a barcoded adapter, or a combination thereof. In some embodiments, the library of (c) is produced with an efficiency of at least 0.5. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a tumor. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a fetus. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the target cell-free nucleic acid is genomic nucleic acid from one or more pathogens. In some embodiments, the pathogen includes bacteria or components thereof. In some embodiments, the pathogen includes a virus or components thereof. In some embodiments, the pathogen includes a fungus or components thereof. In some embodiments, the cell-free nucleic acid includes one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or a combination thereof. In some embodiments, the large-scale multiplex amplification assay is an isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is a polymerase chain reaction (mmPCR). In some embodiments, the biological sample includes a cell type or tissue type with low fetal cell-free nucleic acid compared to peripheral blood. In some embodiments, the method does not consist of performing a venipuncture or withdrawing a biological sample from the venous blood of a subject. In some embodiments, when obtained from a subject, the biological sample has a volume of at most 100 microliters. In some embodiments, when obtained from a subject, the volume is at most 55 microliters. In some embodiments, when obtained from a subject, the volume is at most 50 microliters. In some embodiments, when obtained from a subject, the volume is at most 40 microliters.In some embodiments, when obtained from a subject, the volume is between about 10 microliters to about 40 microliters at most. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains cell-free nucleic acids. In some embodiments, the biological sample contains from about 25 picograms (pg) to about 250 pg of total cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10 4 ~ about 10 7 of cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at a genome equivalent between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15. In some embodiments, the cell-free nucleic acid molecules are cell-free DNA molecules.

[0021] In some embodiments, the aspects disclosed herein are methods for prenatal paternity testing, the method comprising: (a) obtaining a biological sample from a subject during pregnancy with a fetus; (b) optionally tagging at least a portion of the cell-free nucleic acid to generate a library of optionally tagged cell-free nucleic acids; (c) optionally amplifying the optionally tagged cell-free nucleic acid; (d) optionally sequencing at least a portion of the optionally tagged cell-free nucleic acid; (e) receiving paternal genotype information from an individual suspected of being the paternal father of the fetus; and (f) comparing the paternal genotype information to the fetal component of the cell-free nucleic acid to determine whether there is a genotype match between the fetal component and the paternal genotype. In some embodiments, the biological sample comprises cell-free nucleic acid. In some embodiments, the biological sample comprises blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the blood comprises capillary blood. In some embodiments, the capillary blood comprises blood of 40 microliters or less. In some embodiments, the method further comprises pooling two or more biological samples, each sample obtained from a different subject. In some embodiments, the method further comprises contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample obtained from the subject is collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is not collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is collected using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, the biological sample obtained from the subject is collected from the subject by a process comprising: (a) a process of inducing a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process of discarding the first fraction of the biological sample; and (c) a process of collecting a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by lysis of leukocytes.In some embodiments, the method further includes washing the surface of the percutaneous puncture site (e.g., the skin) before obtaining the biological sample from the subject. In some examples, the washing step includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of a finger. In some embodiments, the tagging of (c) is (a)(i) a step of generating blunt ends of cell-free DNA, in some embodiments, using one or more polymerases and one or more exonucleases to remove 5' overhangs or 3' recessed ends; (ii) a step of dephosphorylating the blunt ends of cell-free DNA; (iii) a step of contacting cell-free DNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and cell-free DNA; or (iv) a step of repairing or removing DNA damage in cell-free DNA using a ligase, generating ligation-capable cell-free DNA by one or more steps including; and (b) ligating the ligation-capable cell-free DNA to an adapter oligonucleotide by contacting the ligation-capable cell-free DNA with the adapter oligonucleotide in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer. In some embodiments, the one or more polymerases include T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases include T4 polynucleotide kinase or exonuclease III. In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein.In some embodiments, the ligase includes T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7 ligase fusion protein. In some embodiments, the crowding reagent includes polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof. In some embodiments, the small molecule enhancer includes dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof. In some embodiments, the ligation of (b) includes blunt-end ligation or single-base overhang ligation. In some embodiments, the adapter oligonucleotide includes a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a degradable adapter, a blocked self-ligating adapter, or a barcoded adapter, or a combination thereof. In some embodiments, the library of (c) is produced with an efficiency of at least 0.5. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a tumor. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a fetus. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the target cell-free nucleic acid is genomic nucleic acid from one or more pathogens. In some embodiments, the pathogen includes bacteria or its components. In some embodiments, the pathogen includes a virus or its components. In some embodiments, the pathogen includes fungi or its components. In some embodiments, the cell-free nucleic acid includes one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or a combination thereof. In some embodiments, the large-scale multiplex amplification assay is isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is multiplex polymerase chain reaction (mmPCR). In some embodiments, the biological sample includes cell types or tissue types with low fetal cell-free nucleic acid compared to peripheral blood. In some embodiments, the method does not consist of performing phlebotomy or withdrawing a biological sample from the venous blood of a subject.In some embodiments, when obtained from a subject, the biological sample has a volume of at most 100 microliters. In some embodiments, when obtained from a subject, the volume is at most 55 microliters. In some embodiments, when obtained from a subject, the volume is at most 50 microliters. In some embodiments, when obtained from a subject, the volume is at most 40 microliters. In some embodiments, when obtained from a subject, the volume is between about 10 microliters and about 40 microliters. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains cell-free nucleic acids. In some embodiments, the biological sample contains from about 25 picograms (pg) to about 250 pg of total circulating cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10 4 ~ about 10 7 of cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5 - 6, 6 - 7, 7 - 8, 8 - 9, 9 - 10, 10 - 11, 11 - 12, 12 - 13, 13 - 14, or 14 - 15 genome equivalents. In some embodiments, the cell-free nucleic acid molecules are cell-free DNA molecules.

[0022] Aspects disclosed herein are, in some embodiments, methods of analyzing a biological sample obtained from a subject, the methods comprising: (a) obtaining a biological sample from the subject; (b) optionally tagging at least a portion of the cell-free nucleic acids to generate a library of tagged cell-free nucleic acids; (c) optionally amplifying the tagged cell-free nucleic acids by a large-scale multiplex amplification assay; (d) optionally pooling the optionally tagged cell-free nucleic acids; (e) sequencing at least a portion of the amplified optionally tagged cell-free nucleic acids; and (f) detecting a normal expression, overexpression, or underexpression of at least one target sequence in at least a portion of the optionally tagged cell-free nucleic acids. In some embodiments, the biological sample includes blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the blood includes capillary blood. In some embodiments, the method further comprises pooling two or more biological samples, each sample obtained from a different subject. In some embodiments, the method further comprises contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample obtained from the subject is collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is not collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is collected using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, the biological sample obtained from the subject is collected by: (a) a process that induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method further comprises washing the surface of the percutaneous puncture site (e.g., the skin) before obtaining the biological sample from the subject. In some instances, the washing process includes removing or reducing unwanted contaminants.In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of a finger. In some embodiments, the tagging of (c) is: (a)(i) a step of generating blunt ends of cell-free DNA, in some embodiments, using one or more polymerases and one or more exonucleases to remove 5' overhangs or 3' recessed ends; (ii) a step of dephosphorylating the blunt ends of cell-free DNA; (iii) a step of contacting the cell-free DNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cell-free DNA; or (iv) a step of repairing or removing DNA damage in the cell-free DNA using a ligase, to generate ligation-competent cell-free DNA by one or more steps; and (b) contacting the ligation-competent cell-free DNA with an adapter oligonucleotide in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer to ligate the ligation-competent cell-free DNA to the adapter oligonucleotide. In some embodiments, the one or more polymerases include T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases include T4 polynucleotide kinase or exonuclease III. In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein. In some embodiments, the ligase includes T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7 ligase fusion protein.In some embodiments, the crowding reagent comprises polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof. In some embodiments, the small molecule enhancer comprises dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof. In some embodiments, the ligation of (b) comprises blunt end ligation, or single base overhang ligation. In some embodiments, the adapter oligonucleotide comprises a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a degradable adapter, a blocked self-ligating adapter, or a barcoded adapter, or a combination thereof. In some embodiments, the library of (c) is produced with an efficiency of at least 0.5. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a tumor. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a fetus. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the target cell-free nucleic acid is genomic nucleic acid from one or more pathogens. In some embodiments, the pathogen comprises bacteria or a component thereof. In some embodiments, the pathogen comprises a virus or a component thereof. In some embodiments, the pathogen comprises a fungus or a component thereof. In some embodiments, the cell-free nucleic acid comprises one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or a combination thereof. In some embodiments, the large-scale multiplex amplification assay is isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is multiplex polymerase chain reaction (mmPCR). In some embodiments, the biological sample comprises a cell type or tissue type with low fetal cell-free nucleic acid compared to peripheral blood. In some embodiments, the method does not consist of performing venipuncture or withdrawing a biological sample from the venous blood of a subject. In some embodiments, when obtained from a subject, the biological sample has a volume of at most 100 microliters. In some embodiments, when obtained from a subject, the volume is at most 55 microliters.In some embodiments, when obtained from a subject, the volume is at most 50 microliters. In some embodiments, when obtained from a subject, the volume is at most 40 microliters. In some embodiments, when obtained from a subject, the volume is between about 10 microliters and about 40 microliters. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains cell-free nucleic acids. In some embodiments, the biological sample contains about 25 picograms (pg) to about 250 pg of total cell-free nucleic acid molecules. In some embodiments, the biological sample is at most about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample is at most about 10 4 ~ about 10 7 of cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the cell-free nucleic acid molecules are cell-free DNA molecules.

[0023] In some embodiments, the aspects disclosed herein are methods comprising: (a) obtaining from a subject containing deoxyribonucleic acid (DNA) a biological sample of about 1 to 100 microliters (μl); and (b) detecting epigenetic modifications of the DNA. In some embodiments, epigenetic modifications include DNA methylation at a locus, histone methylation, histone, ubiquitination, histone acetylation, histone phosphorylation, microRNA (miRNA). In some embodiments, DNA methylation includes CpG methylation or CpH methylation. In some embodiments, the locus includes a promoter or regulatory element of a gene. In some embodiments, the locus includes a variable length terminal repeat (LTR). In some embodiments, the locus includes cell-free DNA or a fragment thereof. In some embodiments, the locus includes a single nucleotide polymorphism (SNP). In some embodiments, histone acetylation is indicated by the presence or level of histone deacetylase. In some embodiments, histone modifications are in histones selected from the group consisting of histone 2A (H2A), histone 2B (H2B), histone 3 (H3), and histone 4 (H4). In some embodiments, histone methylation is methylation of H3 lysine 4 (H3K4me2). In some embodiments, histone acetylation is deacetylation at H4. In some embodiments, the miRNA is selected from the group consisting of miR-21, miR-126, mi-R142, mi-R146a, mi-R12a, mi-R181a, miR-29c, miR-29a, miR-29b, miR-101, miRNA-155, and miR-148a. In some embodiments, the biological sample includes blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the method further comprises pooling two or more biological samples, each sample obtained from a different subject. In some embodiments, the biological sample obtained from the subject is collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is not collected by percutaneous puncture.In some embodiments, a biological sample obtained from a subject is collected using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, a biological sample obtained from a subject is collected by: (a) a process that induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method further includes a step of washing the surface of the percutaneous puncture site (e.g., the skin) before obtaining the biological sample from the subject. In some examples, the step of washing includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of a finger. In some embodiments, the method further includes a step of contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the method does not consist of performing a venipuncture or withdrawing a biological sample from the venous blood of the subject. In some embodiments, when obtained from the subject, the biological sample has a volume of at most 100 microliters. In some embodiments, when obtained from the subject, the volume is at most 55 microliters. In some embodiments, when obtained from the subject, the volume is at most 50 microliters. In some embodiments, when obtained from the subject, the volume is at most 40 microliters. In some embodiments, when obtained from the subject, the volume is between about 10 microliters and about 40 microliters. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains cell-free circulating nucleic acids.In some embodiments, the biological sample contains from about 25 picograms (pg) to about 250 pg of total circulating cell-free nucleic acid molecules. In some embodiments, the biological sample contains up to about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample contains up to about 10 4 ~ about 10 7 of cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is up to 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the cell-free nucleic acid molecules are cell-free DNA molecules.

[0024] In some embodiments, the aspects disclosed herein include (a) obtaining a biological sample from a subject, wherein the biological sample contains up to about 10 9A method comprising the steps of: (b) sequencing at least a portion of the cell-free nucleic acid molecules to generate sequencing reads; (c) measuring at least a portion of the sequencing reads corresponding to at least one chromosomal region; and (d) detecting normal, over-expression, or under-expression of at least one chromosomal region. In some embodiments, the method further comprises tagging at least a portion of the cell-free nucleic acid molecules. In some embodiments, the tagging step comprises (a)(i) generating blunt ends of cell-free DNA, wherein in some embodiments, 5' overhangs or 3' recessed ends are removed using one or more polymerases and one or more exonucleases; (ii) dephosphorylating the blunt ends of cell-free DNA; (iii) contacting the cell-free DNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cell-free DNA; or (iv) repairing or removing DNA damage in the cell-free DNA using a ligase, to generate ligation-competent cell-free DNA by one or more steps; and (b) ligating the ligation-competent cell-free DNA to adapter oligonucleotides by contacting the ligation-competent cell-free DNA with adapter oligonucleotides in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer. In some embodiments, the method further comprises pooling two or more biological samples, each sample obtained from a different subject. In some embodiments, the method further comprises contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the one or more polymerases comprise T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases comprise T4 polynucleotide kinase or exonuclease III.In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein. In some embodiments, the ligase comprises T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7 ligase fusion protein. In some embodiments, the crowding reagent comprises polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof. In some embodiments, the small molecule enhancer comprises dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof. In some embodiments, the ligation of (b) comprises blunt-end ligation, or single-base overhang ligation. In some embodiments, the adapter oligonucleotide comprises a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a degradable adapter, a blocked self-ligating adapter, or a barcoded adapter, or a combination thereof. In some embodiments, the biological sample is a biological sample having a volume of less than about 500 microliters (μl). In some embodiments, the biological sample is a biological sample having a volume of about 1 μL to about 100 μl. In some embodiments, the biological sample is a biological sample having a volume of about 5 μL to about 80 μl. In some embodiments, the biological sample comprises blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, when obtained from a subject, the biological sample has a volume of at most 100 microliters. In some embodiments, when obtained from a subject, the volume is at most 55 microliters. In some embodiments, when obtained from a subject, the volume is at most 50 microliters. In some embodiments, when obtained from a subject, the volume is at most 40 microliters.In some embodiments, when obtained from a subject, the volume is between about 10 microliters to about 40 microliters at most. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains circulating cell-free nucleic acids. In some embodiments, the biological sample contains from about 25 picograms (pg) to about 250 pg of total circulating cell-free nucleic acid molecules. In some embodiments, the biological sample contains less than 300 pg of cell-free nucleic acid molecules. In some embodiments, the biological sample contains 3 ng of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10 4 ~ about 10 7It contains cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the cell-free nucleic acid molecule is a cell-free DNA molecule. In some embodiments, the method further comprises the step of separating plasma or serum from a blood sample. In some embodiments, the separating step comprises filtering the blood sample to remove cells, cell fragments, microparticles, or combinations thereof from the blood sample to generate a plasma sample. In some embodiments, obtaining the blood sample includes finger puncture. In some embodiments, the biological sample obtained from the subject is collected using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, the biological sample obtained from the subject is collected by: (a) a process that induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method further comprises the step of washing the surface of the percutaneous puncture site (e.g., skin) before obtaining the biological sample from the subject. In some examples, the washing step includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some embodiments, the subject is a pregnant subject and the cell-free nucleic acid molecule includes cell-free fetal nucleic acid molecules. In some embodiments, the cell-free nucleic acid includes nucleic acid from a tumor in a tissue. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a fetus.In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the target cell-free nucleic acid is genomic nucleic acid from one or more pathogens. In some embodiments, the pathogen includes bacteria or components thereof. In some embodiments, the pathogen includes a virus or components thereof. In some embodiments, the pathogen includes a fungus or components thereof. In some embodiments, the cell-free nucleic acid includes one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or combinations thereof. In some embodiments, the large-scale multiplex amplification assay is an isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is a polymerase chain reaction (mmPCR). In some embodiments, the biological sample includes cell types or tissue types with low fetal cell-free nucleic acid compared to peripheral blood. In some embodiments, the method does not consist of performing a venipuncture or withdrawing a biological sample from the venous blood of a subject.

[0025] In some embodiments, the aspects disclosed herein are methods of prenatal paternity testing, the method comprising: (a) obtaining a biological sample from a subject during pregnancy with a fetus, wherein in some embodiments, the biological sample comprises at most about 10 9 cell-free nucleic acid molecules; (b) sequencing at least a portion of the cell-free nucleic acid to generate sequencing reads; (c) measuring at least a portion of the sequencing reads corresponding to at least one chromosomal region; (d) receiving paternal genotype information from an individual suspected of being the paternal father of the fetus; and (e) comparing the paternal genotype information with the fetal component of the cell-free nucleic acid to determine whether there is a genotype match between the fetal component and the paternal genotype. In some embodiments, the method further comprises amplifying the cell-free nucleic acid. In some embodiments, the method further comprises tagging at least a portion of the cell-free nucleic acid to generate a library of tagged cell-free nucleic acids. In some embodiments, the method further comprises amplifying the tagged cell-free nucleic acid. In some embodiments, the tagging step (a) (i) Generating blunt ends of cell-free DNA, which, in some embodiments, involves removing 5' overhangs or 3' recessed ends using one or more polymerases and one or more exonucleases; (ii) Dephosphorylating the blunt ends of cell-free DNA; (iii) Contacting the cell-free DNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cell-free DNA; or (iv) Repairing or removing DNA damage in the cell-free DNA using a ligase, by one or more steps, to generate ligation-competent cell-free DNA; and (b) Ligating the ligation-competent cell-free DNA to adapter oligonucleotides by contacting the ligation-competent cell-free DNA with adapter oligonucleotides in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer. In some embodiments, the method further includes pooling two or more biological samples, each sample being obtained from a different subject. In some embodiments, the method further includes contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the one or more polymerases include T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases include T4 polynucleotide kinase or exonuclease III. In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein. In some embodiments, the ligase includes T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7 ligase fusion protein. In some embodiments, the crowding reagent includes polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof.In some embodiments, the small molecule enhancer comprises dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof. In some embodiments, the ligation of (b) comprises blunt-end ligation or single-base overhang ligation. In some embodiments, the adapter oligonucleotide comprises a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a degradable adapter, a blocked self-ligating adapter, or a barcoded adapter, or a combination thereof. In some embodiments, the biological sample comprises blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, when obtained from a subject, the biological sample has a volume of up to 500 microliters. In some embodiments, when obtained from a subject, the volume is up to 300 microliters. In some embodiments, when obtained from a subject, the volume is up to 100 microliters. In some embodiments, when obtained from a subject, the volume is up to 55 microliters. In some embodiments, when obtained from a subject, the volume is up to 50 microliters. In some embodiments, when obtained from a subject, the volume is up to 40 microliters. In some embodiments, when obtained from a subject, the volume is between about 10 microliters and about 40 microliters at most. In some embodiments, when obtained from a subject, the volume is between about 10 microliters and about 100 microliters at most. In some embodiments, the biological sample obtained from a subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample comprises cell-free circulating nucleic acids. In some embodiments, the biological sample contains about 25 picograms (pg) to about 250 pg of total cell-free circulating nucleic acid molecules. In some embodiments, the biological sample contains less than 300 pg of cell-free nucleic acid molecules. In some embodiments, the biological sample contains 3 ng of cell-free nucleic acid molecules. In some embodiments, the biological sample is up to about 10 at most.4 ~about 10 9 comprises a cell-free nucleic acid molecule. In some embodiments, the biological sample is up to about 10 4 ~about 10 7It contains cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the cell-free nucleic acid molecule is a cell-free DNA molecule. In some embodiments, the method further comprises the step of separating plasma or serum from a blood sample. In some embodiments, the separating step comprises filtering the blood sample to remove cells, cell fragments, microvesicles, or combinations thereof from the blood sample to produce a plasma sample. In some embodiments, obtaining the blood sample includes finger puncture. In some embodiments, the biological sample obtained from the subject is collected using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, the biological sample obtained from the subject is collected by: (a) a process that induces a first percutaneous puncture to produce a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method further comprises the step of cleaning the surface of the percutaneous puncture site (e.g., skin) before obtaining the biological sample from the subject. In some examples, the cleaning step includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of the finger. In some embodiments, the biological sample is at most about 10 4 to about 10 9It contains cell-free nucleic acid molecules. In some embodiments, the subject is a pregnant subject, and the cell-free nucleic acid molecule contains cell-free fetal nucleic acid molecules. In some embodiments, the cell-free nucleic acid contains nucleic acid from a tumor in a tissue. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a fetus. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the target cell-free nucleic acid is genomic nucleic acid from one or more pathogens. In some embodiments, the pathogen contains bacteria or its components. In some embodiments, the pathogen contains a virus or its components. In some embodiments, the pathogen contains a fungus or its components. In some embodiments, the cell-free nucleic acid contains one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or a combination thereof. In some embodiments, the large-scale multiplex amplification assay is an isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is a polymerase chain reaction (mmPCR). In some embodiments, the biological sample contains cell types or tissue types with low fetal cell-free nucleic acid compared to peripheral blood. In some embodiments, the method does not consist of performing venipuncture or withdrawing a biological sample from the venous blood of the subject.

[0026] In some embodiments, the aspects disclosed herein include: (a) obtaining a biological sample from a subject; (b) amplifying cell-free nucleic acid; (c) optionally tagging at least a portion of the cell-free nucleic acid to generate a library of tagged cell-free nucleic acid; (d) amplifying the optionally tagged cell-free nucleic acid by a large-scale multiplex amplification assay; (e) optionally pooling the optionally tagged cell-free nucleic acid; (f) sequencing at least a portion of the amplified optionally tagged cell-free nucleic acid to generate sequencing reads; (g) measuring at least a portion of the sequencing reads corresponding to at least one chromosomal region; and (h) detecting normal expression, overexpression, or underexpression of at least one chromosomal region. In some embodiments, the tagging step (a) (i) Generating blunt ends of cell-free DNA, which in some embodiments involves removing 5' overhangs or 3' recessed ends using one or more polymerases and one or more exonucleases; (ii) Dephosphorylating the blunt ends of cell-free DNA; (iii) Contacting the cell-free DNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cell-free DNA; or (iv) Repairing or removing DNA damage in the cell-free DNA using a ligase, by one or more steps to generate ligation-competent cell-free DNA; and (b) Ligating the ligation-competent cell-free DNA to an adapter oligonucleotide by contacting the ligation-competent cell-free DNA with the adapter oligonucleotide in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer. In some embodiments, the method further includes pooling two or more biological samples, each sample being obtained from a different subject. In some embodiments, the method further includes contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the one or more polymerases include T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases include T4 polynucleotide kinase or exonuclease III. In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein. In some embodiments, the ligase includes T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7 ligase fusion protein. In some embodiments, the crowding reagent includes polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof.In some embodiments, the small molecule enhancer comprises dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof. In some embodiments, the ligation of (b) comprises blunt end ligation, or single base overhang ligation. In some embodiments, the adapter oligonucleotide comprises a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a degradable adapter, a blocked self-ligating adapter, or a barcoded adapter, or a combination thereof. In some embodiments, the biological sample comprises blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, when obtained from a subject, the biological sample has a volume of at most 300 microliters. In some embodiments, when obtained from a subject, the volume is at most 100 microliters. In some embodiments, when obtained from a subject, the volume is at most 55 microliters. In some embodiments, when obtained from a subject, the volume is at most 50 microliters. In some embodiments, when obtained from a subject, the volume is at most 40 microliters. In some embodiments, when obtained from a subject, the volume is between about 10 microliters to about 40 microliters at most. In some embodiments, when obtained from a subject, the volume is between about 10 microliters to about 100 microliters at most. In some embodiments, the biological sample obtained from a subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample comprises cell-free circulating nucleic acids. In some embodiments, the biological sample contains about 25 picograms (pg) to about 250 pg of total cell-free circulating nucleic acid molecules. In some embodiments, the biological sample contains less than 300 pg of cell-free nucleic acid molecules. In some embodiments, the biological sample contains 3 ng of cell-free nucleic acid molecules. In some embodiments, the biological sample is at most about 10. 4 ~ about 10 9comprises a cell-free nucleic acid molecule. In some embodiments, the biological sample is up to about 10 4 ~ about 10 7It contains cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the method further comprises the step of separating plasma or serum from the blood sample. In some embodiments, the separating step comprises filtering the blood sample to remove cells, cell fragments, microparticles, or combinations thereof from the blood sample to produce a plasma sample. In some embodiments, obtaining the blood sample includes finger puncture. In some embodiments, the biological sample obtained from the subject is collected using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, the biological sample obtained from the subject is collected by (a) a process that induces a first percutaneous puncture to produce a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method further comprises the step of washing the surface of the percutaneous puncture site (e.g., skin) before obtaining the biological sample from the subject. In some examples, the washing step includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of the finger. In some embodiments, the subject is a pregnant subject and the cell-free nucleic acid molecule includes a cell-free fetal nucleic acid molecule. In some embodiments, the cell-free nucleic acid includes nucleic acid from a tumor in the tissue. In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a fetus.In some embodiments, the target cell-free nucleic acid is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the target cell-free nucleic acid is genomic nucleic acid from one or more pathogens. In some embodiments, the pathogen comprises bacteria or a component thereof. In some embodiments, the pathogen comprises a virus or a component thereof. In some embodiments, the pathogen comprises a fungus or a component thereof. In some embodiments, the cell-free nucleic acid comprises one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or a combination thereof. In some embodiments, the large-scale multiplex amplification assay is an isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is a polymerase chain reaction (mmPCR). In some embodiments, the biological sample comprises a cell type or tissue type with low fetal cell-free nucleic acid as compared to peripheral blood. In some embodiments, the method does not consist of performing a venipuncture or withdrawing a biological sample from the venous blood of a subject.

[0027] In some embodiments, the aspects disclosed herein include: (a) obtaining a biological sample from a subject; (b) isolating fetal trophoblasts from the biological sample using an antibody specific for a fetal trophoblast cell surface antigen; (c) lysing the nuclei of the fetal trophoblasts in the fetal trophoblasts; (e) extracting fetal genomic DNA (gDNA) from the lysed fetal trophoblasts; (f) contacting the fetal gDNA with amplification reagents and oligonucleotide primers that anneal to sequences corresponding to a desired sequence to produce an amplification product; (g) detecting (i) the presence or absence of the amplification product, or (ii) the normal, overexpressed, or underexpressed expression of at least one target sequence in at least a portion of the fetal gDNA. In some embodiments, the presence or absence indicates the health status of the fetus. In some embodiments, the method further includes contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample includes blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the biological sample obtained from the subject is collected by the subject using a finger prick. In some embodiments, the biological sample obtained from the subject is collected by the subject without using a finger prick. In some embodiments, the biological sample obtained from the subject is collected by the subject using a device configured to lyse the intercellular junctions of the epidermis of the subject. In some embodiments, the biological sample obtained from the subject is collected by: (a) a process that induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method further includes washing the surface of the percutaneous puncture site (e.g., the skin) before obtaining the biological sample from the subject. In some examples, the washing process includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site.In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of a finger. In some embodiments, the contacting step includes performing isothermal amplification. In some embodiments, the contacting step occurs at room temperature. In some embodiments, the method includes incorporating a tag into the amplification product when amplification occurs, and further includes detecting the presence of the amplification product, and the detecting step includes detecting the tag. In some embodiments, the tag does not contain nucleotides. In some embodiments, the step of detecting the amplification product includes contacting the amplification product with a binding moiety that can interact with the tag. In some embodiments, the method further includes contacting the amplification product with the binding moiety on a lateral flow device. In some embodiments, steps (a) through (c) are performed in less than 15 minutes. In some embodiments, the method is performed by a subject. In some embodiments, the method is performed by an individual who has not received technical training to perform the method. In some embodiments, acquisition, contacting, and detection are performed using a single handheld device. In some embodiments, the health state is selected from the presence or absence of pregnancy. In some embodiments, the health state is selected from the presence or absence of neurological disorders, metabolic disorders, cancer, autoimmune disorders, allergic reactions, and infectious diseases. In some embodiments, the health state is a response to a drug or treatment. In some embodiments, the method does not consist of performing a venipuncture or withdrawing a biological sample from the venous blood of the subject. In some embodiments, when obtained from a subject, the biological sample has a volume of at most 300 microliters. In some embodiments, when obtained from a subject, the volume is at most 100 microliters. In some embodiments, when obtained from a subject, the volume is at most 55 microliters. In some embodiments, when obtained from a subject, the volume is at most 50 microliters. In some embodiments, when obtained from a subject, the volume is at most 40 microliters.In some embodiments, when obtained from a subject, the volume is between about 10 microliters to about 40 microliters at most. In some embodiments, when obtained from a subject, the volume is between about 10 microliters to about 100 microliters at most. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains cell-free nucleic acids in circulation. In some embodiments, the biological sample contains a total of about 25 picograms (pg) to about 250 pg of cell-free nucleic acid molecules. In some embodiments, the biological sample contains less than 300 pg of cell-free nucleic acid molecules. In some embodiments, the biological sample contains 3 ng of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10 4 ~ about 10 7 of cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at a genome equivalent between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15.

[0028] Aspects disclosed herein include: (a) obtaining a biological sample from a subject during pregnancy; (b) contacting at least one cell-free nucleic acid in the biological sample with an amplification reagent and an oligonucleotide primer that anneals to a sequence corresponding to a sex chromosome; and (c) detecting the presence or absence of an amplification product. In some embodiments, the presence or absence indicates the sex of the fetus. In some embodiments, the method further includes contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample is blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, when obtained from the subject, the biological sample has a volume of at most 300 microliters. In some embodiments, when obtained from the subject, the volume is at most 100 microliters. In some embodiments, when obtained from the subject, the volume is at most 55 microliters. In some embodiments, when obtained from the subject, the volume is at most 50 microliters. In some embodiments, when obtained from the subject, the volume is at most 40 microliters. In some embodiments, when obtained from the subject, the volume is between about 10 microliters and about 40 microliters. In some embodiments, when obtained from the subject, the volume is between about 10 microliters and about 100 microliters. In some embodiments, the biological sample obtained from the subject is capillary blood. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the biological sample contains circulating cell-free nucleic acid. In some embodiments, the biological sample contains between about 25 picograms (pg) and about 250 pg of total circulating cell-free nucleic acid molecules. In some embodiments, the biological sample contains less than 300 pg of cell-free nucleic acid molecules. In some embodiments, the biological sample contains 3 ng of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 10 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample contains at most about 104 ~about 10 7 and contains cell-free nucleic acid molecules. In some embodiments, the cell-free nucleic acid in the biological sample is about 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is at most 10 genome equivalents. In some embodiments, the cell-free nucleic acid in the biological sample is between 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 genome equivalents. In some embodiments, the biological sample obtained from the subject is collected by the subject using a finger prick. In some embodiments, the biological sample obtained from the subject is collected by the subject without using a finger prick. In some embodiments, the biological sample obtained from the subject is collected by the subject using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, the biological sample obtained from the subject is collected by: (a) a process that induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method does not consist of performing a venipuncture or withdrawing a biological sample from the venous blood of the subject.

[0029] In some embodiments, the aspects disclosed herein include: (a) obtaining a biological sample from a subject, wherein the volume of the sample is about 300 microliters or less and the biological sample includes fetal trophoblast membranes; (b) isolating fetal trophoblast membranes from the biological sample using a monoclonal antibody specific for a fetal trophoblast cell surface antigen; (c) lysing the fetal trophoblast membranes; (d) optionally purifying the fetal trophoblast nuclei, optionally including lysing the fetal trophoblast nuclei; (f) extracting fetal genomic DNA (gDNA) from the lysed fetal trophoblast membranes; (g) contacting the fetal gDNA with amplification reagents and oligonucleotide primers that anneal to sequences corresponding to a desired sequence to produce an amplification product; and (h) detecting the presence or absence of the amplification product. In some embodiments, the presence or absence indicates the gender of the fetus. In some embodiments, the method further includes contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample is blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the volume of the blood sample is 120 μl or less. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the volume of the plasma sample is 50 μl or less. In some embodiments, the volume of the plasma sample is between about 10 μl and about 40 μl. In some embodiments, the biological sample obtained from the subject is collected by the subject using a finger prick. In some embodiments, the biological sample obtained from the subject is collected by the subject without using a finger prick. In some embodiments, the biological sample obtained from the subject is collected by the subject using a device configured to lyse the intercellular junctions of the subject's epidermis.In some embodiments, a biological sample obtained from a subject is collected by: (a) a process that induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method does not consist of performing a venipuncture or withdrawing a biological sample from a subject's venous blood. The aspects disclosed herein, in some embodiments, include: (a) a step of obtaining a biological sample from a subject, wherein the biological sample contains cell-free nucleic acids; (b) a step of generating a library of nucleic acid molecules fragmented by contacting the cell-free nucleic acids with an endonuclease, thereby fragmenting at least one of the cell-free nucleic acids; (c) optionally amplifying the tagged cell-free nucleic acids; and (d) a step of sequencing at least a portion of the tagged cell-free nucleic acids to detect a desired sequence. In some embodiments, the endonuclease is a Cas enzyme. In some embodiments, the Cas enzyme is Cas9, Cas12, Cascad, and Cas13, or one or more subtypes or orthologs thereof. In some embodiments, the endonuclease is directed to the cell-free nucleic acids by a guide strand that is complementary to at least one of the cell-free nucleic acids. In some embodiments, the method further includes a step of fragmenting the cell-free nucleic acids by the endonuclease. In some embodiments, the method further includes a step of contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample is blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the volume of the blood sample is 300 μl or less. In some embodiments, the volume of the blood sample is 120 μl or less. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the volume of the plasma sample is 50 μl or less. In some embodiments, the volume of the plasma sample is between about 10 μl and about 40 μl.In some embodiments, the biological sample contains from about 25 picograms (pg) to about 250 pg of total circulating cell-free DNA. In some embodiments, the biological sample has a maximum of about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample has a maximum of about 10 4 ~ about 10 7It contains cell-free nucleic acid molecules. In some embodiments, the biological sample contains about 5 to about 100 copies of the desired sequence. In some embodiments, the biological sample obtained from a subject is collected by the subject via finger prick. In some embodiments, the biological sample obtained from a subject is collected by the subject without using finger prick. In some embodiments, the biological sample obtained from a subject is collected by the subject using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, the biological sample obtained from a subject is collected by: (a) a process that induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process that discards the first fraction of the biological sample; and (c) a process that collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method further includes a step of cleaning the surface of the percutaneous puncture site (e.g., skin) before obtaining the biological sample from the subject. In some examples, the step of cleaning includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of the finger. In some embodiments, the method does not consist of performing venipuncture or withdrawing a biological sample from the venous blood of the subject. In some embodiments, detecting the desired sequence includes detecting (i) the presence or absence of the desired sequence, or (ii) the normal expression, overexpression, or underexpression of the desired sequence. In some embodiments, detecting includes large-scale multiplex amplification. In some embodiments, the large-scale multiplex amplification assay is isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is multiplex polymerase chain reaction (mmPCR). In some embodiments, the subject is a pregnant subject, and the cell-free nucleic acid molecule includes cell-free fetal nucleic acid molecules. In some embodiments, the cell-free nucleic acid includes nucleic acid from a tumor in a tissue.In some embodiments, the cell-free nucleic acid is cell-free nucleic acid from a fetus. In some embodiments, the cell-free nucleic acid is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the cell-free nucleic acid is genomic nucleic acid from one or more pathogens. In some embodiments, the pathogen includes bacteria or a component thereof. In some embodiments, the pathogen includes a virus or a component thereof. In some embodiments, the pathogen includes a fungus or a component thereof. In some embodiments, the cell-free nucleic acid includes one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or a combination thereof.

[0030] In some embodiments, the aspects disclosed herein include: (a) obtaining a biological sample from a subject, wherein the biological sample comprises cell-free nucleic acids; (b) generating a library of nucleic acid molecules fragmented by contacting the cell-free nucleic acids with a transposase enzyme, thereby fragmenting at least one of the cell-free nucleic acids and tagging the fragmented cell-free nucleic acids with a composition; (c) optionally amplifying the tagged cell-free nucleic acids; and (d) sequencing at least a portion of the tagged cell-free nucleic acids. In some embodiments, the transposase fragment fragments the cell-free nucleic acids using a cut-and-paste mechanism. In some embodiments, the transposase is Tn5 transposase. In some embodiments, the tag is about 9 base pairs in length. In some embodiments, the method further comprises fragmenting the cell-free nucleic acids with a transposase. In some embodiments, the method further comprises tagging the fragmented cell-free nucleic acids with a transposase. In some embodiments, the method further comprises contacting the biological sample with a leukocyte stabilizer after obtaining the biological sample from the subject. In some embodiments, the biological sample is blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the volume of the blood sample is 300 μl or less. In some embodiments, the volume of the blood sample is 120 μl or less. In some embodiments, the biological sample is a plasma sample from blood. In some embodiments, the volume of the plasma sample is 50 μl or less. In some embodiments, the volume of the plasma sample is between about 10 μl and about 40 μl. In some embodiments, the biological sample contains about 25 picograms (pg) to about 250 pg of total circulating cell-free DNA. In some embodiments, the biological sample contains up to about 10 4 to about 10 9 cell-free nucleic acid molecules. In some embodiments, the biological sample contains up to about 10 4 to about 10 7It contains cell-free nucleic acid molecules. In some embodiments, the biological sample contains about 5 to about 100 copies of the desired sequence. In some embodiments, the biological sample obtained from a subject is collected by the subject using a finger prick. In some embodiments, the biological sample obtained from a subject is collected by the subject without using a finger prick. In some embodiments, the biological sample obtained from a subject is collected by the subject using a device configured to lyse the intercellular junctions of the epidermis of the subject. In some embodiments, the biological sample obtained from a subject is collected by a process comprising: (a) a process of inducing a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process of discarding the first fraction of the biological sample; and (c) a process of collecting a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the method further comprises a step of washing the surface of the percutaneous puncture site (e.g., the skin) before obtaining the biological sample from the subject. In some examples, the step of washing comprises removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of the finger. In some embodiments, the method does not consist of performing a venipuncture or withdrawing a biological sample from the venous blood of the subject. In some embodiments, detecting the desired sequence comprises (i) detecting the presence or absence of the desired sequence, or (ii) detecting the normal expression, overexpression, or underexpression of the desired sequence. In some embodiments, detecting comprises large-scale multiplex amplification. In some embodiments, the large-scale multiplex amplification assay is isothermal amplification. In some embodiments, the large-scale multiplex amplification assay is polymerase chain reaction (mmPCR). In some embodiments, the subject is a pregnant subject and the cell-free nucleic acid molecule comprises a cell-free fetal nucleic acid molecule. In some embodiments, the cell-free nucleic acid comprises nucleic acid from a tumor in a tissue.In some embodiments, the cell-free nucleic acid is cell-free nucleic acid from a fetus. In some embodiments, the cell-free nucleic acid is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the cell-free nucleic acid is genomic nucleic acid from one or more pathogens. In some embodiments, the pathogen includes bacteria or a component thereof. In some embodiments, the pathogen includes a virus or a component thereof. In some embodiments, the pathogen includes a fungus or a component thereof. In some embodiments, the cell-free nucleic acid includes one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or combinations thereof.

[0031] In some embodiments, the aspects disclosed herein provide a system comprising: (a) a sample collection device configured to collect a biological sample from a subject; (b) a sample processor configured to isolate sample components from the biological sample; (c) a nucleic acid detection device configured to detect nucleic acids in the biological sample or the sample components; and (d) a nucleic acid information output unit. In some embodiments, the system further comprises a leukocyte stabilizer. In some embodiments, the sample collection device comprises a percutaneous puncture device. In some embodiments, the percutaneous puncture device comprises at least one of a needle, a lancet, a microneedle, a vacuum, and a microneedle array. In some embodiments, the sample collection device comprises a device configured to lyse the intercellular junctions of the epidermis of the subject. In some embodiments, the sample components are selected from cells, carbohydrates, phospholipids, proteins, nucleic acids, and microvesicles. In some embodiments, the sample components are blood cells. In some embodiments, the sample components do not contain cell-free nucleic acids. In some embodiments, the sample components contain cell-free nucleic acids. In some embodiments, the cell-free nucleic acids are cell-free nucleic acids from a tumor. In some embodiments, the cell-free nucleic acids are cell-free nucleic acids from a fetus. In some embodiments, the cell-free nucleic acids are cell-free nucleic acids from a transplanted tissue or organ. In some embodiments, the cell-free nucleic acids are genomes from one or more pathogens. In some embodiments, the cell-free nucleic acids are derived from a cell type or tissue type with a lower abundance of cell-free nucleic acids compared to peripheral blood. In some embodiments, the pathogen comprises bacteria or its components. In some embodiments, the pathogen comprises a virus or its components. In some embodiments, the pathogen comprises a fungus or its components. In some embodiments, the sample components contain one or more single nucleotide polymorphisms (SNPs), one or more indels, or a combination thereof. In some embodiments, the nucleic acid detection device is configured to perform a genotyping assay. In some embodiments, the genotyping assay comprises quantitative real-time polymerase chain reaction (qPCR), a genotyping array, or automated sequencing. In some embodiments, the qPCR comprises multiplex polymerase chain reaction (mmPCR).In some embodiments, the sample component is plasma or serum. In some embodiments, the sample purification device is configured to isolate plasma from less than 1 milliliter of blood. In some embodiments, the sample purification device is configured to isolate plasma from less than 250 μl of blood. In some embodiments, the volume of the biological sample is 50 μl or less. In some embodiments, the volume of the biological sample is between about 10 μl and about 40 μl. In some embodiments, the biological sample contains about 25 pg to about 250 pg of total circulating cell-free DNA. In some embodiments, the biological sample contains about 5 to about 100 copies of the desired sequence in the biological sample or sample component. In some embodiments, the biological sample is up to about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample is up to about 10 4 ~ about 10 7It contains cell-free nucleic acid molecules. In some embodiments, the biological sample contains less than 300 pg of cell-free nucleic acid molecules. In some embodiments, the biological sample contains 3 ng of cell-free nucleic acid molecules. In some embodiments, the nucleic acid detection device includes a nucleic acid sequencing device. In some embodiments, the system further includes at least one nucleic acid amplification reagent and at least one crowding agent. In some embodiments, the system further includes at least a first tag for generating a library of cell-free nucleic acids from the biological sample and at least one amplification reagent. In some embodiments, the at least one nucleic acid amplification reagent includes primers, polymerases, and combinations thereof. In some embodiments, the nucleic acid detection device generates ligation-capable nucleic acids by one or more steps including: (a) (i) a step of generating blunt ends of nucleic acids, in which, depending on the embodiment, 5' overhangs or 3' recessed ends are removed using one or more polymerases and one or more exonucleases; (ii) a step of dephosphorylating the blunt ends of the nucleic acids; (iii) a step of contacting the nucleic acids with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the nucleic acids; or (iv) a step of using a ligase to repair or remove damaged nucleic acids in the nucleic acids; and (b) ligating the ligation-capable nucleic acids to adapter oligonucleotides by contacting the ligation-capable nucleic acids with adapter oligonucleotides in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer, so as to generate a library of tagged nucleic acids. In some embodiments, the one or more polymerases include T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases include T4 polynucleotide kinase or exonuclease III. In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein.In some embodiments, the ligase includes T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or an Sso7 ligase fusion protein. In some embodiments, the crowding reagent includes polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof. In some embodiments, the small molecule enhancer includes dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof. In some embodiments, the ligation of (b) includes blunt-end ligation or single-base overhang ligation. In some embodiments, the adapter oligonucleotide includes a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a degradable adapter, a blocked self-ligating adapter, or a barcoded adapter, or a combination thereof. In some embodiments, the system is further configured to pool two or more biological samples, and each sample is obtained from a different subject. In some embodiments, the nucleic acid detection device is further configured to count tags to detect the representation of the desired nucleic acid in the sample. In some embodiments, the nucleic acid sequence output is selected from a wireless communication device, a wired communication device, a cable port, and an electronic display. In some embodiments, all components of the system are present at one location. In some embodiments, all components of the system are housed in one device. In some embodiments, the sample collection device is located at a first location, and at least one of the sample purification device and the nucleic acid detection device is at a second location. In some embodiments, the sample collection device, and at least one of the sample purification device and the nucleic acid detection device are at the same location. In some embodiments, the sample purification device includes a filter. In some embodiments, the filter has a pore size of about 0.05 microns to about 2 microns. In some embodiments, the system includes a transport compartment or a storage compartment for transporting or storing at least a portion of the biological sample.In some embodiments, the transport compartment or storage compartment includes an absorbent pad, a fluid container, a sample preservative, or a combination thereof. In some embodiments, the system further includes a nucleic acid amplifier configured to amplify nucleic acids from a sample component or a biological sample, and in some embodiments, the nucleic acid detection device is further configured to detect the amplified nucleic acids in the biological sample or sample component. In some embodiments, the nucleic acid amplifier is a polymerase chain reaction (PCR) device. In some embodiments, the PCR device is a large-scale multiplex PCR device (mmPCR). In some embodiments, the biological sample is not derived from the venous blood of a subject.

[0032] In some embodiments, the aspects disclosed herein provide a system comprising: (a) a sample collection device configured to collect a biological sample of about 1-100 microliters (μl) from a subject; (b) a sample processor configured to isolate sample components from the biological sample; (c) a detector configured to detect epigenetic modifications in the biological sample or sample components; and (d) an information output unit. In some embodiments, epigenetic modifications include DNA methylation, histone methylation, histone, ubiquitination, histone acetylation, histone phosphorylation, microRNA (miRNA) at a locus. In some embodiments, DNA methylation includes CpG methylation or CpH methylation. In some embodiments, the locus includes a promoter or regulatory element of a gene. In some embodiments, the locus includes a variable length terminal repeat (LTR). In some embodiments, the locus includes cell-free DNA or a fragment thereof. In some embodiments, the locus includes a single nucleotide polymorphism (SNP). In some embodiments, histone acetylation is indicated by the presence or level of histone deacetylase. In some embodiments, the histone modification is in a histone selected from the group consisting of histone 2A (H2A), histone 2B (H2B), histone 3 (H3), and histone 4 (H4). In some embodiments, histone methylation is methylation of H3 lysine 4 (H3K4me2). In some embodiments, histone acetylation is deacetylation at H4. In some embodiments, the miRNA is selected from the group consisting of miR-21, miR-126, mi-R142, mi-R146a, mi-R12a, mi-R181a, miR-29c, miR-29a, miR-29b, miR-101, miRNA-155, and miR-148a. In some embodiments, the biological sample includes blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the blood includes capillary blood. In some embodiments, the capillary blood includes blood of 40 microliters or less. In some embodiments, the biological sample obtained from the subject is collected by percutaneous puncture.In some embodiments, the biological sample obtained from the subject was not collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject was collected using a device configured to lyse the intercellular junctions of the subject's epidermis. In some embodiments, the sample collection device is such that the biological sample obtained from the subject is obtained by a process that (a) induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) discards the first fraction of the biological sample; and (c) collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the sample collection device is configured to clean the surface of the percutaneous puncture site (e.g., the skin) prior to obtaining a biological sample from the subject. In some examples, the cleaning step includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissue surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of the finger. In some embodiments, the system further includes a leukocyte stabilizer. In some embodiments, the biological sample is not derived from the subject's venous blood.

[0033] In some embodiments, the aspects disclosed herein are a device comprising: (a) a sample collection device for obtaining a biological sample from a subject in need thereof; (b) a sample purification device for removing cells from the biological sample to generate a cell-depleted sample; and (c) a nucleic acid detection device configured to detect a plurality of cell-free DNA fragments in the cell-depleted sample. In some embodiments, the device further comprises a leukocyte stabilizer. In some embodiments, the sample collection device is configured to lyse the intercellular junctions of the epidermis of the subject. In some embodiments, the sample collection device is configured to collect a sample from a percutaneous puncture. In some embodiments, the first sequence is present on a first cell-free DNA fragment of the plurality of cell-free DNA fragments, the second sequence is present on a second cell-free DNA fragment of the plurality of cell-free DNA fragments, and in some embodiments, depending on the embodiment, the first sequence is at least 80% identical to the second sequence. In some embodiments, at least one of the first sequence and the second sequence is repeated at least twice in the genome of the subject. In some embodiments, the first sequence and the second sequence are each at least 10 nucleotides in length. In some embodiments, the first sequence is on a first chromosome and the second sequence is on a second chromosome. In some embodiments, the first sequence and the second sequence are on the same chromosome but are separated by at least one nucleotide. In some embodiments, the first sequence and the second sequence are functionally linked. In some embodiments, the nucleic acid detection device comprises at least one detection reagent. In some embodiments, the at least one detection reagent comprises an oligonucleotide probe capable of detecting at least one of the plurality of cell-free DNA fragments. In some embodiments, the at least one detection reagent is capable of detecting a fetal epigenetic signature. In some embodiments, the fetal epigenetic signature comprises methylation of nucleic acids. In some embodiments, the methylation is allele-specific.In some embodiments, the device further includes a nucleic acid amplifier configured to amplify nucleic acids from a sample component or a biological sample. In some embodiments, the nucleic acid detection device is further configured to detect the amplified nucleic acids in the biological sample or sample component. In some embodiments, the nucleic acid amplifier is an isothermal polymerase chain reaction (PCR) device. In some embodiments, the isothermal PCR device is a large-scale multiplex PCR device (mmPCR). In some embodiments, the device further includes a genotyper configured to compare a plurality of detected cell-free DNA fragments with known genotypes. In some embodiments, the plurality of cell-free DNA fragments include fetal components and the known genotype is the paternal genotype. In some embodiments, the nucleic acid amplifier includes at least one nucleic acid amplification reagent and a pair of primers for amplifying a first sequence and a second sequence. In some embodiments, the nucleic acid detection device includes a nucleic acid sequencer. In some embodiments, the nucleic acid sequencer includes a signal detector. In some embodiments, the nucleic acid detection device is a lateral flow strip. In some embodiments, the cell-free DNA includes one or more single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or combinations thereof. In some embodiments, the cell-free DNA is cell-free nucleic acid from a tumor. In some embodiments, the cell-free DNA is cell-free nucleic acid from a fetus. In some embodiments, the cell-free DNA is cell-free nucleic acid from a transplanted tissue or organ. In some embodiments, the cell-free nucleic acid is derived from a cell type or tissue type with a lower abundance of cell-free nucleic acid compared to peripheral blood. In some embodiments, the cell-free DNA is derived from one or more pathogens. In some embodiments, the pathogen includes bacteria or components thereof. In some embodiments, the pathogen includes a virus or components thereof. In some embodiments, the pathogen includes a fungus or components thereof. In some embodiments, the sample purification device includes a filter, and in some embodiments, the filter has a pore size of about 0.05 microns to about 2 microns. In some embodiments, the filter is a vertical filter.In some embodiments, the sample purification device comprises a binding moiety selected from antibodies, antigen-binding antibody fragments, ligands, receptors, peptides, small molecules, and combinations thereof. In some embodiments, the binding moiety can bind to extracellular vesicles. In some embodiments, the nucleic acid detection device is configured to generate a library of tagged cell-free DNA by: (a) (i) generating blunt ends of cell-free DNA fragments, which in some embodiments involves removing 5' overhangs or 3' recessed ends using one or more polymerases and one or more exonucleases; (ii) dephosphorylating the blunt ends of the cell-free DNA fragments; (iii) contacting the cell-free DNA fragments with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cell-free DNA fragments; or (iv) repairing or removing DNA damage in the cell-free DNA fragments using a ligase; and (v) ligating the adapter oligonucleotide to the ligation-capable cell-free DNA fragments in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer by contacting the ligation-capable cell-free DNA fragments with the adapter oligonucleotide. In some embodiments, the one or more polymerases include T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases include T4 polynucleotide kinase or exonuclease III. In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein. In some embodiments, the ligase includes T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7 ligase fusion protein.In some embodiments, the crowding reagent includes polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof. In some embodiments, the small molecule enhancer includes dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof. In some embodiments, the ligation of (b) includes blunt-end ligation or single-base overhang ligation. In some embodiments, the adapter oligonucleotide includes a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a degradable adapter, a blocked self-ligating adapter, or a barcoded adapter, or a combination thereof. In some embodiments, the device is further configured to pool two or more biological samples, and each sample is obtained from a different subject. In some embodiments, the nucleic acid detection device is further configured to count tags to detect the representation of the desired nucleic acid in the sample. In some embodiments, the device further includes a nucleic acid sequence output unit including a wireless communication device, a wired communication device, a cable port, or an electronic display. In some embodiments, the device is housed in a single housing. In some embodiments, the device operates at room temperature. In some embodiments, the device can detect multiple biomarkers in a cell-free sample within about 5 minutes to about 20 minutes after receiving the biological fluid. In some embodiments, the device further includes a communication connection portion. In some embodiments, the biological sample includes blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the blood includes capillary blood. In some embodiments, the sample purification device is configured to isolate plasma from less than 250 μl of blood. In some embodiments, the volume of the biological sample is 50 μl or less. In some embodiments, the volume of the biological sample is between about 10 μl and about 40 μl. In some embodiments, the biological sample contains about 25 pg to about 250 pg of total circulating cell-free DNA.In some embodiments, the biological sample contains about 5 to about 100 copies of the desired sequence in the biological sample or sample component. In some embodiments, the biological sample is up to about 10. 4 ~ about 10 9 of cell-free nucleic acid molecules. In some embodiments, the biological sample is up to about 10 4 ~ about 10 7It contains cell-free nucleic acid molecules. In some embodiments, the biological sample contains less than 300 pg of cell-free nucleic acid molecules. In some embodiments, the biological sample contains 3 ng of cell-free nucleic acid molecules. In some embodiments, the sample collection device is configured such that the biological sample obtained from the subject is collected by a process that (a) induces a first percutaneous puncture to generate a first fraction of the biological sample; (b) discards the first fraction of the biological sample; and (c) collects a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis. In some embodiments, the sample collection device is configured to wash the surface of the percutaneous puncture site (e.g., the skin) before obtaining the biological sample from the subject. In some examples, the washing step includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the unwanted contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of the finger. The aspects disclosed herein, in some embodiments, provide a system comprising: (a) a sample collection device configured to collect about 1-100 microliters (μl) of a biological sample from a subject; (b) a sample processor configured to isolate sample components from the biological sample; (c) a detector configured to detect epigenetic modifications in the biological sample or sample components; and (d) an information output unit. In some embodiments, the sample collection device is configured to collect the sample from a percutaneous puncture. In some embodiments, the epigenetic modifications include DNA methylation, histone methylation, histone, ubiquitination, histone acetylation, histone phosphorylation, microRNA (miRNA) at a locus. In some embodiments, the DNA methylation includes CpG methylation or CpH methylation. In some embodiments, the locus includes a promoter or regulatory element of a gene. In some embodiments, the locus includes a variable length terminal repeat (LTR).In some embodiments, the locus comprises cell-free DNA or a fragment thereof. In some embodiments, the locus comprises a single nucleotide polymorphism (SNP). In some embodiments, histone acetylation is indicated by the presence or level of histone deacetylase. In some embodiments, the histone modification is selected from the group consisting of histone 2A (H2A), histone 2B (H2B), histone 3 (H3), and histone 4 (H4). It is in the histone to be selected. In some embodiments, histone methylation is methylation of H3 lysine 4 (H3K4me2). In some embodiments, histone acetylation is deacetylation at H4. In some embodiments, the miRNA is selected from the group consisting of miR-21, miR-126, mi-R142, mi-R146a, mi-R12a, mi-R181a, miR-29c, miR-29a, miR-29b, miR-101, miRNA-155, and miR-148a. In some embodiments, the biological sample includes blood, plasma, serum, urine, interstitial fluid, vaginal cells, vaginal fluid, cervical cells, buccal cells, or saliva. In some embodiments, the blood includes capillary blood. In some embodiments, the capillary blood includes blood of 40 microliters or less. In some embodiments, the device further includes a step of pooling two or more biological samples, and each sample is obtained from a different subject. In some embodiments, the biological sample obtained from the subject is collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is not collected by percutaneous puncture. In some embodiments, the biological sample obtained from the subject is collected using a device configured to lyse the intercellular junctions of the epidermis of the subject. In some embodiments, the biological sample obtained from the subject is collected by: (a) a process of inducing a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process of discarding the first fraction of the biological sample; and (c) a process of collecting a second fraction of the biological sample, thereby reducing or removing fluid contamination. In some embodiments, the device further includes a leukocyte stabilizer. In some embodiments, the sample collection device is such that the biological sample obtained from the subject is collected by: (a) a process of inducing a first percutaneous puncture to generate a first fraction of the biological sample; (b) a process of discarding the first fraction of the biological sample; and (c) a process of collecting a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by leukocyte lysis.In some embodiments, the sample collection device is configured to clean the surface of the percutaneous puncture site (e.g., the skin) before obtaining a biological sample from a subject. In some examples, the cleaning step includes removing or reducing undesirable contaminants. In some examples, the undesirable contaminants include DNA from the percutaneous puncture site. In some examples, the undesirable contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some examples, the percutaneous puncture site is the skin of a finger. In some embodiments, the biological sample is not derived from the venous blood of the subject.

[0034] Aspects disclosed herein include a method of increasing the relative amount of a target nucleic acid in a biological sample obtained from a subject, the method comprising: (a) inducing a percutaneous puncture at a site to generate a first fraction and a second fraction of the biological sample; (b) a process of discarding the first fraction of the biological sample; and (c) collecting the second fraction of the biological sample, thereby reducing or removing contamination or nucleic acid damage of the biological sample, wherein the first fraction contains a lower fraction of the target nucleic acid compared to the fraction of the target nucleic acid in the second fraction. In some embodiments, the method further comprises cleaning the site before inducing the percutaneous puncture, thereby removing or reducing undesirable contaminants. In some embodiments, the undesirable contaminants include DNA from the percutaneous puncture site. In some examples, the undesirable contaminants include DNA from cells or tissues surrounding the percutaneous puncture site. In some examples, the DNA is damaged. In some examples, the DNA is not damaged. In some embodiments, the percutaneous puncture site is the skin of a finger. In some embodiments, the contamination includes nucleic acids from tissues surrounding the site. In some embodiments, the nucleic acid damage includes damage to non-apoptotic DNA in the biological sample. In some embodiments, the biological sample is not derived from the venous blood of the subject.

[0035] Other objects, features, and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating some embodiments of the present disclosure, are by way of illustration only and not restrictive. Many changes and modifications within the scope of the present disclosure may be made without departing from its spirit, and the present disclosure includes all such modifications. Furthermore, aspects of one embodiment may be utilized in various other embodiments. Incorporation by reference

[0036] All patent publications, patents, and patent applications described herein are hereby incorporated by reference to the extent that each patent publication, patent, and patent application is specifically and individually incorporated by reference.

Brief Description of the Drawings

[0037] The new features of the methods, devices, systems, and kits disclosed herein are particularly set forth in detail in the appended claims. A better understanding of the features and advantages of the devices, systems, and kits disclosed herein can be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the devices, systems, and kits disclosed herein are used, and the accompanying drawings.

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[0038] Specific terms The following description is provided to assist in the understanding of the methods, systems, and kits disclosed herein. The following description of the terms used herein is not intended to limit the definitions of these terms. These terms are further described and exemplified throughout this application.

[0039] Generally, the terms “cell-free polynucleotide” and “cell-free nucleic acid” as used interchangeably herein refer to polynucleotides and nucleic acids that can be isolated from a sample without extracting the polynucleotide or nucleic acid from a cell. Cell-free nucleic acids can include DNA. Cell-free nucleic acids can include RNA. Cell-free nucleic acids are nucleic acids that are not encapsulated within a cell membrane, i.e., not enclosed within a cellular compartment. In some embodiments, cell-free nucleic acids are nucleic acids that are not bound by a cell membrane and that circulate or are present in blood or other fluids. In some embodiments, cell-free nucleic acids are cell-free prior to and / or at the time of collection of the biological sample containing them and are not released from cells as a result of intentional or unintentional human manipulation of the sample, including manipulations performed on the sample at the time of or after collection. In some examples, cell-free nucleic acids are produced in cells and are released from cells by physical means including, for example, apoptosis, and non-apoptotic cell death, necrosis, autophagy, spontaneous release (e.g., of DNA / RNA lipoprotein complexes), secretion, and / or mitotic cell death. In some embodiments, cell-free nucleic acids include nucleic acids released from cells by biological mechanisms (e.g., apoptosis, cell secretion, vesicular release). In further or additional embodiments, cell-free nucleic acids are not nucleic acids extracted from cells by human manipulation of cells or sample processing (e.g., cell membrane disruption, lysis, vortexing, shearing, etc.).

[0040] In some instances, the cell-free nucleic acid is cell-free fetal nucleic acid. Generally, as used herein, the term "cell-free fetal nucleic acid" refers to the cell-free nucleic acids described herein, which are derived from cells containing fetal DNA. In a pregnant female, cell-free DNA derived from the placenta may contribute a significant portion of the total amount of cell-free DNA. In most cases, since placental DNA is highly similar to fetal DNA, placental DNA is often an excellent surrogate for fetal DNA. Such applications as chorionic villus sampling have utilized this fact to establish diagnostic applications. Often, much of the cell-free fetal nucleic acid is found in a maternal biological sample as a result of the regular shedding of placental tissue during pregnancy in the subject being pregnant. Often, many of the cells in the shed placental tissue are cells containing fetal DNA. Cells shed from the placenta release fetal nucleic acids. Thus, in some instances, the cell-free fetal nucleic acids disclosed herein are nucleic acids released from placental cells.

[0041] As used herein, the term "cellular nucleic acid" refers to polynucleotides that are contained within cells or released from cells by manipulation of a biological sample. Non-limiting examples of manipulation of a biological sample include subjecting the biological sample, when obtained, to centrifugation, vortexing, shearing, mixing, lysing, and addition of reagents not present in the biological sample (e.g., surfactants, buffers, salts, enzymes). In some instances, cellular nucleic acids are nucleic acids released from cells by disruption or lysis of the cells by a machine, human, or robot. In some instances, cellular nucleic acids (nucleic acids contained by cells) are intentionally or unintentionally released from cells by the devices and methods disclosed herein. However, these are not considered to be "cell-free nucleic acids" as used herein. In some instances, the devices, systems, kits, and methods disclosed herein result in the analysis of cell-free nucleic acids in a biological sample and, in this process, also analyze cellular nucleic acids.

[0042] As used herein, the term "biomarker" generally refers to any marker of the biology or state of a subject. A biomarker can be an index or consequence of a disease or disorder. A biomarker can be an index of health. A biomarker can be an index of a genetic abnormality or genetic disease. A biomarker can be a circulating biomarker (e.g., found in a body fluid such as blood). A biomarker can be a tissue biomarker (e.g., found in a solid organ such as the liver or bone marrow). Non-limiting examples of biomarkers include nucleic acids, epigenetic modifications, proteins, peptides, antibodies, antibody fragments, lipids, fatty acids, sterols, polysaccharides, carbohydrates, virus particles, microbial particles. In some cases, a biomarker may further include whole cells or cell fragments.

[0043] As used herein, the term "tag" generally refers to a molecule that can be used to identify, detect, or isolate a desired nucleic acid. Unless otherwise defined, the term "tag" can be used interchangeably with other terms such as "label", "adapter", "oligo", and "barcode". However, the term "adapter" can be used to ligate the two ends of a nucleic acid or multiple nucleic acids without acting as a tag.

[0044] As used herein, the term "gene information" generally refers to one or more nucleic acid sequences. In some examples, gene information can be a single nucleotide or amino acid. For example, gene information can be the presence (or absence) of a single nucleotide polymorphism. Unless otherwise defined, the term "gene information" may also refer to epigenetic modification patterns, gene expression data, and protein expression data. In some examples, the presence, absence, or amount of a biomarker provides gene information. For example, a cholesterol value can indicate a genetic form of hypercholesterolemia. Thus, gene information should not be limited to nucleic acid sequences.

[0045] As used herein, the term "gene mutation" generally refers to a modification of the nucleotide sequence of the genome. Gene mutations are distinct from natural variations or allelic differences. Gene mutations may be found in less than 10% of the subjects of a species. Gene mutations may be found in less than 5% of the subjects of a species. Gene mutations may be found in less than 1% of the subjects of a species. Gene mutations in a subject may cause a disease or disorder in the subject. Gene mutations may result in a frameshift of the protein-coding sequence. Gene mutations may result in a deletion of at least a portion of the protein-coding sequence. Gene mutations may result in the loss of a stop codon in the protein-coding sequence. Gene mutations may result in a premature stop codon in the protein-coding sequence. Gene mutations may result in a sequence encoding a misfolded protein. Gene mutations may result in a sequence encoding a dysfunctional or non-functional protein (e.g., loss of binding or enzymatic activity). Gene mutations may result in a sequence encoding a hyperactive protein (e.g., increased binding or enzymatic activity). Gene mutations may affect a single nucleotide (e.g., a single nucleotide variation or single nucleotide polymorphism). Gene mutations may affect multiple nucleotides (e.g., frameshift, translocation).

[0046] As used herein, the term "specific for" refers to a sequence or biomarker that is found only in, on, or at the location where it is specific among those for which its sequence or biomarker is specific. For example, if a sequence is specific for the Y chromosome, it means that it is found only on the Y chromosome and not on other chromosomes.

[0047] As used herein, the terms "normal individual" and "normal subject" refer to a subject who does not have a disease or disorder. For example, if the described method or device is being used to detect a type of cancer, a normal subject does not have that type of cancer. A normal subject may not have any cancer at all. In some instances, a normal subject is not diagnosed with any disorder or disease. In some instances, a normal subject does not have a known genetic mutation. In some instances, a normal subject does not have a genetic mutation that results in a detectable phenotype that would distinguish the subject from a normal subject who does not have a known genetic mutation. In some instances, a normal subject is not infected with a pathogen. In some instances, a normal subject is infected with a pathogen but does not have a known genetic mutation.

[0048] As used herein, the term "genomic equivalent" generally refers to the amount of DNA that needs to be present in a purified sample to ensure that all genes are present.

[0049] As used herein, the term "tissue-specific" or the phrase "is specific to a tissue" generally refers to a polynucleotide that is predominantly expressed in a particular tissue. Often, the methods, systems, and kits disclosed herein utilize cell-free tissue-specific polynucleotides. The cell-free tissue-specific polynucleotides described herein are polynucleotides expressed at levels that can be quantified in a body fluid upon injury or disease of the tissue or organ in which they are expressed. In some cases, the presence of the cell-free tissue-specific polynucleotides disclosed herein in a body fluid is due to the release of the cell-free tissue-specific polynucleotides upon injury or disease of the tissue or organ, and not due to fluctuations in the expression of the cell-free tissue-specific polynucleotides. An increase in the level of the cell-free tissue-specific polynucleotides disclosed herein may indicate damage to the corresponding tissue or organ. In some examples, the cell-free polynucleotides disclosed herein are expressed / generated in multiple tissues, but are expressed / generated at tissue-specific levels in at least one of those tissues. In these examples, the absolute or relative amount of the cell-free tissue-specific polynucleotides indicates damage to a particular tissue or organ, a disease of a particular tissue or organ, or a collection of tissues or organs. Alternatively, or additionally, a tissue-specific polynucleotide is a nucleic acid having a tissue-specific modification. A tissue-specific polynucleotide may include RNA. A tissue-specific polynucleotide may include DNA. By way of non-limiting example, the tissue-specific polynucleotides or markers disclosed herein include DNA molecules (e.g., a gene or a portion of a non-coding region) having a tissue-specific methylation pattern. In other words, the polynucleotides and markers may be expressed similarly in many tissues, or throughout a subject, but the modification is tissue-specific. Generally, the tissue-specific polynucleotides or their levels disclosed herein are not disease-specific. Generally, the tissue-specific polynucleotides disclosed herein do not encode proteins involved in disease mechanisms.

[0050] In some examples, a tissue-specific polynucleotide is present in a subject's tissue in a greater amount than it is present in the subject's blood. In some examples, RNA is present in a subject's tissue in a greater amount than it is present in blood cells. In some examples, a tissue-specific polynucleotide is not expressed by blood cells. In some examples, the presence of a tissue-specific polynucleotide is at least two-fold greater in tissue than in blood. In some examples, the presence of a tissue-specific polynucleotide is at least five-fold greater in tissue than in blood. In some examples, the presence of a tissue-specific polynucleotide is at least ten-fold greater in tissue than in blood.

[0051] In some examples, the presence of a tissue-specific polynucleotide is at least three-fold greater in the above tissue than in any other tissue of the subject. In some examples, the presence of a tissue-specific polynucleotide is at least five-fold greater in the above tissue than in any other tissue. In some examples, the presence of a tissue-specific polynucleotide is at least ten-fold greater in the above tissue than in any other tissue. In some examples, the presence of a tissue-specific polynucleotide is at least three-fold greater in two or fewer tissues than in any other tissue. In some examples, the presence of a tissue-specific polynucleotide is at least five-fold greater in two or fewer tissues than in any other tissue. In some examples, the presence of a tissue-specific polynucleotide is at least ten-fold greater in two or fewer tissues than in any other tissue. In some examples, the presence of a tissue-specific polynucleotide is at least three-fold greater in three or fewer tissues than in any other tissue. In some examples, the presence of a tissue-specific polynucleotide is at least five-fold greater in three or fewer tissues than in any other tissue. In some examples, the presence of a tissue-specific polynucleotide is at least ten-fold greater in three or fewer tissues than in any other tissue.

[0052] In some examples, the tissue-specific polynucleotide is specific to the target cell type. In some examples, the presence of the tissue-specific polynucleotide is at least 3-fold more in the target cell type than in non-target cell types. In some examples, the presence of the tissue-specific polynucleotide is at least 5-fold more in the target cell type than in other non-target cell types. In some examples, the presence of the tissue-specific polynucleotide is at least 10-fold more in the target cell type than in non-target cell types. In some examples, the presence of the tissue-specific polynucleotide is at least 3-fold more in at most two target cell types than in non-target cell types. In some examples, the presence of the tissue-specific polynucleotide is at least 5-fold more in at most two target cell types than in non-target cell types. In some examples, the RNA is expressed at least 10-fold more in at most two target cell types than in non-target cell types. In some examples, the presence of the tissue-specific polynucleotide is at least 3-fold more in at most three target cell types than in non-target cell types. In some examples, the presence of the tissue-specific polynucleotide is at least 5-fold more in at most three target cell types than in non-target cell types. In some examples, the presence of the tissue-specific polynucleotide is at least 10-fold more in at most three target cell types than in non-target cell types.

[0053] As used herein, the terms “isolate,” “purify,” “remove,” “capture,” and “separate” may all be used interchangeably unless otherwise specified.

[0054] As used herein, the terms “clinic,” “clinic environment,” “laboratory,” or “laboratory environment” refer to a hospital, clinic, pharmacy, research institution, pathology laboratory, or other for-profit business environment where trained personnel are employed to process and / or analyze biological samples and / or environmental samples. These terms are contrasted with point-of-care, remote, home, school, and non-profit, non-facility environments.

[0055] As used herein, a number followed by the term "about" refers to that number plus or minus 10% of that number. The term "about" when used in the context of a range refers to a range that is minus 10% of the lowest value and plus 10% of the highest value.

[0056] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "sample" includes plural samples, including mixtures thereof.

[0057] The term "accuracy" must be given its broadest definition in view of the specification. However, the term "accuracy" can be used to refer to a statistical measure of how well a binary classification test identifies or excludes conditions. As used herein, the term "accuracy" may further refer to the proportion of correct results (both true positives and true negatives) among all samples tested. As used herein, the term "accuracy" may include accuracy as determined by the "Rand accuracy" or "Rand index".

[0058] As used herein, the terms "homologous", "homology", or "percent homology" describe the sequence similarity of a first amino acid or nucleic acid sequence to a second amino acid or nucleic acid sequence. In some instances, homology can be determined using the equations described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such equations are incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). The percent sequence homology can be determined using the most recent version of BLAST as of the filing date of the present application. In some instances, two or more sequences are homologous if they share at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity when aligned by comparing the sequences over a comparison window or over a specified region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. In some cases, two or more sequences can be homologous if they share at most 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity. Preferably, the % identity or homology is present in a region that is at least 16 amino acids or nucleotides in length, or in some cases, in a region that is about 50 to about 100 amino acids or nucleotides in length. In some cases, the % identity or homology is present in a region that is about 100 to about 1000 amino acids or nucleotides in length.In some cases, two or more sequences may be homologous and may share at least 20% identity over at least 100 amino acids of the sequences. For sequence comparison, generally, one sequence acts as a reference sequence against which a test sequence can be compared. When a sequence comparison algorithm is used to input the test sequence and the reference sequence into a computer, subsequent coordinates can be specified as needed, and the parameters of the sequence algorithm program can be specified. Any suitable algorithm can be used, including but not limited to the Smith-Waterman alignment algorithm, Viterbi, Bayesians, Hidden Markov, etc. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm can be used to calculate the percent sequence identity of the test sequence to the reference sequence based on the program parameters. Any suitable algorithm is used to calculate the percent identity. Some programs calculate the percent identity, for example, as the total number of aligned positions of identical residues divided by the total number of aligned positions. A "comparison window", as used herein, includes reference to any one segment of a number of adjacent or non-adjacent positions that may span from 10 to 600 positions. In some cases, the comparison window may include at least 10, 20, 50, 100, 200, 300, 400, 500, or 600 positions. In some cases, the comparison window may include at most 10, 20, 50, 100, 200, 300, 400, 500, or 600 positions. In some cases, the comparison window may include at least 50 to 200 positions, or at least 100 to 150 positions, where the sequences can be compared to the reference sequence of the same number of adjacent or non-adjacent positions after the two sequences are optimally aligned. Methods of aligning sequences for comparison are well known in the art.Optimal alignment of arrays for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity methods of Pearson and Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al, eds. 1995 supplement)). In some cases, the comparison window can include any subset of the entire alignment, any adjacent positions in the primary sequence, positions that are adjacent in three-dimensional space but not adjacent in the primary sequence, or any other subset of 1 to all residues in the alignment.

[0059] As used herein, the terms overexpression and underexpression generally refer to a difference between the expression in a sample and the control expression of a target nucleic acid. The expression can be significantly less than the control expression and, thus, can be an underevaluation. The expression can be significantly greater than the control expression and, thus, can be an overevaluation. Significance can be statistical. Methods for measuring statistical significance are well known in the art. By way of non-limiting example, statistical significance was performed using a standard two-sided t-test (*: p < 0.05; ** p < 0.01).

[0060] As used herein, the term "cloud" refers to shared or sharable storage of electronic data. The cloud can be used to archive, share, and analyze electronic data.

[0061] Throughout this application, there are references to the phrases "nucleic acid corresponding to a chromosome" and "sequence corresponding to a chromosome." As used herein, these phrases are intended to convey that a "nucleic acid corresponding to a chromosome" is represented by a nucleic acid sequence that is identical or homologous to the sequence found in that chromosome. The term "homologous" is as defined in the foregoing description.

[0062] As used herein, the term "single nucleotide polymorphism (SNP)" refers to a single nucleotide that may differ between the genomes of two members of the same species. The use of this term is not meant to imply any limitation on the frequency at which each variant occurs. In some examples, an SNP is a single allele, two alleles, three alleles, or four alleles.

[0063] As used herein, the term "indel" refers to an insertion or deletion of a nucleobase that may differ between the genomes of two members of the same species. In some examples, an indel is a single allele, two alleles, three alleles, or four alleles. In some examples, an insertion includes one nucleobase, two nucleobases, three nucleobases, four nucleobases, five nucleobases, or more.

[0064] Throughout this application, chromosomal positions are described. These position numbers refer to Genome Build hg38 (UCSC) and GRCh38 (NCBI). A genome build is sometimes referred to in the art as a reference genome or reference assembly. It may be obtained from multiple subjects. It is understood that there are multiple reference assemblies available and that more reference assemblies may be generated over time. However, one of ordinary skill in the art will be able to determine the relative positions provided herein in another genome build or reference genome.

Mode for Carrying Out the Invention

[0065] Conventionally, genetic testing has been performed in a laboratory or clinic setting. However, in many cases where genetic testing is beneficial, access to a laboratory or clinic is unavailable or not practical. Very complex tests such as the analysis of circulating tumor DNA or fetal DNA testing are rare due to limited access to such tests (e.g., requirements for venipuncture, timing, required appointments, distance to the hospital / laboratory), and the cost of such tests (e.g., cost of venipuncture, processing of a few milliliters of sample, sample tubes and reagents, transportation, especially cooled transportation). Therefore, genetic testing that can be performed at the location where it is needed (e.g., locations remote from laboratories and clinics) is desirable. Genetic testing performed at the location where it is needed (e.g., home, school, farm) is preferably cost-effective and can be easily performed by untrained individuals. Genetic testing at the location where it is needed preferably requires only a small amount of biological sample. Conventionally, genetic testing requires venipuncture (phlebotomy) to obtain several milliliters of blood containing sufficient DNA for analysis. However, venipuncture is not practical at the location where it is needed. Ideally, genetic testing would require only an amount of blood achieved, for example, through capillary blood collection by a percutaneous device or other means. This means that devices and methods for genetic testing at the location where it is needed need to be designed to function with ultra-low volume sample input and low-abundance target molecules that are the subject of detection.

[0066] Conventionally, genetic testing also required percutaneous puncture. However, percutaneous puncture can cause inconvenience, discomfort, and in some cases pain to the subject, regardless of the amount of sample. Therefore, a genetic testing device that avoids the need for percutaneous puncture is desired.

[0067] In addition to accommodating ultra - low sample input volumes, it is desirable to perform genetic testing that can analyze cell - free nucleic acids (DNA and RNA) in circulation, such as cell - free fetal DNA, circulating tumor DNA, circulating DNA from transplanted donor organs, and circulating DNA released from specific tissues, as part of health - related issues, disease progression, or treatment response. However, the analysis of cell - free nucleic acids in circulation is very difficult because of their short half - lives and low abundance. Additionally, if care is not taken with the sample to avoid leukocyte lysis, cell - free nucleic acids in the blood can be diluted by DNA released from leukocytes. Leukocyte DNA creates background noise during the detection of cell - free nucleic acids, reducing the sensitivity and specificity of the assay.

[0068] The devices, systems, kits, and methods disclosed herein overcome these challenges by combining gentle and efficient processing of small samples (e.g., less than 1 ml) with unique target region selection and assay design that take advantage of the highly fragmented nature of cell - free DNA (cfDNA). For example, the devices, systems, kits, and methods disclosed herein can provide reliable genetic information from a single finger prick. In some embodiments, the reliable genetic information is obtained from a single finger prick after discarding the first perfusion of capillary blood. In other embodiments, the devices, systems, kits, and methods provide reliable genetic information without the need for percutaneous puncture, for example, by lysing the tight skin bond so that a fluid containing reliable genetic information can be extracted from the skin without the need for percutaneous puncture.

[0069] The devices, systems, kits, and methods disclosed herein provide for the analysis of multiple target regions along a target gene with sufficient spacing such that the target regions are likely to be physically separated when the target gene is fragmented during circulation. Thus, while the above limitations of statistical sampling exist for the individual long DNA fragments conventionally analyzed in genetic testing, the sampling statistics vary favorably for cfDNA fragments. There is only a total of one genomic equivalent present in a capillary blood sample, but there are many individual cfDNA fragments. As a result, amplification of sensitivity can be achieved with ultra-trace amounts of input.

[0070] As an example, if 20 target regions are present along a genomic region and are sufficiently spaced such that they can be independently analyzed and detected when fragmented, the amount of input required to have at least one target region in 99% of the total samples varies from 140 microliters (μl) to 25 μl, significantly increasing sensitivity. In some examples, the target regions contain the same or similar sequences. These target regions can be referred to as copies.

[0071] In other examples, the target regions do not share similar sequences but may share another feature such as a similar epigenetic state. For example, the target regions can have different sequences but all be hypermethylated. Regardless of the basis for similarity between regions, they are appropriately spaced to affect the fragmentation pattern of circulating cell-free DNA, generating many circulating cfDNA fragments where at least one can be detected in small amounts. As a non-limiting example, when a subject has cancer, selected target regions that are sufficiently separated from each other on different cfDNA fragments and are all hypermethylated are detectable by bisulfite sequencing. In a small sample (e.g., blood obtained from a finger prick), it is unlikely that all of these fragments are present (equivalent to non-fragmented DNA), but it is highly likely that at least one fragment is present, enabling the detection of cancer.

[0072] In still other instances, the target region may contain no similar sequences or may contain no similar epigenetic states. In this case, detection may require the preparation of many primer sets or libraries and subsequent amplification using universal primers to detect multiple characteristic target regions. As a non-limiting example, detection of the fetal RHD gene in a pregnant RHD-negative mother can be achieved from a finger-prick volume of blood by using multiple sets of primers to detect multiple different exons of the RHD gene in cell-free fetal DNA fragments. Sensitivity can be increased by selecting primers that amplify regions that are physically distant in the RHD gene and thus are likely to be present in different cell-free DNA fragments. Detection of the fetal RHD gene in a pregnant RHD-negative mother is important for preventing hemolytic disease of the newborn by mothers with antibodies against the infant's blood. The RHD test is currently performed by a full blood draw (8 milliliters of blood) to achieve appropriate and reliable results. This amount is thought to be necessary to achieve reliable results because it is based on the likelihood that the entire RHD gene is present in the sample. Based on this assumption, the likelihood of obtaining the entire RHD gene in a finger-prick volume of blood is low and is likely to easily cause false-negative results.

[0073] Regardless of how the target regions are selected, such regions are present in the sample as individual biomarkers when amplification or detection is performed on cell-free fragmented DNA. The concentration of fragments containing the target regions is higher than that of the corresponding unfragmented DNA or fragments that cannot be analyzed as a group. Thus, there are more signals from the target regions than from the unfragmented DNA or from signals obtained from an assay for one copy of the target region. There are also some that are much more likely to detect target regions present in ultra-trace samples than non-target regions that are not repeated or that do not share multiple common points with another region. By assaying multiple target regions in multiple DNA fragments, the assay sensitivity is increased compared to conventional tests. Blood is a reliable source of cell-free nucleic acids. The methods disclosed herein for analyzing cell-free nucleic acids from blood include the step of isolating a plasma or serum fraction containing the cell-free nucleic acids. The devices, systems, kits, and methods disclosed herein enable gentle processing of blood samples where needed. The devices, systems, kits, and methods disclosed herein, in some embodiments, enable obtaining a blood sample only after discarding the initial blood perfusion after a percutaneous puncture (e.g., finger puncture), thereby removing any white blood cells damaged by the percutaneous puncture. In other embodiments, the methods, devices, and systems enable lysis of the skin's close adhesion and extraction of a fluid containing the same cell-free nucleic acids as capillary blood from the skin without the need for a percutaneous puncture. These methods, systems, and devices can avoid, prevent, or reduce lysis of white blood cells. In some embodiments, the devices, systems, kits, and methods disclosed herein include (a) a process for inducing a first percutaneous puncture to generate a first fraction of a biological sample; (b) a process for discarding the first fraction of the biological sample; and (c) a process for collecting a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by white blood cell lysis, and enable collecting a biological sample obtained from a subject.In some embodiments, a step of cleaning the surface of a percutaneous puncture site (e.g., the skin) is provided herein before obtaining a biological sample from a subject. In some examples, the step of cleaning includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the percutaneous puncture site is the skin of a finger.

[0074] The devices, systems, kits, and methods disclosed herein enable rapid processing of blood samples where needed. This avoids long-term storage and shipping of samples that may cause blood cell lysis. In some examples, the devices disclosed herein perform integrated separation, e.g., immediate isolation of plasma via filtration, to avoid, reduce, or prevent cell lysis. Immediate isolation of cells from cfDNA may be desirable when reagents (e.g., probes, primers, antibodies) or detection methods do not provide sufficient specificity. In some examples, the method is performed on whole blood in an attempt to avoid any white blood cell lysis. Analysis from whole blood may also be more desirable when relatively high specificity can be achieved.

[0075] In addition to requiring only small amounts of sample, the devices, systems, kits, and methods disclosed herein are highly desirable for at least the following reasons. The devices, systems, kits, and methods disclosed herein generally require little or no technical training. Thus, the cost of performing a genetic test is lower than the cost of a test performed by a trained individual, and this test is available to subjects who do not have access to trained personnel. Further, results may be obtained within minutes (e.g., less than one hour), which can be particularly important when testing for infectious diseases. Individuals or animals that test positive for an infectious disease can be quickly isolated and treated to prevent the spread of the infection. Additionally, results may be obtained privately. In some cases, only the patient being tested is made aware of the genetic information obtained. The devices, systems, and kits disclosed herein are generally lightweight and portable, suitable for remote locations, and accessible. Thus, they can be used in other locations when it is not practical or convenient to visit a home, school, workplace, battlefield, farm, or laboratory or clinic setting. Further, because samples can be analyzed at the point of care, there is no need to store or transport the sample, reducing the risk of sample degradation and misidentification (e.g., sample mix-up).

[0076] Figure 1 shows a general flowchart having various paths that can be followed by the methods, devices, and systems disclosed herein. First, a sample is obtained in step (110). In order to gather useful information from the sample, a minimum amount of the sample must be obtained. The sample may be obtained by percutaneous puncture. The sample may also be obtained by means of extracting useful genetic information that does not require percutaneous puncture, as described herein. The sample can be a biological sample disclosed herein. The sample can be a crude, untreated sample (e.g., whole blood, interstitial fluid). The sample can be a processed sample (e.g., plasma). The amount of the sample is likely to be based on the type of the sample. Typically, in step (120), an amplifiable and / or detectable analyte is generated by processing the sample or purifying the analyte (e.g., nucleic acid or other biomarker) from the sample. The processing can include filtration of the sample, binding of components of the sample containing the analyte, binding of the analyte, stabilization of the analyte, purification of the analyte, or combinations thereof. Non-limiting examples of sample components are cells, virus particles, bacteria particles, exosomes, and nucleosomes. In some examples, the analyte is a nucleic acid, which is amplified in step (130) to generate amplicons for analysis. In other examples, the analyte may or may not be a nucleic acid, but is not amplified regardless. The analyte or amplicon is optionally modified (140) prior to detection and analysis. In some examples, the modification occurs during amplification (not shown). For example, the analyte or amplicon may be tagged or labeled. Detection can include sequencing, target-specific probes, isothermal amplification and detection methods, quantitative PCR, or single molecule detection. Figure 1 is provided as an overview of the devices and methods disclosed herein, but the devices and methods disclosed herein are not limited by Figure 1. Devices and methods not shown in Figure 1 may each include additional components and steps.

[0077] In some examples, the devices, systems, kits, and methods disclosed herein are desirable because they can make genetic information user-exclusive. In fact, even the use of the device can be kept secret. Alternatively, the devices, systems, kits, and methods are configured to share information with others or are easily adaptable by the user to share information (e.g., by turning on a Bluetooth® signal). For example, the information can be easily shared with a nurse or a doctor. In some examples, the device or system can send / share test results to medical practitioners or staff in an office or hospital via a secure portal or application programming interface (API). In some examples, the user can choose to personally share the information with a medical practitioner after receiving the results. In some examples, the information can also be shared in real time. This type of communication is desirable, for example, for couples or families separated by military intervention, employment obligations, immigration policies, and health issues.

[0078] The devices, systems, kits, and methods disclosed herein have numerous applications. The devices, systems, kits, and methods disclosed herein enable the diagnosis and monitoring of health conditions. Non-limiting examples of health conditions include autoimmune diseases, metabolic diseases, cancer, and neurological diseases. The devices, systems, kits, and methods disclosed herein enable personalized medicine, including microbiome testing, determination of an individual's appropriate drug dosage, and / or detection of a response to a drug or its dosage. The devices, systems, kits, and methods disclosed herein result in the detection of infectious diseases caused by pathogens and / or the subject's resistance to drugs that can be used to treat the infectious diseases. In almost all cases, little or no technical training or large, expensive experimental equipment is required.

[0079] Figure 1 shows that one of ordinary skill in the art may begin with a small amount (e.g., less than 1 milliliter) of a biological sample from a subject using the devices, systems, kits, or methods disclosed herein. The biological sample typically contains less than 5000 genome equivalents of cell-free DNA. The sample can be processed by filtration, stabilization, purification, or combinations thereof to enable analysis. In some examples, the sample does not require processing such as filtration, stabilization, or purification. A plurality of different analytes in the sample, such as cell-free DNA, cell-free RNA, nucleic acids associated with exosomes, and epigenetic markers on cell-free DNA, can be beneficial. One or more of these analytes can be analyzed. In some examples, the analyte is not amplified. In some examples, the analyte is sequenced without amplification or modification of the analyte. In some examples, the analyte is amplified to generate an amplicon of the analyte (e.g., polymerase-mediated nucleic acid amplification). In some examples, the amplicon is sequenced. In some examples, the amplicon is sequenced without further preparation or modification. In some examples, features such as polymorphisms, mutations, epigenetic marks, or abnormalities within the amplicon or target region are used for detailed analysis.

[0080] In some examples, an analyte, an amplicon, or a combination thereof is converted into a library by labeling the analyte with a label, barcode, or tag. The terms label, barcode, and tag are used interchangeably herein unless otherwise specified. In some examples, library members are amplified to generate amplified library members. In some examples, library members are subjected to whole genome amplification. In some examples, library members are products of whole genome amplification. In some examples, library members are not amplified to generate amplified library members. In some examples, library members are not subjected to whole genome amplification. In some examples, library members are not products of whole genome amplification. In some examples, library members are captured to generate captured library members. In some examples, library members are captured and amplified to generate captured and amplified library members. In some examples, library members are sequenced. In some examples, amplified libraries are sequenced. In some examples, captured library members are sequenced. In some examples, captured and amplified library members are sequenced. In some examples, library members are not sequenced. For example, library members can be detected or quantified by an array of probes or single molecule measurements. In some examples, amplified library members can be detected or quantified by an array of probes or single molecule measurements. In some examples, captured library members can be detected or quantified by an array of probes or single molecule measurements. In some examples, captured and amplified library members can be detected or quantified by an array of probes or single molecule measurements.

[0081] Figure 2 shows that the methods, systems, devices, and kits disclosed herein can be distributed at multiple locations. For example, the methods disclosed herein can be fully implemented in a home environment or other necessary locations. This is particularly important for subjects who do not have access to a laboratory, nucleic acid processing and analysis facilities, or experts or physicians (e.g., physical, financial). In some examples, the sample can be processed in a laboratory (e.g., the required experimental equipment). However, the methods, systems, devices, and kits disclosed herein can also enable sample collection and reporting at home. As an example, a sample is collected at home, sent to a laboratory where processing is performed, and the results are delivered to the subject at home via electronic communication. Thus, even when laboratory processing is required, the methods, systems, devices, and kits disclosed herein only require the user to have a means for sending / transporting their sample and are still convenient for the user. In some examples, it is convenient for data processing to occur on a cloud or server that can communicate the test results to the subject. In some examples, it is convenient to perform data processing in a laboratory and report the results to the subject at home without relying on a cloud or internet server.

[0082] Method Disclosed herein are methods that include obtaining a biological sample and detecting its components. In some examples, the methods disclosed herein are performed using the devices, systems, or kits described herein. In some embodiments, the components include cell-free nucleic acids such as cell-free DNA or cell-free RNA. In some examples, the biological sample includes maternal blood such as capillary blood obtained from the mother. In some examples, the detected component is a fetal cell-free DNA component of maternal blood.

[0083] The acquisition of a biological sample may be performed, for example, by way of non-limiting examples, in a clinical environment or laboratory environment such as a clinic, hospital, scientific research institute, pathology institute, or clinical laboratory. Alternatively, the acquisition may be performed, by way of non-limiting examples, in a location remote from a clinical environment or laboratory environment such as a home, family planning center, workplace, school, farm, or battlefield. In some examples, the acquisition is performed using the devices or systems described herein.

[0084] In some examples, the detection of a component is performed by analyzing a biological sample to detect the presence, absence, or level of a component (e.g., biomarker, cell-free DNA) in the biological sample. In some examples, the method includes determining whether there is overexpression or underexpression of a desired genomic region in the component as compared to the expression of the desired genomic region in at least one control subject.

[0085] The methods disclosed herein include obtaining and analyzing a relatively small amount of biological sample, whether the collection is performed in a clinical environment or remotely. In some examples, the detection is performed in a clinical environment or laboratory environment. In other examples, the detection is performed in a location remote from a clinical environment or laboratory environment. Other steps of the methods disclosed herein, such as amplifying nucleic acids, may be performed in a clinical environment / laboratory environment or remotely. In some examples, the method may be performed by a subject. In some examples, the methods disclosed herein are performed by a user who has not received the technical training necessary to perform the method.

[0086] Sample Acquisition In some examples, the methods disclosed herein include obtaining a biological sample as described herein. The sample may be obtained directly (e.g., a physician collects a blood sample from a subject). The sample may be obtained indirectly (e.g., through transportation, by a technician from a physician or subject). In some examples, the biological sample is a biological fluid. In some examples, the biological sample is a swab sample (e.g., a cheek swab, a vaginal and / or cervical swab). In some examples, the methods disclosed herein include obtaining whole blood, plasma, serum, urine, saliva, interstitial fluid, or vaginal fluid. In some examples, the methods disclosed herein include obtaining a blood sample via finger prick. In some examples, the methods disclosed herein include obtaining a blood sample by a single finger prick. In some embodiments, the methods disclosed herein include obtaining a blood sample with one or fewer finger pricks. In some examples, the blood sample is obtained via finger prick only after discarding the first perfusion of blood (e.g., pricking the finger, wiping away the first blood sample cleanly, and collecting a second blood sample). In some embodiments, the biological sample obtained from a subject is collected by a process that includes (a) inducing a first percutaneous puncture to generate a first fraction of the biological sample; (b) discarding the first fraction of the biological sample; and (c) collecting a second fraction of the biological sample, thereby reducing or removing contamination of the biological sample by lysis of white blood cells. In some embodiments, the surface of the percutaneous puncture site (e.g., the skin) is cleaned before obtaining a biological sample from a subject. In some embodiments, the cleaning includes removing or reducing unwanted contaminants. In some examples, the unwanted contaminants include DNA from the percutaneous puncture site. In some examples, the percutaneous puncture site is the skin of a finger. In some examples, the methods disclosed herein include obtaining capillary blood (e.g., blood obtained from a finger or skin puncture). In some examples, the method includes squeezing or withdrawing blood from the puncture to obtain a desired volume of blood. In other examples, the method does not include squeezing or withdrawing blood from the puncture to obtain a desired volume of blood.Finger pricking is a common method for obtaining capillary blood, but other locations on the body, such as the toes, heels, arms, palms, shoulders, and earlobes, may also be suitable. In some examples, the methods disclosed herein include the step of obtaining a blood sample without performing venipuncture. In some examples, the methods disclosed herein include the step of obtaining capillary blood. In some examples, the methods disclosed herein include the step of obtaining venous blood. In some examples, the methods disclosed herein do not include the step of obtaining venous blood (e.g., blood obtained from a vein). In some examples, the method includes the step of obtaining a biological sample via biopsy. In some examples, the method includes the step of obtaining a body fluid via liquid biopsy.

[0087] In some examples, the methods, systems, and devices described herein include obtaining a biological sample containing reliable genetic information without the need for percutaneous puncture. In some embodiments, the intimate binding of the subject's skin is dissolved, becoming permeable to fluids that may be pushed into the intercellular space and reabsorbed by capillaries, and can be extracted from the permeable skin without performing percutaneous puncture.

[0088] In some examples, the method includes obtaining a sample containing fragmented nucleic acids. The sample may have been exposed to conditions that do not promote the preservation of nucleic acid integrity. As a non-limiting example, the sample may be a forensic sample. Forensic samples are often contaminated and exposed to air, heat, light, etc. The sample may have been frozen and thawed. The sample may also have been exposed to chemicals or enzymes that degrade nucleic acids. In some examples, the method includes obtaining a tissue sample, where the tissue sample contains fragmented nucleic acids. In some embodiments, the method includes obtaining a tissue sample containing nucleic acids and fragmenting the nucleic acids to generate fragmented nucleic acids. In some examples, the tissue sample is a frozen sample. In some examples, the sample is a preserved sample. In some examples, the tissue sample is a fixed sample (e.g., formaldehyde-fixed). The method may include isolating (fragmented) nucleic acids from the sample. The method may include providing the fragmented nucleic acids in solution for genetic analysis.

[0089] In some examples, the methods disclosed herein are performed with a biological fluid sample of 50 μl or less. In some examples, the methods disclosed herein are performed with a biological fluid sample of 75 μl or less. In some examples, the methods disclosed herein are performed with a biological fluid sample of 100 μl or less. In some examples, the methods disclosed herein are performed with a biological fluid sample of 125 μl or less. In some examples, the methods disclosed herein are performed with a biological fluid sample of 150 μl or less. In some examples, the methods disclosed herein are performed with a biological fluid sample of 200 μl or less. In some examples, the methods disclosed herein are performed with a biological fluid sample of 300 μl or less. In some examples, the methods disclosed herein are performed with a biological fluid sample of 400 μl or less. In some examples, the methods disclosed herein are performed with a biological fluid sample of 500 μl or less.

[0090] In some examples, the methods disclosed herein include obtaining an ultra-trace amount of a biological fluid sample, where the ultra-trace amount is within the range of the sample volume. In some examples, the range of the sample volume is from about 5 μl to about 1 milliliter. In some examples, the range of the sample volume is from about 5 μl to about 900 μl. In some examples, the range of the sample volume is from about 5 μl to about 800 μl. In some examples, the range of the sample volume is from about 5 μl to about 700 μl. In some examples, the range of the sample volume is from about 5 μl to about 600 μl. In some examples, the range of the sample volume is from about 5 μl to about 500 μl. In some examples, the range of the sample volume is from about 5 μl to about 400 μl. In some examples, the range of the sample volume is from about 5 μl to about 300 μl. In some examples, the range of the sample volume is from about 5 μl to about 200 μl. In some examples, the range of the sample volume is from about 5 μl to about 150 μl. In some examples, the range of the sample volume is from 5 μl to about 100 μl. In some examples, the range of the sample volume is from about 5 μl to about 90 μl. In some examples, the range of the sample volume is from about 5 μl to about 85 μl. In some examples, the range of the sample volume is from about 5 μl to about 80 μl. In some examples, the range of the sample volume is from about 5 μl to about 75 μl. In some examples, the range of the sample volume is from about 5 μl to about 70 μl. In some examples, the range of the sample volume is from about 5 μl to about 65 μl. In some examples, the range of the sample volume is from about 5 μl to about 60 μl. In some examples, the range of the sample volume is from about 5 μl to about 55 μl. In some examples, the range of the sample volume is from about 5 μl to about 50 μl. In some examples, the range of the sample volume is from about 15 μl to about 150 μl. In some examples, the range of the sample volume is from about 15 μl to about 120 μl. In some examples, the range of the sample volume is from 15 μl to about 100 μl. In some examples, the range of the sample volume is from about 15 μl to about 90 μl. In some examples, the range of the sample volume is from about 15 μl to about 85 μl. In some examples, the range of the sample volume is from about 15 μl to about 80 μl.In some examples, the range of the sample volume is from about 15 μl to about 75 μl. In some examples, the range of the sample volume is from about 15 μl to about 70 μl. In some examples, the range of the sample volume is from about 15 μl to about 65 μl. In some examples, the range of the sample volume is from about 15 μl to about 60 μl. In some examples, the range of the sample volume is from about 15 μl to about 55 μl. In some examples, the range of the sample volume is from about 15 μl to about 50 μl.

[0091] In some examples, the methods disclosed herein include the step of obtaining a ultra-trace amount of biological fluid, where the ultra-trace amount is from about 100 μl to about 500 μl. In some examples, the methods disclosed herein include the step of obtaining a ultra-trace amount of biological fluid, where the ultra-trace amount is from about 100 μl to about 1000 μl. In some examples, the ultra-trace amount is from about 500 μl to about 1 ml. In some examples, the ultra-trace amount is from about 500 μl to about 2 ml. In some examples, the ultra-trace amount is from about 500 μl to about 3 ml. In some examples, the ultra-trace amount is from about 500 μl to about 5 ml.

[0092] In some examples, the methods disclosed herein include the step of obtaining a ultra-trace amount of biological sample, where the biological sample is whole blood. The ultra-trace amount may be from about 1 μl to about 250 μl. The ultra-trace amount may be from about 5 μl to about 250 μl. The ultra-trace amount may be from about 10 μl to about 25 μl. The ultra-trace amount may be from about 10 μl to about 35 μl. The ultra-trace amount may be from about 10 μl to about 45 μl. The ultra-trace amount may be from about 10 μl to about 50 μl. The ultra-trace amount may be from about 10 μl to about 60 μl. The ultra-trace amount may be from about 10 μl to about 80 μl. The ultra-trace amount may be from about 10 μl to about 100 μl. The ultra-trace amount may be from about 10 μl to about 120 μl. The ultra-trace amount may be from about 10 μl to about 140 μl. The ultra-trace amount may be from about 10 μl to about 150 μl. The ultra-trace amount may be from about 10 μl to about 160 μl. The ultra-trace amount may be from about 10 μl to about 180 μl. The ultra-trace amount may be from about 10 μl to about 200 μl.

[0093] In some examples, the methods disclosed herein include obtaining an ultra-trace amount of a biological sample, where the biological sample is plasma or serum. The ultra-trace amount may be from about 1 μl to about 200 μl. The ultra-trace amount may be from about 1 μl to about 190 μl. The ultra-trace amount may be from about 1 μl to about 180 μl. The ultra-trace amount may be from about 1 μl to about 160 μl. The ultra-trace amount may be from about 1 μl to about 150 μl. The ultra-trace amount may be from about 1 μl to about 140 μl. The ultra-trace amount may be from about 5 μl to about 15 μl. The ultra-trace amount may be from about 5 μl to about 25 μl. The ultra-trace amount may be from about 5 μl to about 35 μl. The ultra-trace amount may be from about 5 μl to about 45 μl. The ultra-trace amount may be from about 5 μl to about 50 μl. The ultra-trace amount may be from about 5 μl to about 60 μl. The ultra-trace amount may be from about 5 μl to about 70 μl. The ultra-trace amount may be from about 5 μl to about 80 μl. The ultra-trace amount may be from about 5 μl to about 90 μl. The ultra-trace amount may be from about 5 μl to about 100 μl. The ultra-trace amount may be from about 5 μl to about 125 μl. The ultra-trace amount may be from about 5 μl to about 150 μl. The ultra-trace amount may be from about 5 μl to about 175 μl. The ultra-trace amount may be from about 5 μl to about 200 μl.

[0094] In some examples, the methods disclosed herein include obtaining a ultra-trace biological sample, where the biological sample is urea. Generally, the concentration of DNA in urine is from about 40 ng / ml to about 200 ng / ml. In some examples, the ultra-trace urine is from about 0.25 μl to 1 milliliter. In some examples, the ultra-trace urine is from about 0.25 μl to about 1 milliliter. In some examples, the ultra-trace urine is at least about 0.25 μl. In some examples, the ultra-trace urine is at most about 1 milliliter.In some examples, the ultra-trace amount of urine is from about 0.25 μl to about 0.5 μl, from about 0.25 μl to about 0.75 μl, from about 0.25 μl to about 1 μl, from about 0.25 μl to about 5 μl, from about 0.25 μl to about 10 μl, from about 0.25 μl to about 50 μl, from about 0.25 μl to about 100 μl, from about 0.25 μl to about 150 μl, from about 0.25 μl to about 200 μl, from about 0.25 μl to about 500 μl, from about 0.25 μl to about 1 milliliter, from about 0.5 μl to about 0.75 μl, from about 0.5 μl to about 1 μl, from about 0.5 μl to about 5 μl, from about 0.5 μl to about 10 μl, from about 0.5 μl to about 50 μl, from about 0.5 μl to about 100 μl, from about 0.5 μl to about 150 μl, from about 0.5 μl to about 200 μl, from about 0.5 μl to about 500 μl, from about 0.5 μl to about 1 milliliter, from about 0.75 μl to about 1 μl, from about 0.75 μl to about 5 μl, from about 0.75 μl to about 10 μl, from about 0.75 μl to about 50 μl, from about 0.75 μl to about 100 μl, from about 0.75 μl to about 150 μl, from about 0.75 μl to about 200 μl, from about 0.75 μl to about 500 μl, from about 0.75 μl to about 1 milliliter, from about 1 μl to about 5 μl, from about 1 μl to about 10 μl, from about 1 μl to about 50 μl, from about 1 μl to about 100 μl, from about 1 μl to about 150 μl, from about 1 μl to about 200 μl, from about 1 μl to about 500 μl, from about 1 μl to about 1 milliliter, from about 5 μl to about 10 μl, from about 5 μl to about 50 μl, from about 5 μl to about 100 μl, from about 5 μl to about 150 μl, from about 5 μl to about 200 μl, from about 5 μl to about 500 μl, from about 5 μl to about 1 milliliter, from about 10 μl to about 50 μl, from about 10 μl to about 100 μl, from about 10 μl to about 150 μl, from about 10 μl to about 200 μl, from about 10 μl to about 500 μl, from about 10 μl to about 1 milliliter, from about 50 μl to about 100 μl, from about 50 μl to about 150 μl, from about 50 μl to about 200 μl, from about 50 μl to about 500 μl, from about 50 μl to about 1 milliliter, from about 100 μl to about 150 μl, from about 100 μl to about 200 μl, from about 100 μl to about 500 μl, from about 100 μl to about 1 milliliter, from about 150 μl to about 200 μl, from about 150 μl to about 500 μl, from about 150 μl to about 1 milliliter, from about 200 μl to about 500 μl, from about 200 μl to about 1 milliliter, or from about 500 μl to about 1 milliliter.In some examples, the ultra-trace amount of urine used is about 0.25 μl, about 0.5 μl, about 0.75 μl, about 1 μl, about 5 μl, about 10 μl, about 50 μl, about 100 μl, about 150 μl, about 200 μl, about 500 μl, or 1 milliliter.

[0095] In some examples, the methods disclosed herein include obtaining at least about 5 μL of blood to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 10 μL of blood to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 15 μL of blood to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 20 μL of blood to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 20 μL of blood to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 20 μL of blood to provide test results with at least about 95% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 20 μL of blood to provide test results with at least about 98% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 20 μL of blood to provide test results with at least about 99% reliability or accuracy. In some examples, the methods disclosed herein include obtaining only about 20 μL to about 120 μL of blood to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining only about 20 μL to about 120 μL of blood to provide test results with at least about 95% reliability or accuracy. In some examples, the methods disclosed herein include obtaining only about 20 μL to about 120 μL of blood to provide test results with at least about 97% reliability or accuracy. In some examples, the methods disclosed herein include obtaining only about 20 μL to about 120 μL of blood to provide test results with at least about 98% reliability or accuracy.In some examples, the methods disclosed herein include obtaining only about 20 μL to about 120 μL of blood to provide test results with at least about 99% reliability or accuracy. In some examples, the methods disclosed herein include obtaining only about 20 μL to about 120 μL of blood to provide test results with at least about 99.5% reliability or accuracy.

[0096] In some examples, the biological fluid sample is plasma or serum. Plasma or serum accounts for approximately 55% of whole blood. In some examples, the methods disclosed herein include obtaining at least about 10 μL of plasma or serum to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 10 μL of plasma or serum to provide test results with at least about 98% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 12 μL of plasma or serum to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 12 μL of plasma or serum to provide test results with at least about 95% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 12 μL of plasma or serum to provide test results with at least about 98% reliability or accuracy. In some examples, the methods disclosed herein include obtaining at least about 12 μL of plasma or serum to provide test results with at least about 99% reliability or accuracy. In some examples, the methods disclosed herein include obtaining from slightly about 10 μL to about 60 μL of plasma or serum to provide test results with at least about 90% reliability or accuracy. In some examples, the methods disclosed herein include obtaining from slightly about 10 μL to about 60 μL of plasma or serum to provide test results with at least about 95% reliability or accuracy. In some examples, the methods disclosed herein include obtaining from slightly about 10 μL to about 60 μL of plasma or serum to provide test results with at least about 97% reliability or accuracy. In some examples, the methods disclosed herein include obtaining from slightly about 10 μL to about 60 μL of plasma or serum to provide test results with at least about 98% reliability or accuracy. In some examples, the methods disclosed herein include obtaining from slightly about 10 μL to about 60 μL of plasma or serum to provide test results with at least about 99% reliability or accuracy.In some examples, the methods disclosed herein include obtaining from about 10 μL to about 60 μL of plasma or serum to provide assay results with at least about 99.5% confidence or accuracy.

[0097] In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample contains an amount of cell-free nucleic acid molecules. In some examples, obtaining the biological sample will involve disrupting or lysing the cells in the biological sample. Thus, in some examples, the biological sample contains cellular nucleic acid molecules. In some examples, the cellular nucleic acid molecules account for less than about 1% of the total cellular nucleic acid molecules in the biological sample. In some examples, the cellular nucleic acid molecules account for less than about 5% of the total cellular nucleic acid molecules in the biological sample. In some examples, the cellular nucleic acid molecules account for less than about 10% of the total cellular nucleic acid molecules in the biological sample. In some examples, the cellular nucleic acid molecules account for less than about 20% of the total cellular nucleic acid molecules in the biological sample. In some examples, the cellular nucleic acid molecules account for at least about 50% of the total cellular nucleic acid molecules in the biological sample. In some examples, the cellular nucleic acid molecules account for less than about 90% of the total cellular nucleic acid molecules in the biological sample.

[0098] In some examples, the methods disclosed herein include obtaining a ultra-trace amount of a biological fluid sample from a subject, where the biological fluid sample contains ultra-trace amounts of cell-free nucleic acids. In some examples, the ultra-trace amount is between about 4 pg and about 100 pg. In some examples, the ultra-trace amount is between about 4 pg and about 150 pg. In some examples, the ultra-trace amount is between about 4 pg and about 200 pg. In some examples, the ultra-trace amount is between about 4 pg and about 300 pg. In some examples, the ultra-trace amount is between about 4 pg and about 400 pg. In some examples, the ultra-trace amount is between about 4 pg and about 500 pg. In some examples, the ultra-trace amount is between about 4 pg and about 1 ng. In some examples, the ultra-trace amount is between about 10 pg and about 100 pg. In some examples, the ultra-trace amount is between about 10 pg and about 150 pg. In some examples, the ultra-trace amount is between about 10 pg and about 200 pg. In some examples, the ultra-trace amount is between about 10 pg and about 300 pg. In some examples, the ultra-trace amount is between about 10 pg and about 400 pg. In some examples, the ultra-trace amount is between about 10 pg and about 500 pg. In some examples, the ultra-trace amount is between about 10 pg and about 1 ng. In some examples, the ultra-trace amount is between about 20 pg and about 100 pg. In some examples, the ultra-trace amount is between about 20 pg and about 200 pg. In some examples, the ultra-trace amount is between about 20 pg and about 500 pg. In some examples, the ultra-trace amount is between about 20 pg and about 1 ng. In some examples, the ultra-trace amount is between about 30 pg and about 150 pg. In some examples, the ultra-trace amount is between about 30 pg and about 180 pg. In some examples, the ultra-trace amount is between about 30 pg and about 200 pg. In some examples, the ultra-trace amount is between about 30 pg and about 300 pg. In some examples, the ultra-trace amount is between about 30 pg and about 400 pg. In some examples, the ultra-trace amount is between about 30 pg and about 500 pg. In some examples, the ultra-trace amount is between about 30 pg and about 1 ng. In some examples, the subject is a pregnant subject and the cell-free nucleic acids include cell-free fetal DNA. In some examples, the subject has a tumor and the cell-free nucleic acids include cell-free tumor DNA. In some examples, the subject is an organ transplant recipient and the cell-free nucleic acids include organ donor DNA.

[0099] In some examples, the method includes obtaining cell-free fetal nucleic acid of less than about 1 ng. In some examples, the method includes obtaining cell-free fetal nucleic acid of less than about 500 pg. In some examples, the method includes obtaining cell-free fetal nucleic acid of less than about 100 pg. In some examples, the method includes obtaining cell-free fetal nucleic acid of at least 3.5 pg. In some examples, the method includes obtaining cell-free fetal nucleic acid of at least 10 pg. In some examples, the method includes obtaining cell-free fetal nucleic acid of about 100 pg or less. In some examples, the method includes obtaining cell-free fetal nucleic acid of about 500 pg or less. In some examples, the method includes obtaining cell-free fetal nucleic acid of about 1 ng or less.

[0100] In some examples, the methods disclosed herein include obtaining a biological fluid sample from a subject, wherein the biological fluid sample comprises at least 1 genome equivalent of cell-free DNA. Those skilled in the art understand that a genome equivalent is the amount of DNA necessary to be present in a sample to ensure that all genes are present. The ultra-trace biological fluid samples disclosed herein may contain ultra-trace genome equivalents. In some examples, the biological fluid sample comprises less than 1 genome equivalent of cell-free nucleic acid. In some examples, the biological fluid sample comprises at least 5 genome equivalents of cell-free nucleic acid. In some examples, the biological fluid sample comprises at least 10 genome equivalents of cell-free nucleic acid. In some examples, the biological fluid sample comprises at least 15 genome equivalents of cell-free nucleic acid. In some examples, the biological fluid sample comprises at least 20 genome equivalents of cell-free nucleic acid. In some examples, the biological fluid sample comprises from about 5 to about 50 genome equivalents. In some examples, the biological fluid sample comprises from about 10 to about 50 genome equivalents. In some examples, the biological fluid sample comprises from about 10 to about 100 genome equivalents. In some examples, the biological fluid sample comprises 50 genome equivalents or less of cell-free nucleic acid. In some examples, the biological fluid sample comprises 60 genome equivalents or less of cell-free nucleic acid. In some examples, the biological fluid sample comprises 80 genome equivalents or less of cell-free nucleic acid. In some examples, the biological fluid sample comprises 100 genome equivalents or less of cell-free nucleic acid.

[0101] The ultra-trace biological fluid sample disclosed in this specification may contain an ultra-trace cell equivalent. In some examples, the methods disclosed herein include obtaining a biological fluid sample from a subject, where the biological fluid sample contains at least 1 cell equivalent of cell-free DNA. In some examples, the biological fluid sample contains at least 2 cell equivalents of cell-free nucleic acid. In some examples, the biological fluid sample contains at least 5 cell equivalents of cell-free nucleic acid. In some examples, the biological fluid sample contains at least 5 cell equivalents to 40 cell equivalents of cell-free nucleic acid. In some examples, the biological fluid sample contains at least 5 cell equivalents to about 100 cell equivalents of cell-free nucleic acid. In some examples, the biological fluid sample contains 30 cell equivalents or less of cell-free nucleic acid. In some examples, the biological fluid sample contains 50 cell equivalents or less of cell-free nucleic acid. In some examples, the biological fluid sample contains 80 cell equivalents or less of cell-free nucleic acid. In some examples, the biological fluid sample contains 100 cell equivalents or less of cell-free nucleic acid.

[0102] In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample comprises at least one desired cell-free nucleic acid. By way of non-limiting example, the desired cell-free nucleic acid may be a cell-free fetal nucleic acid, cell-free tumor DNA, or DNA from a transplanted organ. In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample comprises from about 1 to about 5 cell-free nucleic acids. In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample comprises 115 equivalents of cell-free nucleic acid. In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample comprises from about 1 to about 25 cell-free nucleic acids. In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample comprises from about 1 to about 100 cell-free nucleic acids. In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample comprises from about 5 to about 100 cell-free nucleic acids. In some examples, at least one cell-free nucleic acid is represented by a sequence specific to the target chromosome disclosed herein.

[0103] In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample comprises about 10 2 cell-free nucleic acids~about 10 10 cell-free nucleic acids. In some examples, the biological sample comprises about 10 2 cell-free nucleic acids~about 10 9 cell-free nucleic acids. In some examples, the biological sample comprises about 10 2 cell-free nucleic acids~about 10 8 cell-free nucleic acids. In some examples, the biological sample comprises about 10 2 cell-free nucleic acids~about 10 7 cell-free nucleic acids. In some examples, the biological sample comprises about 10 2 cell-free nucleic acids~about 10 6 cell-free nucleic acids. In some examples, the biological sample comprises about 10 2 cell-free nucleic acids~about 10 5 cell-free nucleic acids.

[0104] In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample comprises from about 10 3 cell-free nucleic acids to about 10 10 cell-free nucleic acids. In some examples, the biological sample comprises from about 10 3 cell-free nucleic acids to about 10 9 cell-free nucleic acids. In some examples, the biological sample comprises from about 10 3 cell-free nucleic acids to about 10 8 cell-free nucleic acids. In some examples, the biological sample comprises from about 10 3 cell-free nucleic acids to about 10 7 cell-free nucleic acids. In some examples, the biological sample comprises from about 10 3 cell-free nucleic acids to about 10 6 cell-free nucleic acids. In some examples, the biological sample comprises from about 10 3 cell-free nucleic acids to about 10 5 cell-free nucleic acids.

[0105] In some examples, the methods disclosed herein include obtaining a biological sample from a subject, where the biological sample has a number of cell-free nucleic acids corresponding to the volume of a typical sample type. As a non-limiting example, 4 ml of human blood from a pregnant subject typically contains about 10 10 cell-free fetal nucleic acids. However, the concentration of cell-free fetal nucleic acids in the sample, and thus the amount of sample required to obtain information about the fetus's genetics, varies depending on the sample type. Example 7 provided herein also shows how one of ordinary skill in the art can determine the minimum volume required to obtain a sufficient number of cell-free fetal nucleic acids.

[0106] Sample processing In some examples, the methods disclosed herein include a step of isolating or purifying cell-free nucleic acid molecules from a biological sample. In some examples, the methods disclosed herein include a step of isolating or purifying fetal nucleic acid molecules that do not contain nucleated cells from a biological sample. In some examples, the methods disclosed herein include a step of removing non-nucleic acid components from the biological samples described herein.

[0107] In some examples, the step of isolation or purification includes reducing or removing unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 95% of the unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 97% of the unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 98% of the unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 99% of the unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 95% of the unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 97% of the unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 98% of the unwanted non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes removing at least 99% of the unwanted non-nucleic acid components from the biological sample.

[0108] In some examples, the methods disclosed herein include isolating or purifying nucleic acids from one or more non-nucleic acid components of a biological sample. Non-nucleic acid components may also be considered unwanted substances. Non-limiting examples of non-nucleic acid components include cells (e.g., blood cells), cell fragments, extracellular vesicles, lipids, proteins, or combinations thereof. Additional non-nucleic acid components are described herein and throughout. It should be noted that the method may include the step of isolating / purifying nucleic acids, but the above step may also include analyzing non-nucleic acid components of the sample that are considered unwanted substances in the nucleic acid purification process. The step of isolation or purification may include removing components of the biological sample that inhibit, interfere with, or are otherwise detrimental to subsequent process steps such as amplification or detection of nucleic acids.

[0109] The step of isolation or purification may be performed using the devices or systems disclosed herein. The step of isolation or purification may be performed within the devices or systems disclosed herein. The step of isolation and / or purification may be performed using the sample purification device disclosed herein. In some examples, the step of isolating or purifying nucleic acids includes removing non-nucleic acid components from the biological sample described herein. In some examples, the step of isolating or purifying nucleic acids includes discarding non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes collecting, processing, and analyzing non-nucleic acid components. In some examples, non-nucleic acid components may be considered biomarkers because they provide additional information about the subject.

[0110] In some examples, the step of isolating or purifying a nucleic acid comprises lysing cells. In some examples, the step of isolating or purifying a nucleic acid avoids lysing cells. In some examples, the step of isolating or purifying a nucleic acid does not comprise lysing cells. In some examples, the step of isolating or purifying a nucleic acid does not comprise an active step intended to lyse cells. In some examples, the step of isolating or purifying a nucleic acid does not intentionally comprise lysing cells. Intentionally lysing cells may include mechanically disrupting (e.g., shearing) the cell membrane. Intentionally lysing cells may include contacting the cells with a lysis reagent. Exemplary lysis reagents are described herein.

[0111] In some examples, the step of isolating or purifying a nucleic acid comprises performing lysis, sequence-specific capture of the target nucleic acid by a "bait" in solution, and subsequent binding of the "bait" to a solid support such as magnetic beads (e.g., Legler et al., Specific magnetic bead-based capture of free fetal DNA from maternal plasma, Transfusion and Apheresis Science 40 (2009),153-157). In some examples, the method comprises performing sequence-specific capture in the presence of recombinase or helicase. The use of recombinase or helicase obviates the need for thermal denaturation of the nucleic acid and can speed up the detection step.

[0112] In some examples, the step of isolation or purification includes separating the components of the biological sample disclosed herein. As a non-limiting example, the step of isolation or purification may include separating plasma from blood. In some examples, the step of isolation or purification includes centrifuging the biological sample. In some examples, the step of isolation or purification includes filtering the biological sample to separate the components of the biological sample. In some examples, the step of isolation or purification includes filtering the biological sample to remove non-nucleic acid components from the biological sample. In some examples, the step of isolation or purification includes filtering the biological sample to capture nucleic acids from the biological sample.

[0113] In some examples, the biological sample is blood, and the step of isolating or purifying nucleic acids includes obtaining or isolating plasma from the blood. Obtaining plasma may include separating the plasma from the cellular components of the blood sample. Obtaining plasma may include centrifuging the blood, filtering the blood, or a combination thereof. Obtaining plasma may include subjecting the blood to gravity (e.g., sedimentation). Obtaining plasma may include subjecting the blood to a material that absorbs a portion of the blood from the non-nucleic acid components of the blood. In some examples, the method includes subjecting the blood to vertical filtration. In some examples, the method includes subjecting the blood to a sample purification device that includes a filter matrix for receiving the whole blood, the filter matrix having a pore size through which cells cannot pass while plasma can pass through the filter matrix without being impeded. Such vertical filtration and filter matrices are described for the devices disclosed herein.

[0114] In some examples, the isolation or purification step comprises exposing a biological sample, or a fraction thereof, or a modified form thereof, to a binding moiety. The binding moiety can bind to a component of the biological sample and remove it, thereby generating a modified sample depleted of unwanted or uninteresting cells, cell fragments, nucleic acids, or proteins. In some examples, the isolation or purification step comprises exposing the biological sample to the binding moiety to reduce unwanted substances or non-nucleic acid components in the biological sample. In some examples, the isolation or purification step comprises exposing the biological sample to the binding moiety to generate a modified sample enriched in target cells, target cell fragments, target nucleic acids, or target proteins. By way of non-limiting example, the isolation or purification step may comprise exposing the biological sample to a binding moiety for capturing placenta educated platelets that may contain fetal DNA or RNA fragments. The resulting binding moiety bound to the cells can be captured / concentrated by an antibody or other means, such as low speed centrifugation.

[0115] The isolation or purification step may comprise capturing extracellular vesicles or extracellular microparticles in the biological sample with a binding moiety. In some examples, the extracellular vesicles comprise at least one of DNA and RNA. In some examples, the extracellular vesicles are of fetal / placental origin. The method may comprise the step of capturing extracellular vesicles or extracellular microparticles in a biological sample derived from maternal cells. In some examples, the methods disclosed herein comprise the step of capturing and discarding extracellular vesicles or extracellular microparticles from maternal cells to enrich the sample for fetal / placental nucleic acids.

[0116] In some examples, the method includes capturing nucleosomes in a biological sample and analyzing the nucleic acids attached to the nucleosomes. In some examples, the method includes capturing exosomes in a biological sample and analyzing the nucleic acids attached to the exosomes. The step of capturing nucleosomes and / or exosomes can eliminate the need for a lysis step or reagents, thereby simplifying the method and shortening the time from sample collection to detection.

[0117] In some examples, the method includes exposing a biological sample to a cell-binding moiety for capturing platelets affected by the placenta that may contain fetal DNA or RNA fragments. The capture can include contacting the platelets affected by the placenta with the binding moiety (e.g., an antibody to a cell surface marker), subjecting the biological sample to low-speed centrifugation, or a combination thereof. In some examples, the binding moiety is attached to a solid support disclosed herein, and the method includes separating the solid support from the remainder of the biological sample after the binding moiety has contacted the biological sample.

[0118] In some examples, the methods disclosed herein include removing unwanted non-nucleic acid components from a biological sample. In some examples, the methods disclosed herein include removing and discarding non-nucleic acid components from a biological sample. Non-limiting examples of non-nucleic acid components include cells (e.g., blood cells), cell fragments, extracellular vesicles, lipids, proteins, or combinations thereof. In some examples, the step of removing non-nucleic acid components may include centrifuging the biological sample. In some examples, the step of removing non-nucleic acid components may include filtering the biological sample. In some examples, the step of removing non-nucleic acid components may include contacting the biological sample with a binding moiety described herein.

[0119] In some embodiments, the methods disclosed herein include a step of purifying nucleic acids in a sample. In some examples, the purification does not include washing the nucleic acids with a wash buffer. In some examples, the nucleic acids are cell-free fetal nucleic acids. In some embodiments, the purification includes capturing the nucleic acids with a nucleic acid capture moiety to generate captured nucleic acids. Non-limiting examples of nucleic acid capture moieties are silica particles and paramagnetic particles. In some embodiments, the purification includes passing a sample containing the captured nucleic acids through a hydrophobic phase (e.g., a liquid or wax). The hydrophobic phase retains impurities in the sample that would otherwise inhibit further manipulation of the nucleic acids (e.g., amplification, sequencing).

[0120] In some examples, the methods disclosed herein include a step of removing nucleic acid components from a biological sample described herein. In some examples, the removed nucleic acid components are discarded. As a non-limiting example, the method may include a step of analyzing only DNA. Thus, RNA is unnecessary and causes unwanted background noise or contamination to DNA. In some examples, the methods disclosed herein include a step of removing RNA from a biological sample. In some examples, the methods disclosed herein include a step of removing mRNA from a biological sample. In some examples, the methods disclosed herein include a step of removing microRNA from a biological sample. In some examples, the methods disclosed herein include a step of removing maternal RNA from a biological sample. In some examples, the methods disclosed herein include a step of removing DNA from a biological sample. In some examples, the methods disclosed herein include a step of removing maternal DNA from a biological sample of a pregnant subject. In some examples, the step of removing nucleic acid components includes contacting the nucleic acid components with an oligonucleotide capable of hybridizing to the nucleic acids, where the oligonucleotide is conjugated, attached, or bound to a capture device (e.g., beads, columns, matrices, nanoparticles, magnetic particles, etc.). In some examples, the removed nucleic acid components are discarded.

[0121] In some examples, the step of removing nucleic acid components includes separating the nucleic acid components by size on a gel. For example, cell-free fetal DNA fragments are generally less than 200 base pairs in length. In some examples, the methods disclosed herein include the step of removing cell-free DNA from a biological sample. In some examples, the methods disclosed herein include the step of capturing cell-free DNA from a biological sample. In some examples, the methods disclosed herein include the step of selecting cell-free DNA from a biological sample. In some examples, cell-free DNA has a minimum length. In some examples, the minimum length is about 50 base pairs. In some examples, the minimum length is about 100 base pairs. In some examples, the minimum length is about 110 base pairs. In some examples, the minimum length is about 120 base pairs. In some examples, the minimum length is about 140 base pairs. In some examples, cell-free DNA has a maximum length. In some examples, the maximum length is about 180 base pairs. In some examples, the maximum length is about 200 base pairs. In some examples, the maximum length is about 220 base pairs. In some examples, the maximum length is about 240 base pairs. In some examples, the maximum length is about 300 base pairs. Size-based separation may be useful for other categories of nucleic acids having a limited size range (e.g., microRNA) that are well known in the art.

[0122] In some examples, the process includes enriching the fetal trophoblasts that contain the desired fetal genomic DNA in the biological sample. In some examples, the fetal trophoblasts are enriched by morphology (e.g., size) or marker antigens (e.g., cell surface antigens), or both. In some cases, the enrichment of the trophoblasts is performed using the isolation by size of epithelial tumor cells (ISET) method. In some cases, the enrichment of the trophoblasts in the biological sample includes contacting the biological sample with an antibody or antigen-binding fragment specific for the cell surface antigen of the trophoblasts. Non-limiting examples of trophoblast cell surface antigens include tropomyosin-1 (Trop1), tropomyosin-2 (Trop2), cytotrophoblast and syncytiotrophoblast markers, GB25, human placental lactogen (HPL), and alpha human chorionic gonadotropin (alpha HCG). In some examples, the step of purifying or separating the fetal trophoblasts includes using fluorescence-activated cell sorting (FACS), column chromatography, or magnetic sorting (e.g., Dynabeads). In some examples, the fetal genetic information is extracted from the enriched and / or purified trophoblasts using any suitable DNA extraction method, such as those described herein.

[0123] Amplification of Nucleic Acids In some examples, the methods disclosed herein include amplifying at least one nucleic acid (e.g., cell-free nucleic acid such as cell-free DNA or cell-free RNA) in a sample to generate at least one amplification product. The at least one nucleic acid may be cell-free nucleic acid. The sample may be a biological sample disclosed herein, or a fragment or portion thereof. In some examples, the method includes generating copies of the nucleic acids in the sample and amplifying the copies to generate at least one amplification product. In some examples, the method includes generating a reverse transcript of the nucleic acids in the sample and amplifying the reverse transcript to generate at least one amplification product.

[0124] In some examples, the method includes a step of performing whole genome amplification. In some examples, the method does not include a step of performing whole genome amplification. The term "whole genome amplification" may refer to amplifying all cell-free nucleic acids in a biological sample. The term "whole genome amplification" may refer to amplifying at least 90% of the cell-free nucleic acids in a biological sample. The term "whole genome" may refer to multiple genomes. Whole genome amplification may include amplifying cell-free nucleic acids from a biological sample of a subject, and the biological sample includes cell-free nucleic acids from the subject and foreign tissue. For example, whole genome amplification may include amplifying cell-free nucleic acids from both a subject (host genome) and an organ or tissue transplanted into the subject (donor genome). Also, as a non-limiting example, whole genome amplification may include amplifying cell-free nucleic acids from a biological sample of a pregnant subject, where the biological sample includes cell-free nucleic acids from the pregnant subject and her fetus. Whole genome amplification may include amplifying cell-free nucleic acids from a biological sample of a subject having cancer, where the biological sample includes cell-free nucleic acids from the subject's benign tissue and the subject's tumor. Whole genome amplification may include amplifying cell-free nucleic acids from a biological sample of a subject having an infectious disease, where the biological sample includes cell-free nucleic acids from the subject and a pathogen.

[0125] In some examples, the methods disclosed herein include a step of amplifying a nucleic acid, where the step of amplifying includes performing isothermal amplification of the nucleic acid. Non-limiting examples of isothermal amplification are as follows: loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), and recombinase polymerase amplification (RPA).

[0126] Any suitable nucleic acid amplification method known in the art is contemplated for use in the devices and methods described herein. In some examples, isothermal amplification is used. In some examples, amplification is performed isothermally, except for an initial heating step prior to the start of isothermal amplification. Many isothermal amplification methods are known in the art, each having different considerations and providing different advantages, and are discussed, for example, in Zanoli and Spoto, 2013, “Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices,” Biosensors 3: 18-43, and Fakruddin, et al., 2013, “Alternative Methods of Polymerase Chain Reaction (PCR),” Journal of Pharmacy and Bioallied Sciences 5(4): 245-252, each of which is incorporated herein by reference in its entirety. In some examples, any suitable isothermal amplification method is used. In some examples, the isothermal amplification method used is selected from the following: loop-mediated isothermal amplification (LAMP); nucleic acid sequence-based amplification (NASBA); multiple displacement amplification (MDA); rolling circle amplification (RCA); helicase-dependent amplification (HDA); strand displacement amplification (SDA); nicking enzyme amplification reaction (NEAR); ramification amplification method (RAM); and recombinase polymerase amplification (RPA).

[0127] In some examples, the amplification method used is LAMP (see, e.g., Notomi, et al., 2000, “Loop Mediated Isothermal Amplification” NAR 28(12):e63 i-vii, and U.S. Patent No. 6,410,278 “Process for synthesizing nucleic acid”). LAMP is a one-step amplification system using auto-cycling strand displacement deoxyribonucleic acid (DNA) synthesis. In some examples, LAMP is performed at 60-65° C. for 45-60 minutes in the presence of a thermostable polymerase, such as Bacillus stearothermophilus (Bst) DNA polymerase I, deoxyribonucleotide triphosphates (dNTPs), specific primers, and a target DNA template. In some examples, the template is RNA, and a polymerase having both reverse transcriptase activity and strand displacement DNA polymerase activity, such as Bca DNA polymerase, is used, or a polymerase having reverse transcriptase activity is used in the reverse transcription step and a polymerase not having reverse transcriptase activity is used in the strand displacement-DNA synthesis step.

[0128] In some examples, the amplification method is nucleic acid sequence-based amplification (NASBA). NASBA (also known as 3SR and transcription-mediated amplification) is an isothermal transcription-based RNA amplification system. Single-stranded RNA is generated using three enzymes (avian myeloblastosis virus reverse transcriptase, RNase H, T7 DNA-dependent RNA polymerase). In some cases, NASBA can be used to amplify DNA. The amplification reaction is typically carried out for about 60 to about 90 minutes while maintaining a constant temperature at 41°C (see, e.g., Fakruddin, et al., 2012, “Nucleic Acid Sequence Based Amplification (NASBA) Prospects and Applications,” Int. J. of Life Science and Pharma Res. 2(1):L106-L121, which are incorporated herein by reference).

[0129] In some examples, the NASBA reaction is carried out at about 40°C to about 42°C. In some examples, the NASBA reaction is carried out at 41°C. In some examples, the NASBA reaction is carried out at a maximum of about 42°C. In some examples, the NASBA reaction is carried out at about 40°C to about 41°C, about 40°C to about 42°C, or about 41°C to about 42°C. In some examples, the NASBA reaction is carried out at about 40°C, about 41°C, or about 42°C.

[0130] In some examples, the amplification method is strand displacement amplification (SDA). SDA is an isothermal amplification method that uses four different primers. A primer containing a restriction site (recognition sequence for HincII exonuclease) is annealed to a DNA template. The exonuclease-deficient fragment of Escherichia coli DNA polymerase 1 (exo-Klenow) extends the primer. Each SDA cycle consists of (1) primer binding to the displaced target fragment, (2) extension of the primer / target complex by exo-Klenow, (3) nicking of the resulting hemiphosphothioate HincII site, (4) dissociation of HincII from the nicked site, and (5) extension of the nick and displacement of the downstream strand by exo-Klenow.

[0131] In some examples, the method includes contacting the DNA in the sample with a helicase. In some examples, the amplification method is helicase-dependent amplification (HDA). HDA is an isothermal reaction because a helicase is used to denature the DNA instead of heat.

[0132] In some examples, the amplification method is multiple displacement amplification (MDA). MDA is an isothermal strand displacement method based on using a highly processive and strand-displacing DNA polymerase from bacteriophage Φ29 in combination with modified random primers, and it amplifies the entire genome with high fidelity. It was developed to amplify all the DNA in a sample from a very small amount of starting material. In MDA Φ29, the DNA polymerase is incubated with dNTPs, random hexamers, and denatured template DNA at 30 °C for 16 - 18 hours, and the enzyme must be inactivated at a high temperature (65 °C) for 10 minutes. No iterative recycling is required, but a short initial denaturation step, an amplification step, and a final enzyme inactivation are necessary.

[0133] In some examples, the amplification method is rolling circle amplification (RCA). RCA is a probe DNA sequence at a single temperature, typically about 30 °C for 10 9It is an isothermal nucleic acid amplification method that can amplify more than [X] times. Multiple rounds of isothermal enzymatic synthesis are carried out by Φ29 DNA polymerase, which extends the circle-hybridized primers by continuously progressing around the circular DNA probe. In some examples, the amplification reaction is carried out using RCA at about 28°C to about 32°C.

[0134] Additional amplification methods that can be incorporated into the devices and methods disclosed herein can be found in the art. Ideally, the amplification method is isothermal and faster compared to conventional PCR. In some examples, the amplification involves performing an exponential amplification reaction (EXPAR), which is an isothermal strand displacement reaction where the product of one reaction catalyzes a further reaction that produces the same product. In some examples, the amplification is carried out in the presence of an endonuclease. The endonuclease may be a nicking endonuclease. See, for example, Wu et al., “Aligner-Mediated Cleavage of Nucleic Acids,” Chemical Science (2018). In some examples, the amplification does not require initial heat denaturation of the target DNA. See, for example, Toley et al., “Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis,” The Analyst (2015). Pulse-controlled amplification in an ultra-rapid amplification method developed by GNA Biosolutions GmbH.

[0135] In some examples, the method includes performing a plurality of cycles of nucleic acid amplification using a pair of primers. The number of amplification cycles is important because amplification can introduce bias into the representation of regions. In the case of ultra-low input amounts of nucleic acid, amplification is even more likely to introduce bias, and thus it is important to enhance efficiency prior to amplification for high precision. Since not all regions are amplified with the same efficiency, the overall representation is not uniform, which can affect the accuracy of the analysis. If amplification is required, usually fewer cycles are desirable. In some examples, the method includes performing amplification with less than 30 cycles. In some examples, the method includes performing amplification with less than 25 cycles. In some examples, the method includes performing amplification with less than 20 cycles. In some examples, the method includes performing amplification with less than 15 cycles. In some examples, the method includes performing amplification with less than 12 cycles. In some examples, the method includes performing amplification with less than 11 cycles. In some examples, the method includes performing amplification with less than 10 cycles. In some examples, the method includes performing amplification with at least 3 cycles. In some examples, the method includes performing amplification with at least 5 cycles. In some examples, the method includes performing amplification with at least 8 cycles. In some examples, the method includes performing amplification with at least 10 cycles.

[0136] In some examples, the amplification reaction is carried out for about 5 to about 90 minutes. In some examples, the amplification reaction is carried out for at least 30 minutes. In some examples, the amplification reaction is carried out for a maximum of about 90 minutes. In some examples, the amplification reaction is carried out for about 30 to about 35 minutes, about 30 to about 40 minutes, about 30 to about 45 minutes, about 30 to about 50 minutes, about 30 to about 55 minutes, about 30 to about 60 minutes, about 30 to about 65 minutes, about 30 to about 70 minutes, about 30 to about 75 minutes, about 30 to about 80 minutes, about 30 to about 90 minutes, about 35 to about 40 minutes, about 35 to about 45 minutes, about 35 to about 50 minutes, about 35 to about 55 minutes, about 35 to about 60 minutes, about 35 to about 65 minutes, about 35 to about 70 minutes, about 35 to about 75 minutes, about 35 to about 80 minutes, about 35 to about 90 minutes, about 40 to about 45 minutes, about 40 to about 50 minutes, about 40 to about 55 minutes, about 40 to about 60 minutes, about 40 to about 65 minutes, about 40 to about 70 minutes, about 40 to about 75 minutes, about 40 to about 80 minutes, about 40 to about 90 minutes, about 45 to about 50 minutes, about 45 to about 55 minutes, about 45 to about 60 minutes, about 45 to about 65 minutes, about 45 to about 70 minutes, about 45 to about 75 minutes, about 45 to about 80 minutes, about 45 to about 90 minutes, about 50 to about 55 minutes, about 50 to about 60 minutes, about 50 to about 65 minutes, about 50 to about 70 minutes, about 50 to about 75 minutes, about 50 to about 80 minutes, about 50 to about 90 minutes, about 55 to about 60 minutes, about 55 to about 65 minutes, about 55 to about 70 minutes, about 55 to about 75 minutes, about 55 to about 80 minutes, about 55 to about 90 minutes, about 60 to about 65 minutes, about 60 to about 70 minutes, about 60 to about 75 minutes, about 60 to about 80 minutes, about 60 to about 90 minutes, about 65 to about 70 minutes, about 65 to about 75 minutes, about 65 to about 80 minutes, about 65 to about 90 minutes, about 70 to about 75 minutes, about 70 to about 80 minutes, about 70 to about 90 minutes, about 75 to about 80 minutes, about 75 to about 90 minutes, or about 80 to about 90 minutes. In some examples, the amplification reaction is carried out for about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, or about 90 minutes.

[0137] In some examples, the methods disclosed herein include the step of amplifying a nucleic acid at at least one temperature. In some examples, the methods disclosed herein include the step of amplifying a nucleic acid at a single temperature (e.g., isothermal amplification). In some examples, the methods disclosed herein include the step of amplifying a nucleic acid, and the amplification is performed at two or fewer temperatures. The amplification may occur in one step or multiple steps. Non-limiting examples of the amplification step include double-strand denaturation, primer hybridization, and primer extension.

[0138] In some examples, at least one step of the amplification occurs at room temperature. In some examples, all steps of the amplification occur at room temperature. In some examples, at least one step of the amplification occurs within a temperature range. In some examples, all steps of the amplification occur within a temperature range. In some examples, the temperature range is from about 0°C to about 100°C. In some examples, the temperature range is from about 15°C to about 100°C. In some examples, the temperature range is from about 25°C to about 100°C. In some examples, the temperature range is from about 35°C to about 100°C. In some examples, the temperature range is from about 55°C to about 100°C. In some examples, the temperature range is from about 65°C to about 100°C. In some examples, the temperature range is from about 15°C to about 80°C. In some examples, the temperature range is from about 25°C to about 80°C. In some examples, the temperature range is from about 35°C to about 80°C. In some examples, the temperature range is from about 55°C to about 80°C. In some examples, the temperature range is from about 65°C to about 80°C. In some examples, the temperature range is from about 15°C to about 60°C. In some examples, the temperature range is from about 25°C to about 60°C. In some examples, the temperature range is from about 35°C to about 60°C. In some examples, the temperature range is from about 15°C to about 40°C. In some examples, the temperature range is from about -20°C to about 100°C. In some examples, the temperature range is from about -20°C to about 90°C. In some examples, the temperature range is from about -20°C to about 50°C. In some examples, the temperature range is from about -20°C to about 40°C. In some examples, the temperature range is from about -20°C to about 10°C. In some examples, the temperature range is from about 0°C to about 100°C. In some examples, the temperature range is from about 0°C to about 40°C. In some examples, the temperature range is from about 0°C to about 30°C. In some examples, the temperature range is from about 0°C to about 20°C. In some examples, the temperature range is from about 0°C to about 10°C. In some examples, the temperature range is from about 15°C to about 100°C. In some examples, the temperature range is from about 15°C to about 90°C. In some examples, the temperature range is from about 15°C to about 800°C. In some examples, the temperature range is from about 15°C to about 70°C. In some examples, the temperature range is from about 15°C to about 60°C. In some examples, the temperature range is from about 15°C to about 50°C.In some examples, the temperature range is from about 15°C to about 30°C. In some examples, the temperature range is from about 10°C to about 30°C. In some examples, the methods disclosed herein do not require cooling, freezing, or heating and are performed at room temperature. In some examples, amplifying comprises contacting the sample with random oligonucleotide primers. In some examples, amplifying comprises contacting the cell-free nucleic acid molecules disclosed herein with random oligonucleotide primers. In some examples, amplifying comprises contacting the cell-free fetal nucleic acid molecules disclosed herein with random oligonucleotide primers. In some examples, amplifying comprises contacting the tagged nucleic acid molecules disclosed herein with random oligonucleotide primers. Amplifying with multiple random primers generally results in non-target amplification of multiple nucleic acids of different sequences or overall amplification of most of the nucleic acids in the sample.

[0139] In some examples, amplification includes target amplification (e.g., selector methods (described in U.S. Patent No. 6,558,928), molecular inversion probes). In some examples, the step of amplifying a nucleic acid comprises contacting the nucleic acid with at least one primer having a sequence corresponding to a target chromosomal sequence. Exemplary chromosomal sequences are disclosed herein. In some examples, amplification comprises contacting the nucleic acid with at least one primer having a sequence corresponding to a non-target chromosomal sequence. In some embodiments, amplification comprises contacting the nucleic acid with one or fewer pairs of primers, each primer of the pair of primers comprising a sequence corresponding to a sequence on a target chromosome disclosed herein. In some examples, amplification comprises contacting the nucleic acid with multiple sets of primers, each of the first pair of the first set and each of the pairs of the second set being all different.

[0140] In some examples, amplification comprises contacting a sample with at least one primer having a sequence corresponding to a sequence on a target chromosome disclosed herein. In some examples, amplification comprises contacting a sample with at least one primer having a sequence corresponding to a sequence on a non-target chromosome disclosed herein. In some examples, amplification comprises contacting a sample with one or fewer pairs of primers, wherein each primer of the pair of primers comprises a sequence corresponding to a sequence on a target chromosome disclosed herein. In some examples, amplification comprises contacting a sample with a plurality of sets of primers, wherein each of the first pair of the first set and each of the pairs of the second set are all different.

[0141] In some examples, amplification comprises multiplexing (amplification of multiple nucleic acids in one reaction). In some examples, multiplexing comprises contacting nucleic acids of a biological sample with a plurality of oligonucleotide primer pairs. In some examples, multiplexing comprises contacting a first nucleic acid with a second nucleic acid, wherein the first nucleic acid corresponds to a first sequence and the second nucleic acid corresponds to a second sequence. In some examples, the first sequence and the second sequence are the same. In some examples, the first sequence and the second sequence are different. In some examples, amplification does not comprise multiplexing. In some examples, amplification does not require multiplexing. In some examples, amplification comprises nested primer amplification. The method may comprise multiplex PCR of multiple regions, wherein each region comprises a single nucleotide polymorphism (SNP). Multiplexing may be performed within a single tube. In some examples, the method comprises multiplex PCR of more than 100 regions, wherein each region comprises an SNP. In some examples, the method comprises multiplex PCR of more than 500 regions, wherein each region comprises an SNP. In some examples, the method comprises multiplex PCR of more than 1000 regions, wherein each region comprises an SNP. In some examples, the method comprises multiplex PCR of more than 2000 regions, wherein each region comprises an SNP. In some examples, the method comprises multiplex PCR of more than 300 regions, wherein each region comprises an SNP.

[0142] In some examples, the method includes a step of amplifying nucleic acid in a sample, where the step of amplifying includes contacting the sample with at least one oligonucleotide primer, where the at least one oligonucleotide primer is not activated or not extendable until contacting the sample. In some examples, the step of amplifying includes contacting the sample with at least one oligonucleotide primer, where the at least one oligonucleotide primer is not activated or not extendable until exposed to a selected temperature. In some examples, the step of amplifying includes contacting the sample with at least one oligonucleotide primer, where the at least one oligonucleotide primer is not activated or not extendable until contacting an activation reagent. As a non-limiting example, the at least one oligonucleotide primer may include a blocking group. By using such oligonucleotide primers, primer dimers can be minimized, unused primers can be recognized, and / or false results due to unused primers can be avoided. In some examples, the step of amplifying includes contacting the sample with at least one oligonucleotide primer that includes a sequence corresponding to a sequence on a target chromosome disclosed herein.

[0143] In some examples, the methods disclosed herein include the use of one or more tags. By using one or more tags, at least one of the efficiency, speed, and accuracy of the methods disclosed herein can be improved. In some examples, the oligonucleotide primers include tags, where the tags are not specific to the target sequence. Such tags may be referred to as universal tags. In some examples, the method includes tagging a target sequence or a fragment thereof in a sample with a tag that is not specific to the target sequence. In some examples, tags that are not specific to sequences on human chromosomes are used. Alternatively or additionally, the method includes contacting the sample with a tag and at least one oligonucleotide primer that includes a sequence corresponding to the target sequence, where the tag is separate from the oligonucleotide primer. In some examples, the tag is incorporated into the amplification product generated by the extension of the oligonucleotide primer after the oligonucleotide primer hybridizes to the target sequence. The tag may be an oligonucleotide, a small molecule, or a peptide. In some examples, the tag does not include nucleotides. In some examples, the tag does not include oligonucleotides. In some examples, the tag does not include amino acids. In some examples, the tag does not include peptides. In some examples, the tag is not sequence-specific. In some examples, the tag includes a general sequence that does not correspond to a specific target sequence. In some examples, the tag is detectable when the amplification product is generated, regardless of the amplified sequence. In some examples, at least one of the oligonucleotide primer and the tag includes a peptide nucleic acid (PNA). In some examples, at least one of the oligonucleotide primer and the tag includes a locked nucleic acid (LNA).

[0144] In some examples, the methods disclosed herein include the use of multiple tags, thereby increasing at least one of the accuracy of the method, the speed of the method, and the information obtained by the method. In some examples, the methods disclosed herein include the use of multiple tags, thereby reducing the amount of sample required to obtain reliable results. In some examples, the multiple tags include at least one capture tag. In some examples, the multiple tags include at least one detection tag. In some examples, the multiple tags include a combination of at least one capture tag and at least one detection tag. Capture tags are used to isolate or separate a particular sequence or region from other regions. A typical example of a capture tag is biotin (which can be captured using, for example, a streptavidin-coated surface). Examples of detection tags are digoxigenin and fluorescent tags. Detection tags may be detected directly (e.g., measurement of laser irradiation and / or luminescence), or may be detected indirectly via an antibody that holds or interacts with a secondary detection system such as a luminescence assay or an enzyme assay. In some examples, the multiple tags include a combination of at least one capture tag (a tag used to isolate an analyte) and at least one detection tag (a tag used to detect an analyte). In some examples, a single tag functions as both a detection tag and a capture tag.

[0145] In some examples, the method includes contacting at least one cell-free nucleic acid in a sample with a first tag and a second tag, where the first tag includes a first oligonucleotide complementary to the sense strand of the cell-free nucleic acid and the second capture tag includes a second oligonucleotide complementary to the antisense strand of the cell-free nucleic acid. In some embodiments, the method includes contacting at least one cell-free nucleic acid in a sample with a first tag and a second tag, where the first tag retains the same label as the second tag. In some embodiments, the method includes contacting at least one cell-free nucleic acid in a sample with a first tag and a second tag, where the first tag retains a different label from the second tag. In some examples, the tags are the same and there is a single qualitative / quantitative signal that is an aggregate of all the detected probes / regions. In some examples, the tags are different. One tag may be used for purification and one tag may be used for detection. In some examples, the first oligonucleotide tag is specific for a region (e.g., a cfDNA fragment), retains a fluorescent label, and the second oligonucleotide is specific for an adjacent region and has the same fluorescent label. This is because only the collective signal is desired. In other examples, the first oligonucleotide tag is specific for a region (e.g., a cfDNA fragment), retains a fluorescent label, and the second oligonucleotide is specific for an adjacent region and retains a different fluorescent label, such that two different regions are detected.

[0146] In some examples, the method includes detecting an amplification product, which is generated by amplifying at least a portion of a target chromosome or a fragment thereof disclosed herein. The portion or fragment of the target chromosome may include at least 5 nucleotides. The portion or fragment of the target chromosome may include at least about 10 nucleotides. The portion or fragment of the target chromosome may include at least about 15 nucleotides. In some examples, the step of detecting the amplification product disclosed herein does not include tagging or labeling the amplification product. In some examples, the method detects the amplification product based on its amount. For example, the method may detect an increase in the amount of double-stranded DNA in a sample. In some examples, the detection of the amplification product is performed at least in part based on its size. In some examples, the amplification product has a length of about 50 base pairs to about 500 base pairs.

[0147] In some examples, the step of detecting the amplification product includes contacting the amplification product with a tag. In some examples, the tag includes a sequence complementary to the sequence of the amplification product. In some examples, the tag does not include a sequence complementary to the sequence of the amplification product. Non-limiting examples of tags are described in the foregoing and following disclosures.

[0148] In some examples, regardless of whether it is tagged, the step of detecting the amplification product includes applying the amplification product to a signal detector or an assay assembly of a device, system, or kit disclosed herein. In some examples, the method includes amplification and detection on an assay assembly of a device, system, or kit disclosed herein. In some examples, the assay assembly includes amplification reagents. In some examples, the method includes applying an instrument or reagent to an assay assembly (e.g., a lateral flow assay) disclosed herein, the step of controlling the flow of a biological sample, solution, or combination thereof through the lateral flow assay. In some examples, the instrument is a vacuum, pipette, pump, or combination thereof.

[0149] Array determination In some examples, the methods disclosed herein include the step of sequencing a nucleic acid. The nucleic acid may be a nucleic acid disclosed herein, such as a tagged nucleic acid, an amplified nucleic acid, a cell-free nucleic acid, a cell-free fetal nucleic acid, a nucleic acid having a sequence corresponding to a target chromosome, a nucleic acid having a sequence corresponding to a region of a target chromosome, a nucleic acid having a sequence corresponding to a non-target chromosome, or a combination thereof. In some examples, the nucleic acid is DNA. In some examples, the nucleic acid is RNA. In some examples, the nucleic acid includes DNA. In some examples, the nucleic acid includes RNA.

[0150] In some examples, sequencing includes targeted sequencing. In some examples, sequencing includes whole genome sequencing. In some examples, sequencing includes both targeted sequencing and whole genome sequencing. In some examples, whole genome sequencing includes massively parallel sequencing, also known as next generation sequencing or second generation sequencing in the art. In some examples, whole genome sequencing includes random massively parallel sequencing. In some examples, sequencing includes random massively parallel sequencing of a target region captured from a whole genome library.

[0151] In some examples, the method includes the step of sequencing an amplified nucleic acid disclosed herein. In some examples, the amplified nucleic acid is generated by target amplification (e.g., using primers specific for a desired target sequence). In some examples, the amplified nucleic acid is generated by non-target amplification (e.g., using random oligonucleotide primers). In some examples, the method includes the step of sequencing an amplified nucleic acid, and the sequencing includes massively parallel sequencing.

[0152] In some examples, the method includes performing a genomic sequence alignment using an algorithm. By way of non-limiting example, the algorithm may be designed to recognize the copy number of a chromosome. The algorithm may be designed to reveal the observed number of sequence reads associated with each relevant allele at various SNP loci. The algorithm may use parental genotype and crossover frequency data to in silico generate mono-chromosomal, di-chromosomal, and tri-chromosomal genotypes at the measured loci, which can be used to predict sequencing data for each genotype. Using a Bayesian model, select the sequencing data of maximum likelihood as the copy number and the fetal fraction, and use the accuracy of the calculated likelihood. For each of the two possible alleles of each SNP, different probability distributions are expected and the observed alleles can be compared. This is described in Zimmermann et al., Prenat Diagn (2012) 32:1233-1241. However, Zimmermann et al. considered that samples containing a fetal fraction of less than 4.0% could not be informative and that at least 20 ml of blood volume was required to obtain sufficient cell-free DNA for this type of analysis. In contrast, the method of the present application can employ this analysis with samples containing a fetal fraction of less than 4% and samples that do not require approximately the same amount of sample.

[0153] Library preparation In some examples, the methods disclosed herein include modifying cell-free nucleic acids in a biological sample to generate a library of cell-free nucleic acids for detection. In some examples, the method includes modifying cell-free nucleic acids for nucleic acid sequencing. In some examples, the method includes modifying cell-free nucleic acids for detection, where the detection does not include nucleic acid sequencing. In some examples, the method includes modifying cell-free nucleic acids for detection, where the detection includes counting tagged cell-free nucleic acids based on the occurrence of tag detection. In some examples, the methods disclosed herein include modifying cell-free nucleic acids in a biological sample to generate a library of cell-free nucleic acids, where the method includes amplifying the cell-free nucleic acids. In some examples, the modification is performed prior to amplification. In some examples, the modification is performed after amplification.

[0154] In some examples, the step of modifying cell-free nucleic acid includes repairing the ends of the cell-free nucleic acid that are fragments of nucleic acid. As a non-limiting example, end repair can include restoring a 5' phosphate group, a 3' hydroxy group, or a combination thereof to the cell-free nucleic acid. In some examples, repair includes 5'-phosphorylation, A-tailing, gap filling, closing of nicking sites, or a combination thereof. In some examples, repair can include removing overhangs. In some examples, repair can include filling overhangs with complementary nucleotides. In some examples, the step of modifying cell-free nucleic acid to prepare a library includes the use of an adapter. The adapter may also be referred to herein as a sequencing adapter. In some examples, the adapter aids in sequencing. Generally, an adapter includes an oligonucleotide. As a non-limiting example, the adapter may simplify other steps in the method, such as amplification, purification, and sequencing, because it has a sequence that is universal to a plurality of nucleic acids, if not all, in the sample after modification. In some examples, the step of modifying cell-free nucleic acid includes ligating an adapter to the nucleic acid. Ligation can include blunt-end ligation. In some examples, the step of modifying cell-free nucleic acid includes hybridizing an adapter to the nucleic acid. In some examples, the sequencing adapter includes a hairpin or stem-loop adapter. In some examples, the step of modifying cell-free nucleic acid includes hybridizing a hairpin or stem-loop adapter to the nucleic acid, thereby generating a circular library product that is sequenced or analyzed. In some examples, the sequencing adapter has a blocked 5' end and a nick left at the 3' end. Advantages of this configuration include, but are not limited to, improved library efficiency, reduction of unwanted by-products such as adapter dimers, etc. In further examples, the adapter has a cleavable replication stop site for linearizing the template.

[0155] The efficiency of library preparation steps (such as end repair, tailing, and adapter ligation, etc.) and amplification may benefit from the addition of a crowding agent to the sample or amplification reaction. Enzymatic processes in their natural environment (such as DNA replication in cells) often occur in a crowded environment. Some of these enzymatic processes are more efficient in a crowded environment. For example, a crowded environment can enhance the activity of DNA helicase and the sensitivity of DNA polymerase. Therefore, a crowding agent can be added to mimic a crowded environment. The crowding agent may be a polymer. The crowding agent may be a protein. The crowding agent may be a polysaccharide. Non-limiting examples of crowding agents are polyethylene glycol, dextran, and ficoll. Concentrations that mimic in vivo crowding are often desirable. For example, 4% (40 mg / ml) of PEG 1 kDa provides an approximate crowding effect seen in vivo. In some examples, the concentration of the crowding agent is about 2 to about 20% w / v in the amplification reaction. In some examples, the concentration of the crowding agent is about 2 to about 15% w / v in the amplification reaction. In some examples, the concentration of the crowding agent is about 2 to about 10% w / v in the amplification reaction. In some examples, the concentration of the crowding agent is about 2 to about 8% w / v in the amplification reaction. In some examples, the concentration of the crowding agent is about 3 to about 6% w / v in the amplification reaction.

[0156] In some examples, the step of modifying cell-free nucleic acids to prepare a library involves the use of tags, which may also be referred to herein as barcodes. In some examples, the methods disclosed herein include the step of modifying cell-free nucleic acids having tags corresponding to a desired chromosomal region. In some examples, the methods disclosed herein include the step of modifying cell-free nucleic acids having tags specific to an undesired chromosomal region. In some examples, the methods disclosed herein include the step of modifying a first portion of cell-free nucleic acids having a first tag corresponding to at least one desired chromosomal region and a second portion of cell-free nucleic acids having a second tag corresponding to at least one undesired chromosomal region. In some examples, the step of modifying the nucleic acids includes ligating the tags to the cell-free nucleic acids. The ligation may include blunt-end ligation. In some examples, the step of modifying cell-free nucleic acids includes hybridizing the tags to the nucleic acids. In some examples, the tags include oligonucleotides. In some examples, the tags include non-oligonucleotide markers or labels that are detectable by means other than nucleic acid analysis. By way of non-limiting example, the non-oligonucleotide markers or labels may include fluorescent molecules, nanoparticles, dyes, peptides, or other detectable / quantifiable small molecules.

[0157] In some embodiments, the tagging of (c) comprises one or more steps of: (i) generating blunt ends of cell-free DNA, wherein in some embodiments, 5' overhangs or 3' recessed ends are removed using one or more polymerases and one or more exonucleases; (ii) dephosphorylating the blunt ends of cell-free DNA; (iii) contacting the cell-free DNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cell-free DNA; or (iv) repairing or removing DNA damage in the cell-free DNA using a ligase, to generate ligation-competent cell-free DNA; and (b) ligating the ligation-competent cell-free DNA to adapter oligonucleotides by contacting the ligation-competent cell-free DNA with adapter oligonucleotides in the presence of a ligase, a crowding reagent, and / or a small molecule enhancer. In some embodiments, the one or more polymerases include T4 DNA polymerase or DNA polymerase I. In some embodiments, the one or more exonucleases include T4 polynucleotide kinase or exonuclease III. In some embodiments, the ligase consists of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7-ligase fusion protein. In some embodiments, the ligase includes T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase, Ampligase, E. coli ligase, or Sso7 ligase fusion protein. In some embodiments, the crowding reagent includes polyethylene glycol (PEG), glycogen, or dextran, or a combination thereof. In some embodiments, the small molecule enhancer includes dimethyl sulfoxide (DMSO), polysorbate 20, formamide, or diol, or a combination thereof.In some embodiments, the ligation of (b) includes blunt-end ligation or single-base overhang ligation. In some embodiments, the adapter oligonucleotide includes a Y-shaped adapter, a hairpin adapter, a stem-loop adapter, a cleavable adapter, a blocked self-ligating adapter, or a barcoded adapter, or combinations thereof.

[0158] In some examples, the step of modifying cell-free nucleic acids to prepare a library includes the use of a sample index, which may simply be referred to herein as an index. By way of non-limiting example, the index may include an oligonucleotide, a small molecule, a nanoparticle, a peptide, a fluorescent molecule, a dye, or other detectable / quantifiable moiety. In some examples, a first group of cell-free nucleic acids from a first biological sample is labeled with a first index, and a second group of cell-free nucleic acids from a first biological sample is labeled with a second index, where the first index and the second index are different. Thus, multiple indexes can distinguish cell-free nucleic acids from multiple samples when analyzing multiple samples at once. In some examples, the method discloses a step of amplifying cell-free nucleic acids, where the oligonucleotide primers used to amplify the cell-free nucleic acids include an index.

[0159] DNA loss can occur at all steps of DNA isolation and analysis, but the highest losses generally occur during library preparation. Conventional methods show losses of 80% to 90% of the material. Often, this loss is compensated for by subsequent amplification steps to raise the DNA concentration to the necessary levels required for next-generation sequencing, but amplification cannot compensate for the loss of information that occurred during the previous steps. A library with a drawback of 80% loss of the initial DNA in the sample can be described as a library with an efficiency of 20% or 0.2. In some examples, the methods disclosed herein include the step of achieving a library having an efficiency of at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, or at least about 0.8. In some examples, the methods disclosed herein include the step of producing a library having an efficiency of at least about 0.4. In some examples, the methods disclosed herein include the step of producing a library having an efficiency of at least about 0.5. Methods for producing libraries having such efficiencies can achieve these efficiencies using crowding agents, repairing cell-free DNA fragment ends, ligation methods, purification methods, cycling parameters, and theoretical mixing ratios as described herein. In some examples, the library preparation method does not require nucleic acid amplification of cell-free nucleic acids. In some examples, a library of cell-free nucleic acids is generated using an in vitro CRISPR-Cas targeted cleavage process, whereby a guide RNA (gRNA) binds to a complementary DNA target site and, for example, when an engineered protein is used in combination with Cas9, a blunt-ended double-strand break or a single-strand nick can be generated. This DNA cleavage can be utilized to modify the cleaved DNA to make it suitable for ligation and / or amplification processes, as well as subsequent analysis of these target regions. Thus, CRISPR is used as a target enrichment process for DNA analysis or sequencing.Non-limiting examples of natural and artificial CRISPR-Cas combinations useful for this purpose include Cas9, Cas12, Cascade, and Cas13, or subtypes thereof. In some cases, the Cas enzyme is a Cas ortholog as described in Adrian Pickar-Oliver et al., The next generation of CRISPR-Cas technologies and applications, Nat Rev Mol Cell Biol. 2019 Aug;20(8):490-507, which is incorporated herein by reference.

[0160] In further examples, the gRNA is designed to increase target fragmentation and decrease off-target fragmentation, thereby improving the efficiency of the library without amplification. In some cases, the library is treated with exonuclease, thereby deleting non-target DNA fragments within the library and enriching for target fragments.

[0161] The endonucleases described herein function, in some cases, by initiating single-stranded or double-stranded DNA cleavage. In some cases, the single-stranded or double-stranded DNA cleavage is adjacent to, within, or on either side of the guide RNA binding site. In this way, the endonucleases described herein can be designed to provide specific solutions to current analytical problems. In some examples, library preparation is mediated by a transposase enzyme that fragments double-stranded DNA and ligates synthetic tags to the 3' and 5' ends of the fragments (e.g., oligonucleotides). In some cases, this process is tagmentation-based library construction. In some cases, the transposase operates by a "cut and paste" mechanism. In some cases, the transposase is Tn5 transposase or a variant thereof. In some cases, the tags are oligonucleotides about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs in length. In some cases, the tags are free synthetic ME adapters as provided in the NExtera DNA kit (Illumina). In some cases, the fragmented DNA is amplified by the methods described herein.

[0162] Detection of genetic information Generally, the methods disclosed herein include the step of detecting a biomarker, an analyte, or a modified form thereof. In some examples, the method includes the step of detecting a nucleic acid. In some examples, the method includes the step of detecting a cell-free nucleic acid. In some examples, the method includes the step of detecting a tag of a nucleic acid. In some examples, the method includes the step of detecting an amplicon of a nucleic acid. Alternatively or additionally, the method includes the step of detecting a non-nucleic acid component. As non-limiting examples, the non-nucleic acid components can be selected from proteins, peptides, lipids, fatty acids, sterols, phospholipids, carbohydrates, viral components, microbial components, and combinations thereof. In the case of viral or microbial components, the method can include the steps of releasing, purifying, and / or amplifying nucleic acids from the virus or bacteria prior to detection.

[0163] The detecting step may include sequencing a desired nucleic acid. The detecting step may include detecting a tag on the desired nucleic acid. The detecting step may include detecting a tag on a desired biomarker. The biomarker may be an epigenetic modification. The biomarker may be an epigenetic profile (multiple epigenetic modifications). The biomarker may be an epigenetically modified nucleic acid. The detecting step may include bisulfite sequencing. The detecting step may include performing a chromatin immunoprecipitation (ChIP) assay. The detecting step may include sequencing a tag on a desired biomarker.

[0164] The detecting step may include amplification, as described herein. For example, the amplification may include qPCR where a signal is generated based on the presence or absence of a target analyte. In some examples, the amplification includes PCR. In some examples, the amplification does not include PCR. In some examples, the amplification includes rolling circle amplification (RCA). In some examples, cfDNA is contacted with a DNA ligase and a probe designed to hybridize to the cfDNA. In some examples, cfDNA is first cleaved (e.g., exposed to a restriction enzyme) to produce cfDNA fragments, and the cfDNA fragments are contacted by the ligase and the probe. The ligase creates circularized cfDNA labeled with the probe. Optionally, a backbone oligo is used to circularize the cfDNA or the cfDNA fragment. These circularized fragments are replicated by RCA to produce concatemers. The probe is recognized by a detectable oligonucleotide (e.g., fluorescent) and can be imaged.

[0165] The method may include detecting a gene mutation in the nucleic acid of a biological sample. The method may include detecting multiple gene mutations in the nucleic acid of a biological sample. The method may include detecting a gene mutation in each of multiple nucleic acids of a biological sample. The method may include detecting multiple gene mutations in multiple nucleic acids of a biological sample.

[0166] The method may include a step of detecting epigenetic modifications of nucleic acids in a biological sample. In some examples, the step of detecting epigenetic modifications includes performing bisulfite sequencing. In some examples, the step of detecting epigenetic modifications includes performing a chromatin immunoprecipitation (ChIP) assay. In some examples, the epigenetic modification is a genetic modification. In some examples, the epigenetic modification is a modification that enables a cell to affect transcription in response to one or more environmental stimuli. As a non-limiting example, the epigenetic modification can be methylation of a cytosine or adenine residue. In some examples, the epigenetic modification is the absence of a methyl group. Typically, methylation promotes gene silencing. Epigenetic modifications also include histone acetylation, methylation, ubiquitination, and phosphorylation. Epigenetic modifications can promote, inhibit, interfere with, or reduce biological processes (e.g., immune response, cell proliferation). The method may include a step of detecting multiple epigenetic modifications of nucleic acids in a biological sample. The method may include a step of detecting epigenetic modifications of each of multiple nucleic acids in a biological sample. The method may include a step of detecting epigenetic modifications of multiple nucleic acids in a biological sample. The method may include a step of performing a genome-wide analysis of epigenetic modifications to identify differentially methylated regions between a test sample and a control / reference sample.

[0167] The method may include the step of detecting one or more epigenetic modifications specific to a tissue. For example, the tissue may have a characteristic methylation profile that can be used to trace the origin of cell-free nucleic acids. This may be useful for determining the origin of cell-free nucleic acids. As a non-limiting example, the epigenetic modification may be specific to the brain, and cell-free nucleic acids having such epigenetic modification may indicate a neurodegenerative disease or a brain tumor. The method may further include the step of testing, biopsying, imaging, or treating the tissue if such cell-free nucleic acids are detected. The method may include the step of detecting one or more epigenetic modifications specific to only two tissues. The method may include the step of detecting one or more epigenetic modifications specific to less than three tissues. The method may include the step of detecting one or more epigenetic modifications specific to less than five tissues.

[0168] The method may include the step of detecting a detectable label or a detectable signal of a nucleic acid component or a non-nucleic acid component. The method may include the step of detecting a detectable label or a detectable signal of a binding moiety (e.g., a small molecule, a peptide, an aptamer, an antibody, or an antigen-binding fragment thereof) that binds to a nucleic acid component or a non-nucleic acid component. As a non-limiting example, the detectable label or signal may be a fluorescent molecule, a bioluminescent molecule, a luminescent molecule, a radioactive signal, a magnetic signal, an electrical signal, or a dye. For example, the method may include the step of detecting an interaction between a binding site and a desired protein. As a non-limiting example, the step of detecting may include performing IPCR or PLA.

[0169] The detecting step may include viewing an interface of a device or system disclosed herein where the results of the test are displayed. For example, see FIGS. 4 and 5A-E. The detecting step may include viewing the appearance of a color or a fluorescent signal on a lateral flow device. The detecting step may include receiving the results of the test on a device disclosed herein. The detecting step may include receiving the results of the test on a mobile device, computer, laptop, or other electronic device that communicates with a device of the system disclosed herein.

[0170] Generally, the methods, kits, systems, and devices disclosed herein can provide genetic information in a short time. In some examples, the methods disclosed herein are performed in less than about 1 minute. In some examples, the methods disclosed herein are performed in less than about 2 minutes. In some examples, the methods disclosed herein are performed in less than about 5 minutes. In some examples, the methods disclosed herein are performed in less than about 10 minutes. In some examples, the methods disclosed herein are performed in less than about 15 minutes. In some examples, the methods disclosed herein are performed in less than about 20 minutes. In some examples, the methods disclosed herein are performed in less than about 30 minutes. In some examples, the methods disclosed herein are performed in less than about 45 minutes. In some examples, the methods disclosed herein are performed in less than about 60 minutes. In some examples, the methods disclosed herein are performed in less than about 90 minutes. In some examples, the methods disclosed herein are performed in less than about 2 hours. In some examples, the methods disclosed herein are performed in less than about 3 hours. In some examples, the methods disclosed herein are performed in less than about 4 hours.

[0171] In some examples, the methods disclosed herein require minimal technical training. In some examples, the methods disclosed herein require no technical training. In some examples, the methods disclosed herein require only that an individual practicing the methods disclosed herein follow a simple protocol of carrying and mixing samples and solutions. For example, the methods disclosed herein can be used at home by a subject during pregnancy without the assistance of a technician or healthcare provider. In some examples, the methods disclosed herein are executable by users without medical training or technical training. In some examples, the methods, kits, systems, and devices disclosed herein only require that a user add a biological sample to a system or device and view the results to obtain genetic information.

[0172] Method for detecting a disease or disorder of a subject The method may include a step of detecting the presence of a disease or disorder based on the detection. The method may include a step of detecting the risk of a disease or disorder based on the detection. The method may include a step of detecting the state of a disease or disorder based on the detection. The method may include a step of monitoring the state of a disease or disorder based on the detection. The method may include a step of administering a treatment method based on the detection. The method may include a step of changing the dosage of a drug administered to a subject based on the detection. The method may include a step of monitoring the response of a subject to a treatment based on the detection. For example, the disease may be cancer, and the treatment method may be chemotherapy. Other cancer treatments include, but are not limited to, antibodies, antibody-drug conjugates, antisense molecules, engineered T cells, and radiation. The method may include a step of further examining the subject based on the detection. For example, the disease may be cancer, and the further examination may include, but is not limited to, imaging (e.g., CAT-SCAN, PET-SCAN), and performing a biopsy.

[0173] In some examples, the methods disclosed herein include detecting that there is fetal aneuploidy of at least one target chromosome. In some examples, the methods disclosed herein include detecting that there is fetal aneuploidy of at least one target chromosome when the amount of sequencing reads is detected in a sample disclosed herein. In some examples, the amount of sequencing reads corresponds to sequences from a chromosome or chromosomal region known to exhibit aneuploidy in the human population, as described herein.

[0174] In some examples, the methods disclosed herein include detecting that there is fetal aneuploidy of at least one target chromosome when the ratio of the sequencing reads corresponding to at least one target chromosome to the sequencing reads corresponding to at least one non-target chromosome is different from the respective ratios in a control biological sample from a control subject during pregnancy carrying a euploid fetus. In some examples, the method includes detecting that there is fetal aneuploidy of at least one target chromosome from the fact that the ratio of the sequencing reads corresponding to at least one target chromosome to the sequencing reads corresponding to at least one non-target chromosome is different from the respective ratios in a control biological sample from a control pregnant subject carrying a euploid fetus. In some examples, the method includes detecting that there is no fetal aneuploidy of at least one target chromosome from the fact that the ratio of the sequencing reads corresponding to at least one target chromosome to the sequencing reads corresponding to at least one non-target chromosome is not different from the respective ratios in a control biological sample from a control subject during pregnancy carrying a euploid fetus.

[0175] In some examples, the sequencing reads corresponding to at least one target chromosome include the sequencing reads corresponding to the chromosomal region of at least one target chromosome. In some examples, the sequencing reads correspondin...

Claims

1. 1. A method comprising: a) obtaining a biological sample from a subject, the biological sample comprising cell-free deoxyribonucleic acid (cfDNA) and the biological sample having a volume of up to 120 microliters when obtained from the subject; b) a. i. generating blunt ends of cfDNA, where 5' overhangs or 3' recessed ends are removed using one or more polymerases and one or more exonucleases; ii. dephosphorylating the blunt ends of the cfDNA; iii. Contacting the cfDNA with a crowding reagent, thereby enhancing the reaction between one or more polymerases, one or more exonucleases, and the cfDNA; or iv. Repairing or removing DNA damage in cfDNA using a ligase; generating ligation competent cfDNA by one or more steps; b. Ligating the ligation competent cfDNA to an adaptor oligonucleotide by contacting the ligation competent cfDNA with an adaptor oligonucleotide in the presence of a ligase and one or more of a crowding reagent and a small molecule enhancer; tagging at least a portion of the cfDNA to generate a cfDNA tagged with c) optionally amplifying the tagged cfDNA; d) sequencing at least a portion of the tagged cfDNA; The method includes:

2. 10. The method of claim 1, wherein the volume is up to 100 microliters when obtained from a subject.

3. 3. The method of claim 2, wherein the volume is up to 40 microliters when obtained from a subject.

4. The method of claim 1 , wherein the biological sample obtained from the subject is capillary blood.

5. 5. The method of claim 4, wherein the volume is up to 40 microliters when obtained from a subject.

6. The biological samples are a) inducing a first percutaneous puncture to generate a first fraction of a biological sample; b) discarding a first fraction of the biological sample; c) collecting a second fraction of the biological sample, thereby reducing or eliminating contamination of the biological sample by lysis of white blood cells; The method of claim 4, wherein the antibody is obtained from a subject by

7. 13. The method of claim 1, further comprising detecting normal representation, over-representation, or under-representation of at least one target sequence in at least a portion of the tagged cfDNA.

8. The method of claim 1 , wherein the subject is carrying a fetus.

9. 9. The method of claim 8, wherein the cfDNA component is a fetal cfDNA component from a fetus.

10. 10. The method of claim 9, wherein the cfDNA in the biological sample is about 10 genome equivalents.

11. 10. The method of claim 9, further comprising analyzing the genotype information and the fetal cfDNA component from the individual to determine whether the individual has paternally contributed to the fetus by identifying a genotype match between the fetal cfDNA component and the genotype information.

12. (c) comprising a step of amplifying, wherein generating ligation competent cfDNA comprises: d) generating blunt ends of the cfDNA, where 5' overhangs or 3' recessed ends are removed using one or more polymerases and one or more exonucleases; e) dephosphorylating the blunt ends of the cfDNA; f) contacting the cfDNA with a crowding reagent, thereby enhancing the reaction between the one or more polymerases, the one or more exonucleases, and the cfDNA; g) repairing or removing DNA damage in cfDNA using a ligase; The method of claim 1 , comprising:

13. 2. The method of claim 1, wherein the cfDNA is selected from a subject's tumor, a transplanted tissue or organ, or one or more pathogens.

14. The method of claim 13 , wherein the one or more pathogens comprise a bacterium or a component thereof.

15. The method of claim 13 , wherein the one or more pathogens comprise a virus or a component thereof.

16. The method of claim 13 , wherein the one or more pathogens comprise a fungus or a component thereof.

17. 2. The method of claim 1, comprising amplifying in (c) by a large-scale multiplex amplification assay.

18. The method of claim 17, wherein the massively multiplexed amplification assay is an isothermal amplification.

19. 18. The method of claim 17, wherein the massively multiplexed amplification assay is a massively multiplexed polymerase chain reaction (mmPCR).

20. 10. The method of claim 1, further comprising pooling two or more biological samples, each sample obtained from a different subject.

21. 10. The method of claim 1, further comprising the step of contacting the biological sample with a white blood cell stabilizing agent after obtaining the biological sample from the subject.

22. 10. The method of claim 1, wherein the biological sample obtained from the subject is collected using a device configured to dissolve intercellular junctions in the subject's epidermis.

23. The library h) end repair, 5' phosphorylation, and A-tailing with incubation at 20°C for 30 minutes followed by 65°C for 30 minutes; i) ligating cfDNA to adapter oligonucleotides with incubation at 20° C. for 15 minutes; j) cleaving the ligated adaptor loop from the adaptor oligonucleotide with incubation at 37° C. for 15 minutes to generate ligation competent cfDNA; k) i. denaturing the ligation competent cfDNA at 98°C for 1 minute, followed by 13 cycles of denaturation at 98°C for 10 seconds; ii. annealing the denatured, ligation competent cfDNA to one or more complementary primers from (i) at 65° C. for 75 seconds; and iii. extending the ligation competent cfDNA at 65° C. for 5 minutes to generate an amplified library of ligation competent cfDNA; amplifying ligation-competent cfDNA by: l) purifying the amplified library of ligation-competent cfDNA using SPRI beads; 2. The method of claim 1, wherein when prepared by the method of claim 1, the tagging step in (b) produces a library of tagged cfDNA with an efficiency of at least 0.

5.

24. 1. A method comprising: a) obtaining a biological sample from a pregnant subject carrying a fetus, said biological sample comprising cell-free deoxyribonucleic acid (cfDNA) and having a volume of about 120 microliters or less when obtained from said subject; b) contacting at least one cfDNA in the biological sample with a polynucleotide primer that anneals to a sequence corresponding to the desired sequence and an amplification reagent to generate an amplification product; c) detecting the presence or absence of an amplification product.

25. 25. The method of claim 24, further comprising annealing an oligonucleotide probe having a detectable label to the at least one cfDNA.

26. 26. The method of claim 25, further comprising detecting epigenetic modifications of the cfDNA.

27. 27. The method of claim 26, wherein the epigenetic modification comprises methylation at a locus of cfDNA.

28. 25. The method of claim 24, wherein detecting the presence of the amplification product indicates the sex of the fetus.

29. 29. The method of claim 28, wherein the cfDNA component is of fetal origin.

30. 25. The method of claim 24, further comprising the step of contacting the biological sample with a white blood cell stabilizing agent after obtaining the biological sample from the subject.

31. 25. The method of claim 24, wherein the volume of the biological sample is 50 microliters or less.

32. 32. The method of claim 31, wherein the volume of the biological sample is between about 10 microliters and about 40 microliters.

33. The biological samples are m) inducing a first percutaneous puncture to generate a first fraction of a biological sample; n) discarding a first fraction of the biological sample; o) collecting a second fraction of the biological sample, thereby reducing or eliminating contamination of the biological sample by lysis of white blood cells; The method of claim 24, wherein the antibody is collected from the subject by

34. 1. A method for increasing the relative amount of a target nucleic acid in a biological sample obtained from a subject, the method comprising: p) inducing a percutaneous puncture at a site in the subject to generate a first fraction and a second fraction of the biological sample; q) discarding the first fraction of the biological sample; and r) collecting a second fraction of the biological sample, thereby reducing or eliminating contamination or nucleic acid damage to the biological sample, wherein the first fraction comprises a lower fraction of the target nucleic acid compared to the fraction of the target nucleic acid in the second fraction.

35. 35. The method of claim 34, further comprising the step of cleaning the site prior to inducing a percutaneous puncture, thereby removing or reducing unwanted contaminants.

36. 36. The method of claim 35, wherein the unwanted contaminants include DNA from the percutaneous puncture site.

37. 35. The method of claim 34, wherein the nucleic acid damage comprises damage to non-apoptotic DNA in the biological sample.

38. 35. The method of claim 34, wherein the biological sample is capillary blood.

39. 35. The method of claim 34, further comprising detecting the target nucleic acid in the second fraction of the biological sample using an assay selected from massively multiplexed polymerase chain reaction (mmPCR) or nucleic acid sequencing.

40. A device, the device comprising: a) a sample collection device for obtaining a biological sample from a subject, the biological sample comprising a volume of up to 120 microliters, the biological sample comprising target cell-free DNA (cfDNA); b) a sample purification device for removing cells from the biological sample to generate a cell-depleted sample; and c) a nucleic acid detection apparatus configured to detect target cfDNA in the cell depletion sample.

41. The method further comprises the steps of: a) a ligation formulation for generating a ligation-competent target cfDNA, comprising: 1) one or more exonucleases adapted to generate blunt ends of the target cfDNA and to remove 5' overhangs or 3' recessed ends of the blunt ends of the target cfDNA; 2) blunt-end cfDNA dephosphorylating agent; 3) crowding reagent, 4) a DNA damage repair agent, or 5) a DNA ligase; and and b) one or more adaptor oligonucleotides ligated to the ligation-competent target cfDNA.

42. 41. The device of claim 40, further comprising a white blood cell stabilizing agent.

43. The device of claim 40, wherein the nucleic acid detection device is a massively multiplexed PCR device (mmPCR).

44. Nucleic acid ligation is s) one or more exonucleases adapted to generate blunt ends of the target cfDNA and to remove 5' overhangs or 3' recessed ends of the blunt ends of the target cfDNA; t) blunt-end cfDNA dephosphorylating agent; u) a DNA damage repair agent, and The device of claim 40, further comprising: v) a DNA ligase.

45. The device of claim 40 , wherein the nucleic acid detection apparatus comprises a nucleic acid sequencer or a lateral flow strip.

46. The device of claim 45 , wherein the nucleic acid sequencer comprises a signal detector.

47. 41. The device of claim 40, wherein the sample purification apparatus comprises a filter, the filter having a pore size of about 0.05 microns to about 2 microns.

48. 48. The device of claim 47, wherein the filter is a vertical filter.

49. 41. The device of claim 40, wherein the sample purification apparatus comprises a binding moiety selected from an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, and combinations thereof.

50. 41. The device of claim 40, wherein the sample collection apparatus is configured to dissolve intercellular junctions in the epidermis of the subject to obtain the biological sample.

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