Correcting for deamination-induced sequence errors

Abstract

To correct deamination-induced sequence errors.SOLUTION: Sequencing nucleic acids can identify variations associated with presence, susceptibility or prognosis of disease. However, the value of such information can be compromised by errors introduced by or before the sequencing process including preparing nucleic acids for sequencing. Blunting single-stranded overhangs on nucleic acids in a sample can introduce deamination-induced sequencing errors. The disclosure provides methods of identifying and correcting such deamination-induced sequencing errors and distinguishing them from real sequence variations.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Application No. 62 / 581,309, filed November 3, 2017, which is hereby incorporated by reference in its entirety for all purposes. [Background technology]

[0002] background A tumor is an abnormal proliferation of cells. When cells, such as tumor cells, die, fragmented DNA is often released into bodily fluids. Thus, some of the cell-free DNA in bodily fluids is tumor DNA. Tumors can be benign or malignant. Malignant tumors are often referred to as cancer.

[0003] Cancer is the leading cause of disease worldwide. Each year, tens of millions of people worldwide are diagnosed with cancer, and more than half will ultimately die from cancer. In many countries, cancer ranks as the second most common cause of death after cardiovascular disease. For many cancers, early detection is associated with improved outcomes.

[0004] Cancer is caused by the accumulation of mutations and / or epigenetic variations in an individual's normal cells, at least some of which lead to inappropriate control of cell division. Such mutations generally include copy number variations (CNVs), copy number abnormalities (CNAs), single nucleotide polymorphisms (SNVs), gene fusions, and indels, and epigenetic variations include modifications to the fifth atom of the six-atom ring of cytosine and its association with chromatin and transcription factors in DNA.

[0005] Cancer is often detected by tumor biopsy followed by analysis of cells, markers, or DNA extracted from cells. However, it has been recently proposed that cancer can also be detected from cell-free nucleic acids in bodily fluids, such as blood or urine (see, for example, Siravegna et al., Nature Reviews 2017). Such tests have the advantage of being non-invasive and being able to be performed without identifying suspected cancer cells by biopsy. However, such tests are complicated by the fact that the amount of nucleic acid in bodily fluids is very low and, depending on what nucleic acid is present, it needs to be processed into a more homogenous form before sequencing can be performed. Summary of the Invention [Means for solving the problem]

[0006] Abstract One aspect of the disclosure is a method for identifying variant nucleotides in a population of nucleic acids, comprising: (a) contacting a population of nucleic acids comprising double-stranded molecules having single-stranded overhangs at one or both ends with a protein having 5'-3' polymerase activity and 3'-5' exonuclease activity, where the protein digests the 3' overhangs and fills in the 5' overhangs with a complementary nucleic acid at one or both ends to generate double-stranded blunt-ended nucleic acids; (b) determining the sequence of the double-stranded blunt-ended nucleic acids to obtain sequenced nucleic acids; and (c) for each designated position in a reference sequence, (i) identifying a subset of sequenced nucleic acids that includes the designated position, and (ii) identifying a subset of sequenced nucleic acids within the subset in which the designated position is occupied by a variant nucleotide. and (d) calling the presence of a variant nucleotide at each designated position for which a sequenced nucleic acid in the subset having a variation supports the call, except that the presence of a variant nucleotide at a designated position is not called if (i) the variant is a C to T or G to A variation compared to the reference nucleotide, and (ii) the variant nucleotide is classified as a deamination error based on (1) the nucleotide context surrounding the designated position, and / or (2) the distance of the C to T variation at the designated position from the 5' end of a sequenced nucleic acid in the subset or the distance of the G to A variation at the designated position from the 3' end of a sequenced nucleic acid in the subset.

[0007] In some embodiments, step (c)(ii) identifies the number of consensus sequences in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in steps (d)(i) and (ii), the presence of a variant nucleotide at each designated position is called when the number of consensus sequences in the subset having variations meets a threshold.

[0008] In some embodiments, a variant nucleotide is classified as a deamination error based on the occurrence of a C to T variation at a designated position within a defined proximity of the 5' end in sequenced nucleic acids in the subset, or a G to A variation at a designated position within a defined proximity of the 3' end in sequenced nucleic acids in the subset.

[0009] In some embodiments, (ii) of (c) further comprises identifying the number of sequenced nucleic acids in the subset in which the designated position is occupied by a reference nucleotide.

[0010] In some embodiments, (b) comprises determining the sequence of both strands of the double-stranded blunt-ended nucleic acid.

[0011] In some embodiments, (c) is performed for at least one designated position, where the sequenced nucleic acids in the subset having a variation include a sequence of both strands of the sequenced nucleic acid of a double-stranded blunt-ended nucleic acid.

[0012] In some embodiments, (b) comprises determining the sequence from both ends of the strand.

[0013] In some embodiments, the method further comprises ligating the double-stranded blunt-ended nucleic acid to an adaptor comprising a barcode and amplifying the nucleic acid primed from a primer molecule that binds to the adaptor, where (b) comprises determining a sequence of the amplified nucleic acid molecule and classifying the sequences of the amplified nucleic acid molecule into families, members of the family having the same start and end points on the nucleic acid and the same barcode, and determining for the family a consensus nucleotide at each of the plurality of positions from the sequences of its respective members. A consensus sequence is not determined for families having only one member.

[0014] In some embodiments, the population of nucleic acids is derived from a cell-free nucleic acid sample of a subject. The cell-free nucleic acid sample can be derived from the bodily fluid of a subject who has cancer or has signs or symptoms consistent with having cancer. The bodily fluid can be selected from the group consisting of blood, plasma, saliva, urine, and cerebrospinal fluid. Blood and blood products (e.g., plasma and serum) contain cell-free nucleic acids that are particularly useful for the uses described herein.

[0015] In some embodiments, a C to T variation at a designated position is classified as a deamination error if its occurrence is at least 50% in a first proportion of the subset in which the designated position is within a defined proximity of the 5' end, or a G to A variation at a designated position is classified as a deamination error if its occurrence is at least 50% in a second proportion of the subset in which the designated position is within a defined proximity of the 3' end. A C to T variation at a designated position may be classified as a deamination error based on the variation having at least twice as many occurrences in a first proportion of the subset in which the designated position is within a defined proximity of the 5' end than in other sequenced nucleic acids in the subset, or a G to A variation at a designated position is classified as a deamination error based on the variation having at least twice as many occurrences in a second proportion of the subset in which the designated position is within a defined proximity of the 3' end than in other sequenced nucleic acids in the subset.

[0016] In some embodiments, the threshold is that the variation is present in at least 1% of the sequenced nucleic acids in the subset.

[0017] In some embodiments, a C to T or G to A transition is classified as a deamination error based at least on the surrounding context being TCG to TTG or CGA to CAA.

[0018] In some embodiments, the defined proximity to the 5' end is defined as being within 20 nucleotides or less to the 5' end, and the defined proximity to the 3' end is defined as being within 20 nucleotides or less to the 3' end. The defined proximity to the 5' end can be defined as being within 20 nucleotides to the 5' end, and the defined proximity to the 3' end is defined as being within 20 nucleotides to the 3' end.

[0019] In some embodiments, the protein is Klenow.

[0020] In some embodiments, (c) and (d) are performed in a computer-implemented system that performs these steps, etc. In these embodiments, the disclosure thus relates to computer-implemented methods for identifying variant nucleotides in a population of nucleic acids.

[0021] In some embodiments, the reference sequence is a sequence of the human genome. The reference sequence may be a sequence of a human chromosome. The reference sequence may include non-contiguous regions of the human genome.

[0022] In some embodiments, at least one of the called variant nucleotides is known to be associated with cancer.

[0023] In some embodiments, the methods can be performed on a population of nucleic acids derived from samples obtained from a population of subjects having or suspected of having cancer, where subjects within the population subsequently receive different treatments depending on which variant nucleotides were called in the individual subjects.

[0024] In some embodiments, variant nucleotides classified as deamination errors are at least 1% of the called variant nucleotides.

[0025] In some embodiments, the variant nucleotides classified as deamination errors are at least 10% of the called variant nucleotides.

[0026] In some embodiments, the presence of a variant is not called if at least five variant nucleotides are classified as deamination errors.

[0027] In some embodiments, the population of nucleic acids is derived from a solid tissue.

[0028] In some embodiments, the bodily fluid is plasma.

[0029] In some embodiments, the adapter comprising the barcode linked to the 5' end is different from the adapter comprising the barcode linked to the 3' end.

[0030] In some embodiments, the frequency of deamination errors is at least 1%.

[0031] In some embodiments, the frequency of deamination errors is at least 10%.

[0032] In some embodiments, a variant nucleotide is classified as a deamination error based on the average distance of a C to T transition at a specified position from the 5' end of the sequenced nucleic acids in the subset being less than the average distance of a reference nucleotide at the specified position, or the average distance of a G to A transition at a specified position from the 3' end of the sequenced nucleic acids in the subset being less than the average distance of a reference nucleotide at the specified position.

[0033] In some embodiments, the variant nucleotide is a single nucleotide variant (SNV).

[0034] One aspect of the disclosure is a method for identifying variant nucleotides in a nucleic acid, comprising: (a) contacting a double-stranded nucleic acid having a single-stranded overhang with a protein having 5'-3' polymerase activity and 3'-5' exonuclease activity, thereby producing a double-stranded, blunt-ended nucleic acid; (b) determining the sequence of the double-stranded, blunt-ended nucleic acid; and (c) comparing the determined sequence to a reference sequence, wherein the determined sequence is selected from the group consisting of at least one C at at least one designated position within 20 nucleotides or less of the 5' end of the determined sequence. and (d) calling the sequence of the nucleic acid as the determined sequence, except that in at least one of the positions where a C to T variation is within 20 nucleotides or less of the 5' end of the determined sequence or a G to A variation is within 20 nucleotides or less of the 3' end of the determined sequence, the nucleotide occupying the reference sequence is called at the designated position.

[0035] In some embodiments, the C to T or G to A variation occurs in the surrounding context of TCG to TTG or CGA to CAA.

[0036] One aspect of the disclosure is a method of identifying variant nucleotides in a population of nucleic acids comprising: (a) contacting a population of nucleic acids of overlapping sequence, at least one of which is a double-stranded molecule having a single-stranded overhang on one or both ends, with a protein having 5'-3' polymerase activity and 3'-5' exonuclease activity, where the protein digests the 3' overhang and fills in the 5' overhang to generate a double-stranded blunt-ended nucleic acid; (b) ligating the double-stranded blunt-ended nucleic acid to an adapter comprising a barcode and amplifying the nucleic acid primed from a primer molecule that binds to the adapter; (c) determining a sequence of the amplified nucleic acid molecules and classifying the sequences of the amplified nucleic acid molecules into families, members of a family having the same start and end points on the nucleic acid and the same adapters, and determining a consensus sequence for the family from the sequences of its respective members; and (d) each designation in a reference sequence. For a position, determining a subset of families having a consensus sequence that includes the specified position and identifying consensus sequences in which the specified position is occupied by a variant nucleotide; and (e) calling the presence of a variant nucleotide at each specified position where a consensus sequence in the subset having a variant nucleotide supports the call, except that the presence of a variant nucleotide at a specified position is not called if (i) the variant nucleotide is a C to T or a G to A variation compared to the reference nucleotide, and (ii) the variant nucleotide is classified as a deamination error based on (1) the nucleotide context surrounding the specified position, and / or (2) the distance of the C to T variation at the specified position of the consensus sequence in the subset from the 5' end or the distance of the G to A variation at the specified position of the consensus sequence from the 3' end.

[0037] In some embodiments, step (c) identifies the number of consensus sequences in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in (i) and (ii) of step (d), the presence of a variant nucleotide at each designated position is called when the number of consensus sequences in the subset having variations meets a threshold.

[0038] In one aspect, the disclosure provides a method for identifying false positive variant nucleotides in a population of nucleic acids, the method comprising: (a) contacting a population of overlapping sequence nucleic acids, at least one of which is a double-stranded molecule having a single-stranded overhang on one or both ends, with a protein having 5'-3' polymerase activity and 3'-5' exonuclease activity, where the protein digests the 3' overhang and fills in the 5' overhang with a complementary nucleic acid at one or both ends to generate a double-stranded blunt-ended nucleic acid; (b) determining the sequence of the double-stranded blunt-ended nucleic acid to obtain sequenced nucleic acids; and (c) for each designated position in a reference sequence, identifying a subset of sequenced nucleic acids that includes the designated position, and identifying within the subset in which the designated position is occupied by a reference nucleotide. and (d) calling the presence of a false positive variant nucleotide at each designated position where sequenced nucleic acids having a C to T or G to A variation at the designated position support the call, wherein the variation is classified as a deamination error based on (1) the nucleotide context surrounding the designated position, and / or (2) an over-representation of a C to T conversion in sequenced nucleic acids in a first proportion of the subset where the designated position is within a defined proximity of the 5' end or an over-representation of a G to A conversion in sequenced nucleic acids in a second proportion of the subset where the designated position is within a defined proximity of the 3' end.

[0039] In some embodiments, step (c) identifies the number of consensus sequences in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in (i) and (ii) of step (d), the presence of a variant nucleotide at each designated position is called when the number of consensus sequences in the subset having variations meets a threshold.

[0040] In one aspect, the disclosure relates to a method of determining the minor allele frequency of a "C" to "T" or "G" to "A" variant at a designated position in a reference sequence in a population of sequenced nucleic acids that map to the designated position, where the minor allele frequency is determined by comparing the number of sequenced nucleic acids that map to the designated position that contain the variant ("variant count") to the total number of sequenced nucleic acids that map to the designated position, the method comprising adjusting the variant count of the T or A variant at the designated position for the probability of a deamination error, where the probability of the error is a function of the distance of the variant from the 5' end of the molecule in the case of "T" and from the 3' end of the molecule in the case of "A".

[0041] In some embodiments, a C to T variant located within a selected distance from the 5' end of a sequenced polynucleotide, or a G to A variant located within a selected distance from the 3' end of a sequenced nucleic acid, is not counted in the variant count.

[0042] In some embodiments, if the ratio of C to T variants located within a selected distance from the 5' end of the sequenced polynucleotide to C to T variants located outside a selected distance from the 5' end of the sequenced nucleic acid is greater than a predetermined ratio (e.g., greater than 50%), or if the ratio of G to A variants located within a selected distance from the 3' end of the sequenced nucleic acid to G to A variants located outside a selected distance from the 3' end of the sequenced nucleic acid is greater than a predetermined ratio (e.g., greater than 50%), all C to T variants are discounted from the variant count.

[0043] In some embodiments, the number of variants is determined as the sum of the probabilities that each C to T variant or each G to A variant is a true variant.

[0044] In one aspect, the disclosure relates to a method comprising administering to a subject determined to have a cancer marker by a method according to any of the preceding paragraphs, a therapeutic intervention effective to treat the cancer characterized by the cancer marker.

[0045] The disclosure further provides a method comprising receiving data regarding the identity of one or more variant nucleotides in the cell-free nucleic acid of a subject by performing a method according to any of the preceding paragraphs, determining the presence of a cancer marker from the one or more variant nucleotides, and administering a therapeutic intervention effective to treat a cancer characterized by the cancer marker.

[0046] In another aspect, the present disclosure relates to a system. (1) One such system is (2) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; (3) a computer in communication with the communications interface, the computer having one or more computer processors and, when executed by the one or more computer processors, (a) receiving, via a communications network, sequencing reads generated by a nucleic acid sequencer; (b) for each specified position in the reference sequence, (i) identifying a subset of sequencing reads that includes a specified position; (ii) identifying sequencing reads within the subset in which the designated position is occupied by a variant nucleotide; (c) calling the presence of a variant nucleotide at each designated position for which the sequencing reads in the subset having variation support the call, wherein the presence of the variant nucleotide at the designated position supports: (i) the variant is a C to T or G to A variation compared to the reference nucleotide; and (ii) the variant nucleotide is (1) the nucleotide context surrounding the specified position, and / or (2) a sequence error is classified as a deamination error based on the distance of a C to T transition at a specified position from the 5' end of a sequenced nucleic acid in the subset, or the distance of a G to A transition at a specified position from the 3' end of a sequenced nucleic acid in the subset, except that the sequence error is not called if the sequence error is classified as a deamination error based on the distance of a C to T transition at a specified position from the 5' end of a sequenced nucleic acid in the subset, or the distance of a G to A transition at a specified position from the 3' end of a sequenced nucleic acid in the subset, and a computer readable medium including machine executable code for implementing the method comprising: Includes.

[0047] In some embodiments, step (c) identifies the number of consensus sequences in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in (i) and (ii) of step (d), the presence of a variant nucleotide at each designated position is called when the number of consensus sequences in the subset having variations meets a threshold.

[0048] The present disclosure further relates to (1) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; (2) a computer in communication with the communications interface, the computer having one or more computer processors, and, when executed by the one or more computer processors, (a) receiving, via a communications network, sequencing reads generated by a nucleic acid sequencer; (b) comparing the determined sequence to a reference sequence, wherein the determined sequence contains at least one C to T variation at at least one designated position within 20 nucleotides or less of the 5' end of the determined sequence, or at least one G to A variation within 20 nucleotides or less of the 3' end of the determined sequence; and (c) calling the sequence of the nucleic acid as the determined sequence, except that in at least one of the positions where a C to T variation is within 20 nucleotides or less of the 5' end of the determined sequence or a G to A variation is within 20 nucleotides or less of the 3' end of the determined sequence, the nucleotide occupying the reference sequence is called at the designated position.

[0049] The present disclosure further relates to (1) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; (2) a computer in communication with the communications interface, the computer having one or more computer processors, and, when executed by the one or more computer processors, (a) receiving, via a communications network, sequencing reads generated by a nucleic acid sequencer; (b) classifying sequences of the sequencing reads into families, where members of a family have the same start and end points on the nucleic acid and the same adaptors, and determining a consensus sequence for the family from the sequences of its respective members; (c) for each designated position in the reference sequence, determining a subset of families having consensus sequences that include the designated position, and identifying consensus sequences in which the designated position is occupied by a variant nucleotide; (d) calling the presence of a variant nucleotide at each designated position, wherein a consensus sequence within the subset having the variant nucleotide supports the call, but the presence of the variant nucleotide at the designated position is (i) the variant nucleotide is a C to T or G to A variation compared to the reference nucleotide; and (ii) the variant nucleotide is (1) the nucleotide context surrounding the specified position, and / or (2) classified as a deamination error based on the distance from the 5' end of a C to T transition at a specified position of the consensus sequence in the subset, or the distance from the 3' end of a G to A transition at a specified position of the consensus sequence in the subset; In addition, the step and and a computer readable medium including machine executable code for implementing the method comprising: The present invention provides a system including:

[0050] In some embodiments, step (c) identifies the number of consensus sequences in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in (i) and (ii) of step (d), the presence of a variant nucleotide at each designated position is called when the number of consensus sequences in the subset having variations meets a threshold.

[0051] The present disclosure further relates to (1) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; (2) a computer in communication with the communications interface, the computer having one or more computer processors, and, when executed by the one or more computer processors, (a) receiving, via a communications network, sequencing reads generated by a nucleic acid sequencer; (b) for each designated position in the reference sequence, identifying a subset of sequencing reads that includes the designated position, and identifying sequenced nucleic acids within the subset in which the designated position is occupied by a reference nucleotide and sequenced nucleic acids within the subset in which the designated position is occupied by a variant nucleotide; (c) calling the presence of a false positive variant nucleotide at each designated position where a sequenced nucleic acid having a C to T or G to A variation at the designated position supports the call, wherein the variation is: (1) the nucleotide context surrounding the specified position, and / or (2) an over-representation of C to T conversions in a first proportion of sequenced nucleic acids in a subset in which the designated position is within a defined proximity to the 5' end, or an over-representation of G to A conversions in a second proportion of sequenced nucleic acids in a subset in which the designated position is within a defined proximity to the 3' end. and a computer readable medium comprising machine executable code for implementing the method, the method comprising the steps of: The present invention provides a system including:

[0052] In some embodiments, step (c) identifies the number of consensus sequences in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in (i) and (ii) of step (d), the presence of a variant nucleotide at each designated position is called when the number of consensus sequences in the subset having variations meets a threshold.

[0053] The present disclosure further relates to (1) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; (2) a computer in communication with the communications interface, the computer having one or more computer processors, and, when executed by the one or more computer processors, (a) receiving, via a communications network, sequencing reads generated by a nucleic acid sequencer; (b) adjusting the number of T or A variants in the sequencing reads based on the probability of deamination errors, where the probability of error is a function of the distance of the variant from the 5' end of the molecule for "T" and from the 3' end of the molecule for "A".

[0054] Any of the above-mentioned systems may further comprise a nucleic acid sequencer. Optionally, the nucleic acid sequencer sequences a sequencing library obtained from cell-free DNA molecules derived from a subject, where the sequencing library comprises cell-free DNA molecules and an adaptor, and the adaptor comprises a barcode. Optionally, the nucleic acid sequencer performs sequencing by synthesis on the sequencing library to generate sequencing reads. Optionally, the nucleic acid sequencer performs pyrosequencing, single molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing by ligation, or sequencing by hybridization on the sequencing library to generate sequencing reads. Optionally, the nucleic acid sequencer uses a clonal single molecule array derived from the sequencing library to generate sequencing reads. Optionally, the nucleic acid sequencer comprises a chip with an array of microwells for sequencing the sequencing library to generate sequencing reads.

[0055] In some systems, the computer-readable medium comprises a memory, a hard drive, or a computer server. In some systems, the communication network comprises a remote communication network, the Internet, an extranet, or an intranet. In some systems, the communication network comprises one or more computer servers capable of distributed computing, for example, cloud computing. In some systems, the computer is located on a computer server that is remotely located from the nucleic acid sequencer. In some systems, the sequencing library further comprises a sample barcode that distinguishes the sample from one or more samples.

[0056] Some systems further include an electronic display in communication with the computer over a network, the electronic display including a user interface, e.g., a graphical user interface (GUI) or a web-based user interface, for displaying results from performing (a)-(c). In some systems, the electronic display is present on a personal computer. In some systems, the electronic display is present on an Internet-enabled computer, optionally remote from the computer.

[0057] In some embodiments, the results of the systems and methods disclosed herein are used as input to generate a report in paper format, which can provide, for example, an indication of called variants and / or variants that are considered to be deamination errors.

[0058] Various steps of the methods disclosed herein or steps performed by the systems disclosed herein may be performed at the same or different times, in the same or different geographic locations, e.g., countries, and / or by the same or different people. [Brief description of the drawings]

[0059] [Figure 1] FIG. 1 shows an overview of end repair by Klenow polymerase.

[0060] [Diagram 2] FIG. 2 shows the C to T deamination scheme.

[0061] [Diagram 3] FIG. 3 shows the preference for C to T conversions at the 5' end of the molecule and G to A conversions at the 3' end of the molecule.

[0062] [Figure 4]FIG. 4 shows plots comparing the frequency of errors of C to T and G to A transitions with those of other transitions with distance from the end of the molecule. The error frequency of C to T and G to A transitions is higher near the end of the molecule, while that of other transitions is independent of position relative to the end of the molecule. The points labeled "C to T or G to A" show the average C to T error rate stratified by measured distance from the 5' end and the average G to A error rate stratified by measured distance from the 3' end, and the points labeled "other errors" show the average C to A+C to G error rate stratified by measured distance from the 5' end and the average G to T+G to C error rate stratified by measured distance from the 3' end.

[0063] [Diagram 5] FIG. 5 shows a computer system.

[0064] [Figure 6] FIG. 6 shows five sequencing families that contain G to A substitutions classified as deamination errors.

[0065] [Figure 7] FIG. 7 shows five sequencing families that contain G to A substitutions classified as bona fide mutations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] definition A subject refers to an animal, such as a mammalian species (preferably human) or an avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, such as a mammal, such as a mouse, a primate, a monkey, or a human. Animals include livestock animals, sport animals, and pets. A subject can be a healthy individual, an individual with symptoms or signs, or suspected of having a disease or a tendency to a disease, or an individual in need of treatment or suspected of needing treatment.

[0067] Genetic variant refers to an alteration, variant, or polymorphism in a nucleic acid sample or genome of a subject. Such alteration, variant, or polymorphism can be relative to a reference genome, which can be the reference genome of a species (e.g., hG19 or hG38 for humans), a subject, or another individual. Variations include one or more single nucleotide polymorphisms (SNVs), insertions, deletions, repeats, short insertions, short deletions, short repeats, structural variant junctions, variable length tandem repeats, and / or flanking sequences, and copy number variants (CNVs), transversions, gene fusions, and other rearrangements are also forms of genetic variation. Variations can be base changes, insertions, deletions, repeats, copy number polymorphisms, transversions, or combinations thereof.

[0068] Cancer markers are genetic variants associated with the presence of cancer or the risk of developing cancer.Cancer markers can provide an indication that a subject has cancer or has a higher risk of developing cancer than the same species of subjects of age and sex that do not have the cancer marker.Cancer markers may or may not be the cause of cancer.

[0069] Barcodes are short nucleic acids (e.g., less than 500, less than 100, less than 50, or less than 10 nucleotides long) that are used to label nucleic acid molecules to distinguish them from different types or differently processed samples (e.g., presenting a sample index), or different nucleic acid molecules in the same sample (e.g., presenting a barcode). The tags can be single-stranded, double-stranded, or at least partially double-stranded. The tags can have the same length or can have varying lengths. The tags can be blunt-ended or have an overhang. The tags can be attached to one or both ends of the nucleic acid. When decoded, the barcodes can indicate information such as the sample of origin, form, or processing of the nucleic acid. Tags can be used to enable pooling and parallel processing of multiple samples containing nucleic acids with different barcodes and / or sample indexes, with the nucleic acids later deconvoluted by reading the barcodes. Barcodes can also be referred to as molecular identifiers, sample identifiers, index tags, and / or tags. Additionally or alternatively, barcodes can be used to distinguish different molecules in the same sample. This includes uniquely barcoding each different molecule in a sample, or non-uniquely barcoding each molecule. In the case of non-unique barcoding, a limited number of barcodes may be used to barcode each molecule so that different molecules can be distinguished based on their start / end positions mapped onto a reference genome in combination with at least one tag. Thus, typically, a sufficient number of different barcodes are used so that the probability that any two molecules with the same start / end also have the same barcode is low (e.g., less than 10%, less than 5%, less than 1%, or less than 0.1%). Some barcodes include multiple molecular identifiers to label multiple samples, multiple molecular forms within one sample, and multiple molecules within one form with the same start and end points. Such barcodes can exist in the format A1i, where the letter indicates the type of sample, the Arabic numerals indicate the form of the molecule within the sample, and the Roman numerals indicate the molecule within the form.

[0070] Adapters are usually short nucleic acids (e.g., less than 500, 100, or 50 nucleotides long) that are at least partially double-stranded for ligation to either one or both ends of a sample nucleic acid molecule. Adapters may include primer binding sites that allow amplification of the nucleic acid molecule adjacent to the adapter at both ends, and / or sequencing primer binding sites, including primer binding sites for next generation sequencing (NGS). Adapters may also include binding sites for capture probes, such as oligonucleotides attached to a flow cell support. Adapters may also include a barcode as described above. Preferably, the barcode is positioned relative to the primer and sequencing primer binding sites so that it is included in the amplicon and sequencing reads of the nucleic acid molecule. Identical or different adapters can be ligated to each end of the nucleic acid molecule. Identical adapters may be ligated to each end, except that the barcodes are different. A preferred adapter is a Y-shaped adapter, one end of which is blunt-ended or tailed as described herein for joining to a nucleic acid molecule that is also blunt-ended or tailed with respect to one or more complementary nucleotides. Another preferred adaptor is a bell-shaped adaptor, which also has a blunt or tailed end for joining to the nucleic acid to be analyzed.

[0071] As used herein, the term "sequencing" refers to any of several techniques used to determine the sequence of a biomolecule, e.g., a nucleic acid molecule, such as DNA or RNA. Exemplary sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon sequencing, electron microscope-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy end sequencing, whole genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, and the like. Sequencing includes, for example, sequencing, emulsion PCR, co-amplification PCR at low denaturation temperature (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired-end sequencing, short-term sequencing, exonuclease sequencing, ligation sequencing, short-read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa genome analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, and combinations thereof.In some embodiments, sequencing can be performed by genetic analyzer, for example, the genetic analyzer commercially available from Illumina or Applied Biosystems.

[0072] The expression "next-generation sequencing" or NGS refers to sequencing technologies that have increased throughput compared to traditional Sanger and capillary electrophoresis-based techniques, for example, with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next-generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization.

[0073] The expression "sequencing run" refers to any step or portion of a sequencing experiment that is performed to determine some information about at least one biological molecule (e.g., a nucleic acid molecule such as DNA or RNA).

[0074] DNA (deoxyribonucleic acid) is a nucleotide chain that contains four nucleotides; adenine (A), thymine (T), cytosine (C), and guanine (G). RNA (ribonucleic acid) is a nucleotide chain that contains four nucleotides; A, uracil (U), G, and C. Certain pairs of nucleotides bind specifically to each other in a complementary manner (called complementary base pairing). In DNA, adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U) and cytosine (C) pairs with guanine (G). When a first nucleic acid strand binds to a second nucleic acid strand that is composed of nucleotides that are complementary to the nucleotides in the first strand, the two strands combine to form a duplex. As used herein, "nucleic acid sequencing data", "nucleic acid sequencing information", "nucleic acid sequence", "nucleotide sequence", "genomic sequence", "gene sequence", or "fragment sequence", or "nucleic acid sequencing read" refers to any information or data that displays the order of nucleotide bases (e.g., adenine, guanine, cytosine, and thymine or uracil) in a nucleic acid molecule such as DNA or RNA (e.g., a whole genome, a whole transcriptome, an exome, an oligonucleotide, a polynucleotide, or a fragment). It should be understood that the teachings of the present invention contemplate sequence information obtained using all available variations of techniques, platforms, or technologies, including, but not limited to, capillary electrophoresis, microarrays, ligation-based systems, polymerase-based systems, hybridization-based systems, direct or indirect nucleotide identification systems, pyrosequencing, ion or pH-based detection systems, and electronic signature-based systems.

[0075] A "polynucleotide", "nucleic acid", "nucleic acid molecule", or "oligonucleotide" refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by internucleoside linkages. Typically, a polynucleotide contains at least three nucleosides. Oligonucleotides often range in size from a small number of monomeric units, e.g., 3-4, to several hundred monomeric units. Whenever a polynucleotide is represented by a letter sequence, such as "ATGCCTG", it will be understood that the nucleotides are in 5' to 3' order from left to right, unless otherwise noted, with "A" indicating deoxyadenosine, "C" indicating deoxycytidine, "G" indicating deoxyguanosine, and "T" indicating thymidine. As is standard in the art, the letters A, C, G, and T can be used to refer to the bases themselves, the nucleosides that contain the bases, or the nucleotides.

[0076] Reference sequence is a known sequence that is used to compare with experimentally determined sequence.For example, known sequence can be the whole genome, chromosome, or any segment thereof.Reference typically comprises at least 20, 50, 100, 200, 250, 300, 350, 400, 450, 500, 1000 or more nucleotides.Reference sequence can be aligned with a single continuous sequence of genome or chromosome, or can comprise non-contiguous segments that are aligned with different regions of genome or chromosome.Reference human genome includes, for example, hG19 and hG38.

[0077] The term "designated position" in a reference sequence refers to a genomic coordinate in the reference sequence.

[0078] A first single-stranded nucleic acid sequence overlaps with a second single-stranded nucleic acid sequence if the first nucleic acid sequence or its complement and the second nucleic acid sequence or their complements are aligned in an overlapping manner, excluding non-homologous segments of a contiguous reference sequence, such as the sequence of a human chromosome. A wholly or partially double-stranded nucleic acid overlaps with another wholly or partially double-stranded nucleic acid if either of its strands overlaps with a strand of the other nucleic acid.

[0079] A "C" to "T" variant or conversion refers to the presence of the base "T" in the sequenced polynucleotide at a coordinate position occupied by the base "C" in the reference sequence. A "G" to "A" variant or conversion refers to the presence of the base "A" in the sequenced polynucleotide at a coordinate position occupied by the base "G" in the reference sequence.

[0080] A nucleic acid molecule can be conceptually divided into a 5' end, an internal portion, and a 3' end. The ends can be written based on a certain number of nucleotides from the end. For example, the 5' end is written, for example, by the 20 terminal nucleotides to the 5' end. The 3' end is written, for example, by the 20 terminal nucleotides to the 3' end. Alternatively, a nucleic acid molecule can be divided into the terminal portion as described and the remainder.

[0081] The term "minor allele frequency" refers to the frequency with which a minor allele (e.g., one that is not the most common allele) occurs in a given nucleic acid population, e.g., sample. A genetic variant with a low minor allele frequency may have a relatively low frequency of occurrence in a sample.

[0082] "Minor allele fraction" (MAF) refers to the fraction of DNA molecules with allelic alterations in a given sample. The MAF of a somatic variant can be less than 0.5, less than 0.1, less than 0.05, or less than 0.01. For example, the MAF of a somatic variant is less than 0.05.

[0083] The terms "processing", "calculating" and "comparing" are used interchangeably. This term can refer to determining differences, such as differences in numbers or sequences. For example, gene expression, copy number variation (CNV), indels, and / or single nucleotide variation (SNV) values ​​or sequences can be processed.

[0084] Adapters are artificially synthesized sequences that can be linked to nucleic acid molecules or polynucleotide sequences by any approach, including ligation, hybridization, and / or amplification. Adapters are usually short nucleic acids (e.g., less than 500 nucleotides long, less than 100 nucleotides long, or less than 50 nucleotides long) that are at least partially double-stranded for linking to either or both ends of a sample nucleic acid molecule. Adapters can include primer binding sites that allow amplification of the nucleic acid molecule flanked by the adapter at both ends, and / or sequencing primer binding sites, including primer binding sites for next-generation sequencing (NGS). Adapters can also include binding sites for capture probes, such as oligonucleotides bound to flow cell supports. Adapters can also include barcodes as described above. The tags are preferably positioned relative to the primer and sequencing primer binding sites, so that the tags are included in the amplicons and the sequencing reads of the nucleic acid molecules. The same or different adapters can be linked to each end of a nucleic acid molecule. The same adapters can be linked to each end, but with different tags. A preferred adaptor is a Y-shaped adaptor, in which one end is blunt-ended or has a tail for attachment to a nucleic acid molecule as described herein, which also has a blunt end or a tail with one or more complementary nucleotides. Another preferred adaptor is a bell-shaped adaptor, which also has a blunt end or an end with a tail for attachment to the nucleic acid to be analyzed. I. Overview

[0085] Sequencing nucleic acids can identify variations associated with the presence, susceptibility, or prognosis of disease. However, the value of such information can be compromised by errors introduced by the sequencing process, including preparing nucleic acids for sequencing, or other factors, such as environmental conditions that affect the quality of nucleic acid samples during transportation and / or initial laboratory processing. Environmental conditions that affect quality include temperature and length of storage before processing. The present disclosure is based on the observation that, in certain embodiments, smoothing single-stranded overhangs in nucleic acids in a sample has a significant tendency to introduce deamination-induced sequencing errors, in which cytosine (C) is changed to thymine (T) at the 5' end of a nucleic acid strand, resulting in a complementary base change from guanine (G) to adenine (A) at the 3' end of a complementary nucleic acid strand. The present disclosure provides a method for identifying such deamination-induced sequencing errors and distinguishing them from actual sequence variations that may be associated with cancer or other diseases. II. Methods for identifying and correcting deamination-induced errors

[0086] Nucleic acids can be subjected to deamination, in which the base "C" is converted to the base "T". In this case, in a double-stranded molecule, one strand will have a "T" and the complementary strand will have a "G". Such errors can be detected by sequencing, by following the sequences of the different strands.

[0087] The method can be performed on any nucleic acid that is partially double-stranded with at least a single-stranded overhang, or on a population that includes such nucleic acids. Typically, the method is performed on a population of nucleic acids where at least a portion of the population is partially double-stranded with a single-stranded overhang at one or both ends. The method can be performed on a population that includes, for example, at least 2, 10,000, 1,000,000, 1,000,000,000, 10,000,000,000, or more different such nucleic acids. Usually, at least some of the nucleic acids in the population, including those with single-stranded overhangs, are of overlapping sequences. Such a population can be naturally occurring or can be the result of fragmentation during sample preparation, or can be generated by enzymes, for example, by partial restriction digestion.

[0088] The preferred form of nucleic acid population is cell-free nucleic acid, for example, that present in blood and other body fluids.Such nucleic acid is typically in heterogeneous form, including double-stranded DNA with single-stranded overhangs at one or both ends, as well as single-stranded DNA and RNA.Double-stranded blunt-ended DNA can also be present.

[0089] A nucleic acid population can be prepared for sequencing by enzymatic blunting of double-stranded nucleic acids with single-stranded overhangs at one or both ends. The population can be treated with a protein with 5'-3' DNA polymerase activity and 3'-5' exonuclease activity in the presence of nucleotides (e.g., A, C, G, and T or U). Exemplary proteins are DNA polymerases, such as Klenow large fragment and T4 DNA polymerase. In a 5' overhang, the protein extends the recessed 3' end in the complementary strand until it is the same as the 5' end, resulting in a blunt end. In a 3' overhang, the protein digests from the 3' end to the 5' end of the opposite strand, and sometimes beyond the 5' end. If digestion proceeds beyond the 5' end of the opposite strand, the gap can be filled by polymerase activity, as in a 5' overhang. Blunting of double-stranded nucleic acids facilitates adapter attachment and subsequent amplification.

[0090] However, the blunting process can also introduce deamination errors, as shown in Figures 1-3. Figure 1 shows a scheme in which Klenow enzyme fills in the 5' overhang and digests the 3' overhang. Figures 2 and 3 show a scheme in which a CT deamination-induced error is introduced at the 5' end of the Watson strand and a complementary GA error is introduced at the 3' end of the complementary Crick strand. The deamination-induced C to T conversion is indicated by a circled T. The circled A represents the corresponding change in the complementary strand. The deamination-induced error in the 5' Watson strand is reproduced at the 3' end of the Crick strand as a complementary nucleotide due to the extension of the 3' end based on the 5' overhang of the Watson strand, e.g., a C to T conversion in the Watson strand and a G to A conversion in the Crick strand. Deamination-induced errors in double-stranded regions are not reproduced by the filling or digestion process, and the two strands have non-complementary nucleotides at that position or nucleotide. Deamination-induced errors at the 3' end of the Watson strand are removed by digestion. Deamination-induced errors near the 5' end of the Crick strand can be retained if the 3' end of the Watson strand is digested back, requiring filling with a nucleotide complementary to the deamination-induced error. In some embodiments, only the C to T variation at the 5' end of the strand and the G to A variation at the 3' end of the strand appear in both strands of the nucleic acid molecule.

[0091] Thus, in the case of end-repaired double-stranded molecules, a "C" to "T" conversion located in the 5' overhang in the Watson strand of the original molecule is represented by a T error and is propagated in all amplified molecules as an A in the complementary strand. A "C" to "T" conversion located in the double-stranded portion of the original molecule is represented by a G in one strand and an A in the complementary strand. During amplification, the error is likely to be propagated as a "T" in one strand, e.g., the Watson strand, and a mixture of "A" and "G" in the same position in the complementary strand, e.g., the Crick strand. A "C" to "T" conversion located in the 3' overhang in the Watson strand of the original molecule is digested and removed from the overhang to form a blunt-ended double-stranded molecule. A "C" to "T" conversion located near the 5' end of the Crick strand of a molecule with a 3' overhang on the Watson strand may appear as a T / A in the Watson / Crick strand when the 5' overhang has been digested back and filled in. This is likely to be propagated as a T / A in all amplified molecules. Thus, when sequenced, a "C" to "T" conversion in the double-stranded portion of the original molecule may be detected as an error, since reads from the original Watson strand contain a T, but reads from the original Crick strand contain a G. In contrast, a "C" to "T" conversion located in the 5' overhang in the Watson strand of the original molecule results in a complementary T / A in the Watson / Crick strand, respectively. Thus, conversion of nucleotides in both the 5' and 3' overhangs typically provides neither obvious errors nor double-stranded support, e.g., A / T (Watson / Crick) or C / G (Watson / Crick).

[0092] The population of nucleic acids can be subjected to further processing, such as converting single stranded nucleic acids to double stranded and / or converting RNA to DNA. These forms of nucleic acids can also be ligated to adaptors and amplified.

[0093] The nucleic acid that is subjected to blunting as described above, with or without previous amplification, and other nucleic acids in the sample as necessary, are sequenced to obtain sequenced nucleic acid. Sequenced nucleic acid can refer to either the sequence of a nucleic acid, including the sequence reads produced after redundantly sequencing the nucleic acid (e.g., by single molecule amplification or re-reading), or the nucleic acid that has been sequenced. Sequencing is performed so that the sequence data of each nucleic acid molecule in the sample is obtained from the consensus sequence of the amplification products of each nucleic acid molecule in the sample, either directly or indirectly.

[0094] In some methods, the double-stranded nucleic acid with single-stranded overhang in the sample after blunting is ligated (ligated or joined by primer extension) at both ends to an adapter containing a barcode or tag, and the nucleic acid sequence and the barcode in the adapter are determined by sequencing. The blunt-ended DNA molecule can be ligated at the blunt end to the blunt end of an at least partially double-stranded adapter (e.g., a Y-shaped or bell-shaped adapter). Alternatively, the blunt ends of the sample nucleic acid and the adapter can be tailed with complementary nucleotides to facilitate ligation. For example, the adapter can have at least one nucleotide attached or linked to the tail, e.g., one of the strands, which is complementary to the overhang introduced into the nucleic acid molecule of interest. The tail of the adapter can be any one or more of the nucleotides A, T, C, or G.

[0095] The sample may be contacted with a sufficient number of adapters such that the probability that any two instances of the same nucleic acid will receive the same barcode combination from the adapters ligated to one or both ends is low (e.g., less than 1% or less than 0.1%). Using adapters in this manner allows sequences with the same start and end points on the reference nucleic acid and ligated to the same barcode combination to be grouped into families of reads generated from the same original molecule. Such families represent the sequences of the amplification products of the nucleic acid in the sample before amplification. The sequences of the family members can be compiled to derive a consensus nucleotide or a complete consensus sequence of the nucleic acid molecule in the original sample modified by blunting and adapter ligation. In other words, a nucleotide occupying a particular position of the nucleic acid in the sample is determined to be the consensus of the nucleotides occupying its corresponding position in the family member sequences. The consensus nucleotide can be determined by methods such as voting or confidence scores, to name two methods. A family may include sequences of one or both strands of a double-stranded nucleic acid. When a family member comprises both strands of sequences from double-stranded nucleic acid, the sequence of one strand is converted to its complement for the purpose of compiling all sequences to derive consensus nucleotide or sequence.Some families may only comprise a single member sequence.In this case, this sequence can be interpreted as the sequence of the nucleic acid in the sample before amplification.Alternatively, families with only a single member sequence may be excluded from subsequent analysis.

[0096] Nucleotide variations in sequenced nucleic acids can be determined by comparing the sequenced nucleic acids with a reference sequence. The reference sequence is often a known sequence, for example, a known full or partial genomic sequence from a subject, the entire genomic sequence of a human subject. The reference sequence can be hG19. The sequenced nucleic acid can represent a sequence determined directly for a nucleic acid in a sample, or a consensus of sequences of an amplification product of such a nucleic acid, as described above. Comparison can be performed at one or more designated positions in the reference sequence. A subset of sequenced nucleic acids can be identified that includes a position corresponding to a designated position of the reference sequence when the respective sequences are maximally aligned. Within such a subset, it can be determined which, if any, sequenced nucleic acids include a nucleotide variation at a designated position, and optionally which, if any, include a reference nucleotide (i.e., the same as in the reference sequence) and / or the number of sequences in the subset that include a reference nucleotide. A variant can be called when supported by a sequenced nucleic acid that includes a nucleotide variation. For example, if the number of sequenced nucleic acids in the subset containing nucleotide variants exceeds a threshold, a variant nucleotide can be called at the designated position. The threshold can be a simple number, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequenced nucleic acids in the subset containing nucleotide variants, or the threshold can be a ratio, for example, at least 0.5, 1, 2, 3, 4, 5, 10, 15, or 20%, among other possibilities, of sequenced nucleic acids in the subset containing nucleotide variants. The comparison can be repeated for any desired designated position in the reference sequence. Often, the comparison can be performed for designated positions occupying at least 20, 100, 200, or 300 consecutive positions on the reference sequence, for example, 20-500 or 50-300 consecutive positions.A C to T or G to A variation that is supported by sequenced nucleic acids in the subset with the same confidence used to call other variations may nevertheless contain a deamination-induced sequencing error.

[0097] Deamination-induced sequencing errors will be unintentionally included in the called variant nucleotides unless steps are taken to exclude them from the called variant nucleotides. Deamination-induced errors can be recognized by either or both of two basic criteria. First, deamination errors are context-dependent. Deamination of cytosine to thymine occurs more frequently when cytosine is flanked by thymine and guanine (i.e., as TCG) than by other nucleotides. Similarly, guanine to adenine (in the complementary strand) variation occurs more frequently when guanine is flanked by C and A as CGA than by other nucleotides. Thus, deamination-induced errors can be called when C to T or G to A variation occurs in the context of TCG to TTG or CGA to CAA, respectively. In some methods, about 90% of deamination errors occur in these contexts.

[0098] Second, deamination-induced errors depend on the distance between the designated position and the end of the sequenced nucleic acid, or in other words, the number of nucleotides between these positions. For example, deamination-induced errors occurring in the internal part of the sequence are likely to be detectable as a "T" in the read from one strand and a "G" in the read from the complementary strand. However, deamination-induced errors occurring near the end (terminus) of the nucleic acid being sequenced may not be obvious because such errors may be introduced by the process of blunt-end repair, resulting in two perfectly complementary strands. More specifically, sequence reads containing cytosine to thymine deamination may occur more frequently near the 5' end of the sequenced nucleic acid, and guanine to adenine deamination may occur more frequently near the 3' end. Therefore, the average distance between the C to T variation caused by deamination at the designated position and the 5' end of the sequenced nucleic acid is shorter than the average distance between the reference nucleotide at the designated position and the 5' end of the sequenced nucleic acid. Similarly, the average distance between the G to A variation caused by deamination at the designated position and the 3' end of the sequenced nucleic acid is shorter than the average distance between the reference nucleotide at the designated position and the 3' end of the sequenced nucleic acid. The greater the difference in average distance, the higher the probability of deamination error. In contrast, if the G to A or C to T variation at the designated position represents a real variation rather than a sequencing error, there should be no systematic difference that may be caused by random factors in the average distance between these variations and the end of the sequenced nucleic acid compared to that of the reference nucleotide at the designated position.

[0099] In certain aspects, provided herein are methods for determining the minor allele frequency of a "C" to "T" or "G" to "A" variant at a specified position in a reference sequence in a population of sequenced polynucleotides that map to the specified position, where the minor allele frequency is determined by comparing the number of sequenced polynucleotides that map to the specified position that contain the variant ("variant count") to the total number of sequenced polynucleotides that map to the specified position, where the method includes adjusting the variant count of the T or A variant at the genomic coordinate for the probability of deamination error, where the probability of error is a function of the distance of the variant from the 5' end of the molecule for "T" and from the 3' end of the molecule for "A."

[0100] As shown in Figure 4, the likelihood that a deamination error will result in a "T" variant in a molecule is a function of the distance from the 5' end of the molecule where the variant is located. More specifically, the closer the variant is to the 5' end of the molecule, the more likely it is that the variant is a C to T transversion. This is because errors are propagated where there is a 5' overhang to be filled in, and shorter overhangs at the 5' end are more likely than longer overhangs. Similarly, a G to A variant at the 3' end of a molecule is more likely the closer the location is to the 3' end of the molecule for similar reasons.

[0101] In any sample, a statistical determination of C to T variants can be made as a function of distance from the ends of the molecule. This function reaches an asymptote at some interior distance of the molecule. The asymptotic amount represents the overall deamination rate. This rate can vary from sample to sample.

[0102] The relative proximity to the end of the sequenced nucleic acid where deamination-induced errors are likely to occur corresponds roughly to the length of the single-stranded overhang in the sequenced nucleic acid population, but may be slightly longer in the case of 3' overhang due to digestion beyond the end of the complementary strand and subsequent filling.Proximity can be defined as, for example, 30 nucleotides or less, 25 nucleotides or less, 20 nucleotides or less, 15 nucleotides or less, 10 nucleotides or less, or 5 nucleotides or less from the 3' or 5' end of the sequenced nucleic acid strand ("terminal proximity").Proximity can be defined the same or differently at the 3' or 5' end.

[0103] As described above, a subset of sequenced nucleic acids is identified that includes a position that corresponds to a designated position of the reference sequence when the respective sequences are maximally aligned. A portion of the sequenced nucleic acids in this subset has a designated position that occurs within a defined proximity of the 5' end. These sequenced nucleic acids can be referred to as a first ratio of the subset. A portion of the sequenced nucleic acids in the subset has a designated position that occurs within a defined proximity of the 3' end. These sequenced nucleic acids can be referred to as a second ratio of the subset. A "C" to "T" conversion can then be recognized by its occurrence in the sequenced nucleic acids that make up the first ratio, and a "G" to "A" conversion can be recognized by its occurrence in the sequenced nucleic acids that make up the second ratio. An occurrence can simply be defined as the number of sequenced nucleic acids present that include a C to T or G to A variation at a designated position within the relevant ratio. For example, a C to T deamination error can be called if a certain number, e.g., at least 1, 2, 3, 4, 5, or 6, of sequenced nucleic acids of a first ratio contain a C to T variation at a specified position. Similarly, a G to A deamination error can be called if a certain number, e.g., at least 1, 2, 3, 4, 5, or 6, of sequenced nucleic acids of a second ratio contain a G to A variation at a specified position.

[0104] Occurrence can also be defined by the proportion of nucleic acids within a first or second ratio that contain a C to T or G to A variation at a specified position compared to the proportion outside the first or second ratio, respectively. For example, a deamination error can be called if the occurrence of a C to T or G to A variation at a specified position of the relevant ratio is at least 25, 30, 40, 50, 60, or 70% of the sequenced nucleic acids within the relevant ratio. Over-occurrence can also be defined by the relative proportion of sequenced nucleic acids within the relevant ratio that have a C to T or G to A variation at a specified position compared to the corresponding proportion of sequenced nucleic acids within the same subset but outside the ratio that have a C to T or G to A variation. A higher occurrence of sequenced nucleic acids with a C to T or G to A variation within the relevant ratio than outside the ratio is an indication that the variation is a deamination error. For example, if 50% of the sequenced nucleic acids within a first proportion of the subset and only 1% of the nucleic acids within the subset but outside the proportion (where the specified position is not within a defined proximity of the 5' end) contain a C to T transition at a specified position, then the C to T transition is likely a deamination-induced error.

[0105] Determining the minor allele ratio can include calculating the ratio of molecules that contain a particular variant that is mapped to a specified position to all molecules that are mapped to a specified position. So, for example, if 100 molecules are mapped to a genomic coordinate, and 13 of them contain a variant, the minor allele frequency can be calculated as 13%. However, if certain variants are considered to be the result of deamination errors, they can be discounted from the count. So, for example, if 7 of 13 variants are designated as errors, the ratio can be calculated as 6 / 93, or 6.4%. In certain cases, for example, if the ratio of variants at a coordinate located at the 5' end of a molecule accounts for more than 50% of all variants at the coordinate, all variants at the specified position can be discounted.

[0106] Deamination-induced errors can be classified based on either context or occurrence, or both. For example, if a C to T or G to A transition occurs in a context suggestive of a deamination error as shown above, the degree of over-representation in the relevant proportion of the subset required to classify the transition as a deamination error can be reduced compared to what would be required if the classification was based on over-representation alone.

[0107] Whether an apparent variant is called as a deamination error may be based on several factors. The presence of a variant at a locus may be true if the absolute number of variant molecules is above a certain threshold (or ratio or percentage). The presence of a variant may also be reported off if the allelic ratio (percentage of molecules mapping to the locus that has the variant) is above a threshold determined, for example, by the ratio expected in a control sample. When reporting off, both the presence of the variant and the minor allelic ratio of the variant may be reported off. When reporting off, deamination errors may be handled in any of several different ways. In one embodiment, any "T" variant located within a predetermined terminal proximity may simply be attributed and discounted as an error. In this case, only "T" variants outside the predetermined terminal proximity are counted as real variants and subject to reporting requirements. In another method, the ratio of "T" variants located within a predetermined terminal proximity to those located outside the predetermined terminal proximity is determined. If the amount is above a certain threshold amount, e.g., above 20%, above 30%, above 40%, above 50%, the error rate is considered high enough not to report the variant at that position. If the amount is below the threshold level, the variant is subject to normal reporting requirements. Alternatively, if the minor allele ratio is above the expected overall error rate, the variant is reported out regardless of the presence of an error, which may or may not be corrected. In another embodiment, at selected positions (which may all be within the neighborhood zone or may include variants outside the neighborhood zone), the "T" variant is scored as the probability of the variant being an error, and the scores at all positions are added to get a number that is incorporated into the minor allele ratio. So, for example, the probability that a variant at the first (terminal) 5' nucleotide is a true variant may be 50%. The probability of a variant at the 10th 5' nucleotide may be 75%. The probability of a variant at any 5' nucleotide beyond the 20th may be 95%.If a sample exhibits one variant at position 1, one at position 10, and one at position 50, these can be scored as 0.5+0.75+0.95=2.2, counting with a variant of 2.2 relative to the minor allele ratio. Such probabilities can be empirically determined, for example, by testing at least 10, at least 50, at least 100, or at least 500 control samples.

[0108] When sequencing is performed by identifying families that represent amplified copies of a single sequenced nucleic acid in the original sample, typically each family member within the family, including families that represent both strands of the nucleic acid in the original sample, contains a deamination error. If the different strands have different nucleotides, the error is self-evident.

[0109] The number of designated positions in a reference sequence at which a variant nucleotide is classified as a deamination error in a particular sample can vary. For example, the number of such designated positions can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, among other possibilities. III. Computer Implementation

[0110] The method can be implemented on a computer, such that any or all of the steps described herein or in the appended claims, other than the wet chemistry steps, can be implemented on a suitable programmed computer. The computer can be a mainframe, a personal computer, a tablet, a smart phone, the cloud, online data storage, remote data storage, etc. The computer can be operated in one or more locations.

[0111] The computer program for analyzing a population of nucleic acids may include code for performing any of the non-wet chemistry steps described herein or in the appended claims, e.g., code for determining the sequence of the double-stranded blunt-ended nucleic acid to obtain sequenced nucleic acids; code for identifying a subset of the sequenced nucleic acids that includes a designated position, the designated position being identified as the number of sequenced nucleic acids in the subset that are occupied by a variant nucleotide at each designated position in the reference sequence; and code for identifying the number of sequenced nucleic acids in the subset that are occupied by a variant nucleotide at each designated position in the reference sequence when the number of sequenced nucleic acids in the subset that have a variation meets a threshold value. The computer program may also include code for accepting sequence data from a database or a sequencing device and outputting calculated data, such as the variant nucleotide or deamination-induced sequencing error, to a display or printer.

[0112] The method can be implemented in a system (e.g., a data processing system) for analyzing a nucleic acid population. The system can also include the steps described herein or in the appended claims, such as the following: determining the sequence of the double-stranded blunt-ended nucleic acid to obtain a sequenced nucleic acid; identifying a subset of the sequenced nucleic acid that includes a designated position, and identifying the number of sequenced nucleic acids in the subset in which the designated position is occupied by a variant nucleotide at each designated position in the reference sequence; and calling the presence of a variant nucleotide at each designated position when the number of sequenced nucleic acids in the subset with variation meets a threshold value, wherein the presence of a variant nucleotide at a designated position is determined by: The system may also include a processor, a system bus, a main memory, and optionally an auxiliary memory, coupled together to perform one or more of the steps except that (i) if the variant is a C to T or G to A variation compared to the reference nucleotide, and (ii) if the variant nucleotide is classified as a deamination error based on (1) the nucleotide context around the designated position, and / or (2) the distance of the C to T variation at the designated position from the 5' end of the sequenced nucleic acid in the subset, or the distance of the G to A variation at the designated position from the 3' end of the sequenced nucleic acid in the subset. The system may also include a display or printer for outputting the results, e.g., variant nucleotides and deamination-induced errors, a keyboard and / or a pointer for providing user input, e.g., for setting thresholds or defined proximity, among other ancillary equipment. The system may also include a sequencing device coupled to the memory to provide raw sequencing data.

[0113] The various steps of the method can utilize information and / or programs stored on a computer readable medium (e.g., a hard drive, an auxiliary memory, an external memory, a server; a database, a portable memory device (e.g., a CD-R, a DVD, a ZIP disk, a flash memory card), etc. to generate results. For example, information used in the method and results generated by the method that can be stored on a computer readable medium include reference sequences, thresholds or defined proximities for calling nucleotide variants or deamination-induced errors, raw sequencing data, sequenced nucleic acids, variant nucleotides and their association with diseases, and deamination-induced errors.

[0114] The disclosure also includes an article of manufacture for analyzing a nucleic acid population, the article including a machine-readable medium containing one or more programs that, when executed, perform the steps of the methods.

[0115] The present disclosure can be implemented in hardware and / or software. For example, different aspects of the present disclosure can be implemented in either client-side logic or server-side logic. The present disclosure or its components can be embodied in a fixed-medium program component that contains logic instructions and / or data that, when loaded into a properly configured computing device, causes the device to perform according to the present disclosure. The fixed medium containing the logic instructions can be delivered to the fixed-medium viewer for physical loading into the viewer's computer, or the fixed medium containing the logic instructions can reside on a remote server that the viewer accesses via a communication medium to download the program component.

[0116] The present disclosure provides a computer control system programmed to perform the methods of the present disclosure. FIG. 5 shows a computer system 901 programmed to perform or otherwise configured to perform the methods of the present disclosure. The computer system 901 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 905, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 901 also includes memory or memory locations 910 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 915 (e.g., hard disk), a communication interface 920 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 925, such as cache memory, other memory, data storage, and / or electronic display adapters. The memory 910, storage unit 915, interface 920, and peripheral devices 925 may be configured to:

[0117] The computer system 901 communicates with the CPU 905 through a communication bus (solid line) such as a motherboard. The storage unit 915 may be a data storage unit (or data repository) for storing data. The computer system 901 may be operatively connected to a computer network ("network") 930 with the aid of a communication interface 920. The network 930 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. In some cases, the network 930 is a telecommunications and / or data network. The network 930 may be a local area network. The network 930 may include one or more computer servers, which may enable distributed computing such as cloud computing. The network 930 may implement a peer-to-peer network, which may enable devices connected to the computer system 901 to function as clients or servers with the aid of the computer system 901 in some cases.

[0118] The CPU 905 can execute sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 910. The instructions may be directed to the CPU 905, which may then be programmed or otherwise configured to perform the methods of the present disclosure. Examples of operations performed by the CPU 905 may include fetching, decoding, executing, and writing back.

[0119] The CPU 905 may be part of a circuit, for example, an integrated circuit. One or more other components of the system 901 may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0120] The storage unit 915 can store files such as drivers, libraries, and saved programs. The storage unit 915 can store user data, such as user preferences and user programs. The computer system 901 may, in some cases, include one or more additional data storage units external to the computer system 901, such as located on a remote server that communicates with the computer system 901 over an intranet or the Internet.

[0121] The computer system 901 can communicate with one or more remote computer systems through the network 930. For example, the computer system 901 can communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., Apple® iPad®, Samsung® Galaxy Tab), a phone, a smartphone (e.g., Apple® iPhone®, Android® enabled device, Blackberry®), or a personal digital assistant. A user can access the computer system 901 through the network 930.

[0122] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 901, such as in memory 910 or on the electronic storage unit 915. The machine executable or machine readable code may be provided in the form of software. In use, the code may be executed by the processor 905. In some cases, the code may be retrieved from the storage unit 915 and stored on the memory 910 for easy access by the processor 905. In some situations, the electronic storage unit 915 may be omitted and the machine executable instructions are stored on the memory 910.

[0123] The code may be precompiled and configured for use on a machine having a processor adapted to execute the code, or it may be compiled during run-time. The code may be provided in a programming language that may be selected to allow the code to execute in a precompiled or compiled manner.

[0124] Aspects of the systems and methods provided herein, such as computer system 901, may be embodied in programming. Various aspects of the technology may be thought of as an "article of manufacture" or "article of manufacture," typically in the form of machine (or processor) executable code and / or associated data carried or embodied in a type of machine-readable medium. The machine executable code may be stored on an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory), or a hard disk.

[0125] A "storage" type medium may include any or all of the tangible memory of a computer, processor, or the like, or their associated modules (various semiconductor memories, tape drives, disk drives, etc.), which may provide non-transitory storage of software programming at all times. All or a portion of the software may be communicated from time to time over the Internet or various other telecommunications networks. For example, such communication may enable loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, another type of medium that may have software elements includes optical waves, radio waves, and electromagnetic waves used across physical interfaces between local devices, through wired and optical terrestrial communication networks, and on various wireless links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media bearing software. As used herein, non-transitory tangible

[0126] Unless limited to "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0127] Thus, a machine-readable medium such as a computer executable code can take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices, such as any computer, that can be used to implement, for example, the databases shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media can take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium having a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, a cable or link transporting such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0128] The computer system 901 may include or be in communication with an electronic display 935 that includes a user interface (UI) 940, for example, to provide reports. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0129] The methods and systems of the present disclosure may be implemented using one or more algorithms, which may be implemented by software when executed by the central processing unit 905. IV. General Characteristics of the Method 1. Sample

[0130] The sample can be any biological sample isolated from a subject. Samples include body tissues, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, feces, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial or extracellular fluid, fluid in the space between cells, including gingival crevicular fluid, bone marrow, pleural fluid, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, and urine. The sample is preferably a body fluid, in particular blood and its proportions, and urine. Such samples include nucleic acids shed from tumors. Nucleic acids can include DNA and RNA, and can be in double-stranded and / or single-stranded form. Sample may be in the form originally isolated from subject, or may be further processed to remove or add components, such as cells, to enrich one component with respect to another, or to convert one form of nucleic acid into another, such as RNA to DNA, or single-stranded nucleic acid to double-stranded.Thus, for example, the body fluid for analysis is plasma or serum, which contains cell-free nucleic acid, such as cell-free DNA (cfDNA).

[0131] The volume of plasma may depend on the desired read depth of the region being sequenced. Exemplary volumes are 0.4-40 ml, 5-20 ml, and 10-20 ml. For example, the volume may be 0.5 ml, 1 ml, 5 ml, 10 ml, 20 ml, 30 ml, or 40 ml. The volume of plasma sampled may be 5-20 ml.

[0132] A sample can contain various amounts of nucleic acid, including genome equivalents. For example, a sample of about 30 ng of DNA can contain about 10,000 (10 4) haploid human genome equivalents, and in the case of cfDNA, approximately 200 billion (2×10 4 Similarly, a sample of about 100 ng of DNA may contain about 30,000 haploid human genome equivalents, and in the case of cfDNA, about 600 billion individual molecules.

[0133] The sample may comprise nucleic acid from different sources, for example, cells and acellular. The sample may comprise nucleic acid with mutation. For example, the sample may comprise DNA with germline mutation and / or somatic mutation. The sample may comprise DNA with cancer-associated mutation (e.g., cancer-associated somatic mutation).

[0134] Exemplary amounts of cell-free nucleic acid in a sample prior to amplification range from about 1 fg to about 1 μg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, the amount can be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. The amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules. The amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules. The method can include obtaining between 1 femtogram (fg) and 200 ng.

[0135] A cell-free nucleic acid sample refers to a sample that contains cell-free nucleic acid. Cell-free nucleic acid is nucleic acid that is not contained within or bound to cells, or in other words, nucleic acid that remains in a sample after removal of intact cells. Cell-free nucleic acid can refer to all unencapsulated nucleic acid obtained from a body fluid (e.g., blood, urine, CSF, etc.) from a subject. Cell-free nucleic acid includes DNA (cfDNA), RNA (cfRNA), and hybrids thereof, including genomic DNA, mitochondrial DNA, circulating DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-coding RNA (long ncRNA), or fragments of any of these. Cell-free nucleic acid can be double-stranded, single-stranded, or hybrids thereof. Cell-free nucleic acid can be released into body fluids through secretion or cell death processes, such as cell necrosis and apoptosis. Some cell-free nucleic acid, such as circulating tumor DNA (ctDNA), is released into body fluids from cancer cells. Others are released from healthy cells. ctDNA can be non-encapsulated tumor-derived fragmented DNA. Cell-free fetal DNA (cffDNA) is fetal DNA that circulates freely in the maternal bloodstream.

[0136] The cell-free nucleic acid or its associated proteins may have one or more epigenetic modifications. For example, the cell-free nucleic acid may be acetylated, 5-methylated, ubiquitinated, phosphorylated, sumoylated, ribosylated, and / or citrullinated.

[0137] The cell-free nucleic acids have an exemplary size distribution of about 100 to 500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of the molecules, with the mode in humans being about 168 nucleotides, and a second minor peak in the range of 240 to 440 nucleotides. The cell-free nucleic acids can be about 160 to about 180 nucleotides, or about 320 to about 360 nucleotides, or about 440 to about 480 nucleotides.

[0138] Cell-free nucleic acid can be isolated from body fluids by a partitioning step, which separates the cell-free nucleic acid found in solution from intact cells and other insoluble components of the body fluid. Partitioning can include techniques such as centrifugation or filtration. Alternatively, cells in the body fluid can be lysed and the cell-free and cellular nucleic acids can be processed together. In general, after the addition of buffer and a washing step, the cell-free nucleic acid can be precipitated with alcohol. Additional washing steps, such as silica-based columns, can be used to remove contaminants or salts. To optimize certain aspects of the procedure, such as yield, for example, non-specific bulk carrier nucleic acid can be added throughout the reaction.

[0139] After such processing, the sample may contain nucleic acids in various forms, including double-stranded DNA, single-stranded DNA, and single-stranded RNA. If necessary, single-stranded DNA and RNA may be converted to double-stranded form for inclusion in subsequent processing and analysis steps. 2. Amplification

[0140] The sample nucleic acid flanked by adaptors can be amplified by PCR and other amplification methods that typically prime by binding primers to the primer binding sites in the adaptors flanking the DNA molecules to be amplified.The amplification method can include cycles of extension, denaturation, and annealing as a result of thermal cycling, or can be isothermal as in transcription-mediated amplification.Other amplification methods include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustaining sequence-based replication.

[0141] Using conventional nucleic acid amplification methods, one or more rounds of amplification can be applied to introduce barcodes to nucleic acid molecules. Amplification can be performed in one or more reaction mixtures. Molecular tags and sample indexes / tags can be introduced simultaneously or in any sequential order. Molecular tags and sample indexes / tags can be introduced before and / or after sequence capture. In some cases, only molecular tags are introduced before probe capture, while sample indexes / tags are introduced after sequence capture. In some cases, both molecular tags and sample indexes / tags are introduced before probe capture. In some cases, sample indexes / tags are introduced after sequence capture. Typically, sequence capture involves introducing a single-stranded nucleic acid molecule complementary to a target sequence, e.g., a coding sequence of a genomic region, where mutations in such regions are associated with cancer types. Typically, amplification produces multiple non-uniquely or uniquely tagged nucleic acid amplicons with molecular tags and sample indexes / tags in sizes ranging from 200nt to 700nt, 250nt to 350nt, or 320nt to 550nt. In some embodiments, the amplicon has a size of about 300 nt. In some embodiments, the amplicon has a size of about 500 nt. 3. Barcode

[0142] Barcodes can be incorporated into or otherwise attached to adapters by chemical synthesis, ligation, overlap extension PCR, among other methods. In general, assignment of unique or non-unique barcodes in reactions follows the methods and systems described in U.S. Patent Application Nos. 20010053519, 20110160078, and U.S. Patent Nos. 6,582,908 and 7,537,898, and US 9,598,731.

[0143] The tags can be linked to the sample nucleic acid randomly or non-randomly. In some cases, they are introduced into the microwells in a predicted identifier ratio (e.g., a combination of barcodes). The barcode population can be unique, e.g., all barcodes have the same nucleotide sequence. The barcode population can be non-unique, e.g., some of the barcodes have the same nucleotide sequence and some of the barcodes have different nucleotide sequences. For example, identifiers may be loaded such that more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 20, more than 50, more than 100, more than 500, more than 1000, more than 5000, more than 10000, more than 50,000, more than 100,000, more than 500,000, more than 1,000,000, more than 10,000,000, more than 50,000,000, or more than 1,000,000,000 identifiers are loaded per genomic sample. In some cases, identifiers may be loaded such that fewer than 2, fewer than 3, fewer than 4, fewer than 5, fewer than 6, fewer than 7, fewer than 8, fewer than 9, fewer than 10, fewer than 20, fewer than 50, fewer than 100, fewer than 500, fewer than 1000, fewer than 5000, fewer than 10000, fewer than 50,000, fewer than 100,000, fewer than 500,000, fewer than 1,000,000, fewer than 10,000,000, fewer than 50,000,000, or fewer than 1,000,000,000 identifiers are loaded per genomic sample. In some cases, the average number of identifiers loaded per sample genome is less than or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 identifiers per genome sample.

[0144] A preferred format uses 20-50 different tags ligated to both ends of a target molecule, generating 20-50 x 20-50 tags, e.g., 400-2500 tags. Such a number of tags is sufficient to ensure that different molecules with the same start and end points will receive different combinations of tags with a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%).

[0145] In some cases, the identifier may be an oligonucleotide of a predetermined or random or semi-random sequence. In other cases, multiple barcodes may be used, where the barcodes are not necessarily unique to each other among the multiple. In this example, the barcode may be attached (e.g., by ligation or PCR amplification) to individual molecules such that the combination of the barcode and the sequence to which it may be attached creates a unique sequence that can be tracked individually. As described herein, detection of non-uniquely tagged barcodes in combination with sequence data at the beginning (start) point and end (end) portion of the sequence read may allow a unique identity to be assigned to a particular molecule. The length or number of base pairs of individual sequence reads may also be used to assign a unique identity to such molecules. As described herein, fragments derived from a single strand of nucleic acid that have been assigned a unique identity may thereby allow identification of subsequent fragments derived from the parent strand and / or the complementary strand. 4. Sequencing

[0146] The sample nucleic acid flanked by adapters can be subjected to sequencing, whether or not it is previously amplified.Sequencing methods include, for example, Sanger sequencing, high throughput sequencing, pyrosequencing, sequencing by synthesis, single molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing by ligation, sequencing by hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), next generation sequencing, single molecule sequencing by synthesis (SMSS) (Helicos), massively parallel sequencing, clonal single molecule array (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent or Nanopore platform. The sequencing reactions can be performed in a variety of sample processing units, which can be multi-lane, multi-channel, multi-well, or other means of processing multiple sample sets substantially simultaneously. Sample processing units can also include multiple sample chambers that allow multiple runs to be processed simultaneously.

[0147] The sequencing reaction can be performed on one or more fragment types that are known to contain markers of cancer of other diseases. The sequencing reaction can also be performed on any nucleic acid fragment present in the sample. The sequencing reaction can provide at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% sequence coverage of the genome. In other cases, the sequence coverage of the genome can be less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 95%, less than 99%, less than 99.9%, or less than 100%.

[0148] Multiplex sequencing can be used to carry out simultaneous sequencing reactions.In some cases, cell-free polynucleotides can be sequenced at least 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times, 9000 times, 10000 times, 50000 times, 100,000 times sequencing reactions.In other cases, cell-free polynucleotides can be sequenced less than 1000 times, less than 2000 times, less than 3000 times, less than 4000 times, less than 5000 times, less than 6000 times, less than 7000 times, less than 8000 times, less than 9000 times, less than 10000 times, less than 50000 times, less than 100,000 times sequencing reactions.Sequencing reactions can be carried out sequentially or simultaneously. Subsequent data analysis can be performed on all or part of the sequencing reactions. In some cases, data analysis can be performed on at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. In other cases, data analysis can be performed on less than 1000, less than 2000, less than 3000, less than 4000, less than 5000, less than 6000, less than 7000, less than 8000, less than 9000, less than 10000, less than 50000, less than 100,000 sequencing reactions. An exemplary read depth is 1000 to 50000 reads per locus (base). 5.Analysis

[0149] The methods can be used to diagnose the presence of a condition, particularly cancer, in a subject, to characterize the condition (e.g., to determine the stage of the cancer or determine the heterogeneity of the cancer), to monitor the response to treatment of the condition, or to achieve a prognosis of the risk of developing the condition or the subsequent course of the condition.

[0150] Various cancers can be detected using this method. Cancer cells, like most cells, can be characterized by turnover rate, where old cells die and are replaced by new cells. In general, dead cells can release DNA or fragments of DNA into the bloodstream when they come into contact with the vasculature in a given subject. This is also true for cancer cells at various stages of disease. Cancer cells can also be characterized by various genetic abnormalities, such as copy number variations and rare mutations, depending on the stage of disease. This phenomenon can be used to detect the presence or absence of cancer in an individual using the methods and systems described herein.

[0151] The types and number of cancers that can be detected include blood cancer, brain cancer, lung cancer, skin cancer, nasal cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, colon cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, heterogeneous tumors, homogeneous tumors, etc.

[0152] Cancer can be detected by genetic variations including mutations, rare mutations, indels, copy number variations, transversions, translocations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural alterations, gene fusions, chromosomal fusions, gene truncations, gene amplifications, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, and abnormal changes in epigenetic patterns.

[0153] Genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous, both in composition and stage determination. Genetic profile data can allow characterization of specific subtypes of cancer, which may be important for the diagnosis or treatment of that particular subtype. This information can also provide clues to the subject or practitioner regarding the prognosis of a particular cancer type, allowing either the subject or practitioner to adapt treatment options to the progression of the disease. Some cancers become more aggressive and genetically unstable as they progress. Other cancers may remain benign, inactive, or dormant. The systems and methods of the present disclosure can be useful in determining disease progression.

[0154] This analysis is also useful for determining the effectiveness of a particular treatment option. A successful treatment option may increase the amount of copy number variation or rare mutations detected in the subject's blood, since more cancers may die and DNA may be released if the treatment is successful. In other cases, this may not occur. In another example, perhaps a particular treatment option may correlate with the genetic profile of the cancer over time. This correlation may be useful for selecting a treatment method. In addition, if the cancer is observed to be in remission after treatment, the method can be used to monitor residual disease or recurrence of the disease.

[0155] The method can also be used to detect genetic variations in conditions other than cancer. Immune cells, such as B cells, can undergo rapid clonal expansion in the presence of certain diseases. Clonal expansion can be monitored using copy number polymorphism detection to monitor certain immune conditions. In this example, copy number polymorphism analysis can be performed over time to obtain a profile of how a certain disease may progress. Copy number polymorphism or even rare mutation detection can be used to determine how a pathogen population is changing during the course of infection. This can be particularly important in chronic infections, such as HIV / AIDs or hepatitis infections, whereby the virus can change life cycle state and / or mutate into a more virulent form during the course of infection. The method can be used to determine or profile the host body's rejection activity when immune cells try to destroy the transplanted tissue to monitor the state of the transplanted tissue, as well as to change the course of treatment or prevent rejection.

[0156] Furthermore, the method of the present disclosure can be used to characterize the heterogeneity of abnormal conditions in a subject, the method includes generating a genetic profile of extracellular polynucleotides in the subject, where the genetic profile includes a plurality of data obtained from the analysis of copy number variation and rare mutations. In some cases, diseases, including but not limited to cancer, can be heterogeneous. Disease cells may not be identical. In the example of cancer, some tumors are known to contain different types of tumor cells, and some cells are at different stages of cancer. In other examples, heterogeneity may constitute a multifocal disease. Furthermore, in the example of cancer, there may be multiple tumor foci, where one or more foci are probably the result of metastasis spreading from the primary site.

[0157] The method can be used to generate a profile, fingerprint, or set of data that is a compilation of genetic information from different cells in a heterogeneous disease. The set of data can include copy number variation and rare mutation analysis, either alone or in combination.

[0158] The methods can be used to diagnose, prognose, monitor, or observe cancer or other diseases of fetal origin. That is, these techniques can be used in pregnant subjects to diagnose, prognose, monitor, or observe cancer or other diseases in prenatal subjects where DNA and other polynucleotides may be co-circulating with maternal molecules. 6. Treatment

[0159] The number and type of variant nucleotides in a sample can provide an indication of the suitability of the subject who provided the sample for treatment, i.e., therapeutic intervention. For example, the presence of a large number of variant nucleotides is a positive indication for immunotherapy, since the presence of such mutations is associated with neoepitopes that form the target of immunotherapy. Immunotherapy can include the use of antibodies against any of PD-1, PD-2, PD-L1, PD-L2, CTLA-40, OX40, B7.1, B7He, LAG3, CD137, KIR, CCR5, CD27, or CD40, among other treatments. Other exemplary agents for immunotherapy include proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α. Other exemplary agents are by activated T cells against tumors, such as the expression of chimeric antigens that target tumor antigens from T cells. Immunotherapy stimulates the immune system to attack tumor antigens that are differentiated from their wild-type counterparts by the presence of mutations.

[0160] Other variant nucleotides provide targets for existing drugs or show resistance to such drugs.By eliminating false positives caused by deamination-induced sequencing errors, the number and type of variant nucleotides can be determined with increased accuracy.Therefore, the subject analyzed by this method can then be subjected to different treatment regimens according to the nucleotide variants found.Therefore, for example, the subjects whose number of variant nucleotides determined is at or above a threshold value are more likely to receive immunotherapy than the subjects whose number of variant nucleotides determined is below the threshold value.

[0161] Nucleotide mutations in sequenced nucleic acids can be determined by comparing sequenced nucleic acids with a reference sequence. The reference sequence is often a known sequence, for example, a known full genome sequence or partial genome sequence from a subject, a full genome sequence of a human subject. The reference sequence may be hG19. The sequenced nucleic acid can represent a sequence determined directly for a nucleic acid in a sample, or a consensus of sequences of an amplification product of such a nucleic acid, as described above. The comparison can be performed at one or more designated positions of the reference sequence. A subset of sequenced nucleic acids that includes a position corresponding to a designated position of the reference sequence can be identified when each sequence is maximally sequenced. Within such a subset, it can be determined which sequenced nucleic acids, if any, include a nucleotide mutation at a designated position, and, if necessary, which include a reference nucleotide (i.e., the same as in the reference sequence). If the number of sequenced nucleic acids in the subset that includes a nucleotide mutation exceeds a threshold value, a variant nucleotide can be called at the designated position. The threshold may be a simple number, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequenced nucleic acids in the subset that contain the nucleotide variant, or may be a ratio, e.g., at least 0.5, where 1, 2, 3, 4, 5, 10, 15, or 20 of the sequenced nucleic acids in the subset contain the nucleotide variant, among other possibilities. The comparison may be repeated for any designated position of interest in the reference sequence. The comparison may also be performed for designated positions that occupy at least 20, 100, 200, or 300 consecutive positions in the reference sequence, e.g., 20-500, or 50-300 consecutive positions.

[0162] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be so incorporated by reference. Where various sequence versions relate to accession numbers at different times, this refers to the version associated with the accession number at the effective filing date of this application. Effective filing date, where applicable, refers to the earlier of the actual filing date or the filing date of the priority application that refers to the accession number. Similarly, where different versions of publications, websites, etc. have been published at different times, this refers to the version published closest to the effective filing date of this application, unless otherwise indicated. Any configuration, step, element, embodiment, or aspect of the present disclosure can be used in combination with any other, unless otherwise specifically indicated. The present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, but it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. EXAMPLES

[0163] Example 1 FIG. 6 shows sequencing read families of cell-free DNA. The sequencing reads are mapped to various segments of the ALK gene (CD246) on human chromosome 2. The reference sequence of the relevant region of the ALK gene is shown at the bottom of the figure (gaps in the sequence represent additional nucleotides that are not shown for the sake of brevity of the figure). The figure shows five sequencing read families, from top to bottom, with 2, 3, 6, 3, and 6 reads, respectively. Reads from one orientation are shown in black and reads from the other orientation are shown in white. Each of the families shows a G to A mismatch in each read of the family. Viewed separately, these sequencing read families provide sufficient evidence to call the G to A mutation. However, the figure shows that the location of the G to A mutation is, relative to the 3' end of the sequence read, as follows: (1) Family 1: 1st strand: 2 reads, 2nd strand: no reads, G to A mutation located 70 bases from the 3' end (2) Family 2: 1st strand: no reads, 2nd strand: 3 reads, G to A mutation located 2 bases from the 3' end (3) Family 3: 1st strand: 2 reads, 2nd strand: 4 reads, G to A mutation located 6 bases from the 3' end (4) Family 4: 1st strand: 1 read, 2nd strand: 2 reads, G to A mutation located 1 base from the 3' end (5) Family 5: 1st strand: 5 reads, 2nd strand: 1 read, G to A mutation located 3 bases from the 3' end It changes when it is considered

[0164] In four of the five families (80%) where a G to A substitution was found, the substitution occurred within 20 bases from the 3' end of the sequencing read. The figure of 80% is greater than 50% of families with a G to A substitution that have a substitution within 20 bases from the 3' end of the sequencing read. Thus, the substitution is classified as a deamination-induced error with no functional significance other than as a true mutation (which may have implications for cancer diagnosis or prognosis). Example 2

[0165] Figure 7 is presented in a similar format to Figure 6, showing sequencing reads from five families with 8, 4, 2, 5, and 4 members, respectively. Again, each of the five families has an apparent G to A substitution in each of its reads. However, in this case the relative location of the substitution to the 3' end of the sequencing read is different, as shown below. (1) Family 1: 1st strand: 8 reads, 2nd strand: no reads, G to A mutation located 62 bases from the 3' end (2) Family 2: 1st strand: 2 reads, 2nd strand: 2 reads, G to A mutation located 2 bases from the 3' end (3) Family 3: 1st strand: 2 reads, 2nd strand: no reads, G to A mutation located 72 bases from the 3' end (4) Family 4: 1st strand: 1 read, 2nd strand: 4 reads, G to A mutation located 63 bases from the 3' end (5) Family 5: 1st strand: no reads, 2nd strand: 4 reads, G to A mutation located 79 bases from the 3' end

[0166] In this case, only one in five families (20%) has an apparent G to A substitution within 20 bases from the 3' end of its sequencing read. 20% do not exceed the 50% cutoff for classifying a mutation as a deamination error. Therefore, the apparent G to A substitution is classified as a true mutation. The present invention provides, for example, the following items. (Item 1) 1. A method for identifying variant nucleotides in a population of nucleic acids, comprising: (a) contacting a population of nucleic acids comprising double-stranded molecules having single-stranded overhangs at one or both ends with a protein having 5'-3' polymerase activity and 3'-5' exonuclease activity, wherein the protein digests the 3' overhangs and fills in the 5' overhangs with complementary nucleic acid at one or both ends to generate double-stranded blunt-ended nucleic acids; (b) determining the sequence of the double-stranded blunt-ended nucleic acid to obtain a sequenced nucleic acid; (c) for each specified position in the reference sequence, (i) identifying a subset of sequenced nucleic acids that includes the specified position; (ii) identifying sequenced nucleic acids within said subset in which said designated position is occupied by a variant nucleotide; (d) calling the presence of a variant nucleotide at each designated position where the sequenced nucleic acid in step (c)(ii) supports said call, wherein the presence of a variant nucleotide at a designated position is (i) the variant is a C to T or G to A variation compared to the reference nucleotide; and (ii) the variant nucleotide is (1) the nucleotide context surrounding the specified position; and / or (2) if a sequenced nucleic acid in said subset is classified as a deamination error based on the distance of said C to T transition at said designated position from the 5' end of the sequenced nucleic acid in said subset or the distance of said G to A transition at said designated position from the 3' end of the sequenced nucleic acid in said subset, then said sequenced nucleic acid in said subset is not called as a deamination error. A method comprising: (Item 2) 2. The method of claim 1, wherein step (c)(ii) identifies the number of nucleic acids in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in step (d)(i) and (ii), the presence of a variant nucleotide at each designated position is called if the number of sequenced nucleic acids in the subset having the variation meets a threshold. (Item 3) 2. The method of claim 1, wherein the variant nucleotide is classified as a deamination error based on the occurrence of the C to T variation at the designated position within a defined proximity of the 5' end in the sequenced nucleic acids in the subset, or the occurrence of the G to A variation at the designated position within a defined proximity of the 3' end in the sequenced nucleic acids in the subset. (Item 4) The method of any of the preceding items, wherein step (c)(ii) further comprises identifying the number of sequenced nucleic acids in the subset in which the designated position is occupied by a reference nucleotide. (Item 5) The method of any of the preceding items, wherein step (b) comprises determining the sequence of both strands of the double-stranded blunt-ended nucleic acid. (Item 6) 6. The method of claim 5, wherein step (c) is performed for at least one designated position, and the sequenced nucleic acids in the subset having the variation comprise the sequence of both strands of the sequenced nucleic acid of the double-stranded blunt-ended nucleic acid. (Item 7) The method of any of the preceding items, wherein step (b) comprises determining sequences from both ends of the strands. (Item 8) ligating the double stranded blunt ended nucleic acid to an adaptor comprising a barcode; amplifying the nucleic acid primed from a primer molecule that binds to the adaptor; The method of any of the preceding items, wherein step (b) comprises determining sequences of the amplified nucleic acid molecules and grouping the sequences of the amplified nucleic acid molecules into families, members of a family having the same start and end points on the nucleic acid and the same barcode, and determining for the family a consensus nucleotide at each of a plurality of positions from the sequences of its respective members. (Item 9) The method of any of the preceding items, wherein the population of nucleic acids is derived from a sample containing cell-free nucleic acid of a subject. (Item 10) 10. The method of claim 9, wherein the cell-free nucleic acid is derived from a bodily fluid of a subject having cancer or having signs or symptoms consistent with having cancer. (Item 11) 11. The method of claim 10, wherein the body fluid is selected from the group consisting of blood, plasma, saliva, urine, and cerebrospinal fluid. (Item 12) The method of any of the preceding items, wherein the C to T variation at the designated position is classified as a deamination error if its occurrence is at least 50% in a first proportion of the subset in which the designated position is within a defined proximity of the 5' end, or the G to A variation at the designated position is classified as a deamination error if its occurrence is at least 50% in a second proportion of the subset in which the designated position is within a defined proximity of the 3' end. (Item 13) 13. The method of claim 12, wherein the C to T variation at the designated position is classified as a deamination error based on the variation having at least twice as much occurrence in a first proportion of the subset in which the designated position is within a defined proximity of the 5' end than in other sequenced nucleic acids in the subset, or the G to A variation at the designated position is classified as a deamination error based on the variation having at least twice as much occurrence in a second proportion of the subset in which the designated position is within a defined proximity of the 3' end than in other sequenced nucleic acids in the subset. (Item 14) The method of any of the preceding items, wherein the threshold is that the variation is present in at least 1% of the sequenced nucleic acids in the subset. (Item 15) The method of any of the preceding items, wherein the C to T or G to A variation is classified as a deamination error based at least on the surrounding context being TCG to TTG or CGA to CAA. (Item 16) 4. The method of claim 3, wherein the defined proximity to the 5' end is defined as being within 20 nucleotides or less to the 5' end, and the defined proximity to the 3' end is defined as being within 20 nucleotides or less to the 3' end. (Item 17) 17. The method of claim 16, wherein the defined proximity to the 5' end is defined as being within 20 nucleotides of the 5' end and the defined proximity to the 3' end is defined as being within 20 nucleotides of the 3' end. (Item 18) Item 11. The method of any of the preceding items, wherein the protein is Klenow. (Item 19) The method of any of the preceding items, wherein steps (c) and (d) are performed in a system implemented by a computer that performs these steps. (Item 20) 2. The method of any of the preceding items, wherein the reference sequence is a sequence of the human genome. (Item 21) 21. The method of claim 20, wherein the reference sequence is a sequence of a human chromosome. (Item 22) 21. The method of claim 20, wherein the reference sequence comprises a non-contiguous region of the human genome. (Item 23) The method of any of the preceding items, wherein at least one of the called variant nucleotides is known to be associated with cancer. (Item 24) The method of any of the preceding items, performed on a population of nucleic acids derived from samples of a population of subjects having or suspected of having cancer, subjects within said population then receive different treatments depending on which variant nucleotides were called in said individual subjects. (Item 25) 2. The method of any of the preceding items, wherein variant nucleotides classified as deamination errors are at least 1% of the called variant nucleotides. (Item 26) 2. The method of any of the preceding items, wherein variant nucleotides classified as deamination errors are at least 10% of the called variant nucleotides. (Item 27) The method of any of the preceding items, wherein the presence of a variant is not called for if at least five variant nucleotides are classified as deamination errors. (Item 28) 2. The method of any of the preceding items, wherein the population of nucleic acids is derived from a solid tissue. (Item 29) 12. The method of claim 11, wherein the body fluid is plasma. (Item 30) 9. The method of claim 8, wherein the adapter comprising the barcode linked to the 5' end is different from the adapter comprising the barcode linked to the 3' end. (Item 31) Item 11. The method of any of the preceding items, wherein the frequency of deamination errors is at least 1%. (Item 32) Item 11. The method of any of the preceding items, wherein the frequency of deamination errors is at least 10%. (Item 33) The method of any of the preceding items, wherein the variant nucleotide is classified as a deamination error based on the average distance of the C to T transition at the designated position being less than the average distance of the reference nucleotide at the designated position from the 5' end of the sequenced nucleic acids in the subset, or the average distance of the G to A transition at the designated position being less than the average distance of the reference nucleotide at the designated position from the 3' end of the sequenced nucleic acids in the subset. (Item 34) 2. The method of any of the preceding items, wherein the variant nucleotide is a single variant (SNV). (Item 35) 1. A method for identifying variant nucleotides in a nucleic acid, comprising: (a) contacting a double-stranded nucleic acid having single-stranded overhangs with a protein having 5'-3' polymerase activity and 3'-5' exonuclease activity, thereby producing a double-stranded blunt-ended nucleic acid; (b) determining the sequence of the double-stranded blunt-ended nucleic acid; (c) comparing the determined sequence to a reference sequence, wherein the determined sequence contains at least one C to T variation at at least one designated position within 20 nucleotides or less of the 5' end of the determined sequence, or at least one G to A variation within 20 nucleotides or less of the 3' end of the determined sequence; (d) calling the sequence of said nucleic acid as said determined sequence, except that in at least one of said positions where a C to T transition occurs within 20 nucleotides or less of the 5' end of said determined sequence or a G to A transition occurs within 20 nucleotides or less of the 3' end of said determined sequence, the nucleotide occupying said reference sequence is called at said designated position; A method comprising: (Item 36) 36. The method of claim 35, wherein the C to T or G to A variation occurs in the surrounding context of TCG to TTG or CGA to CAA. (Item 37) 1. A method for identifying variant nucleotides in a population of nucleic acids, comprising: (a) contacting a population of nucleic acids of overlapping sequences, at least one of which is a double-stranded molecule having a single-stranded overhang on one or both ends, with a protein having 5'-3' polymerase activity and 3'-5' exonuclease activity, wherein the protein digests the 3' overhangs and fills in the 5' overhangs to generate double-stranded blunt-ended nucleic acids; (b) ligating the double-stranded blunt-ended nucleic acid to an adapter comprising a barcode and amplifying the nucleic acid primed from a primer molecule that binds to the adapter; (c) determining the sequence of the amplified nucleic acid molecule and classifying the sequences of the amplified nucleic acid molecule into families, members of a family having the same start and end points on the nucleic acid and the same barcode, and determining a consensus sequence for the family from the sequences of its respective members; (d) for each designated position in a reference sequence, determining a subset of families having consensus sequences that include the designated position, wherein the consensus sequences within the subset are occupied by a variant nucleotide; (e) calling the presence of a variant nucleotide at a designated position where the consensus sequence within the subset having the variant nucleotide supports the call, wherein the presence of a variant nucleotide at a designated position is supported by: (i) the variant nucleotide is a variation of C to T or G to A compared to the reference nucleotide; and (ii) the variant nucleotide is (1) the nucleotide context surrounding the specified position; and / or (2) is classified as a deamination error based on the distance from the 5' end of the C to T transition at the specified position of a consensus sequence in the subset or the distance from the 3' end of the G to A transition at the specified position of a consensus sequence in the subset. In addition, the step and A method comprising: (Item 38) 38. The method of claim 37, wherein step (c)(ii) identifies the number of nucleic acids in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in step (d)(i) and (ii), the presence of a variant nucleotide at each designated position is called when the number of sequenced nucleic acids in the subset having the variation meets a threshold. (Item 39) 1. A method for identifying false positive variant nucleotides in a population of nucleic acids, comprising: (a) contacting a population of overlapping sequence nucleic acids, at least one of which is a double-stranded molecule having a single-stranded overhang on one or both ends, with a protein having 5'-3' polymerase activity and 3'-5' exonuclease activity, wherein the protein digests the 3' overhangs and fills in the 5' overhangs with complementary nucleic acid at one or both ends to generate double-stranded blunt-ended nucleic acids; (b) determining the sequence of the double-stranded blunt-ended nucleic acid to obtain a sequenced nucleic acid; (c) for each designated position in a reference sequence, identifying a subset of sequenced nucleic acids that includes the designated position, and identifying the number of sequenced nucleic acids in the subset in which the designated position is occupied by a reference nucleotide and the number of sequenced nucleic acids in the subset in which the designated position is occupied by a variant nucleotide; (d) calling the presence of a false positive variant nucleotide at each designated position, wherein the sequenced nucleic acid having a C to T or G to A variation at the designated position supports the call, and wherein the variation is (1) the nucleotide context surrounding the specified position; and / or (2) classifying as a deamination error based on an over-representation of the C to T conversion in sequenced nucleic acids in a first proportion of the subset where the designated position is within a defined proximity of the 5' end, or an over-representation of the G to A conversion in sequenced nucleic acids in a second proportion of the subset where the designated position is within a defined proximity of the 3' end; A method comprising: (Item 40) 40. The method of claim 39, wherein step (c)(ii) identifies the number of nucleic acids in the subset in which the designated position is occupied by a variant nucleotide, and except as specified in step (d)(i) and (ii), the presence of a variant nucleotide at each designated position is called when the number of sequenced nucleic acids in the subset having the variation meets a threshold. (Item 41) 1. A method of determining the minor allele frequency of a "C" to "T" or "G" to "A" variant at a designated position in a reference sequence in a population of sequenced nucleic acids that map to said designated position, wherein the minor allele frequency is determined by comparing the number of sequenced nucleic acids that map to said designated position that contain said variant ("number of variants") to the total number of sequenced nucleic acids that map to said designated position, said method comprising adjusting the number of T or A variants at said designated position for the probability of a deamination error, wherein the probability of error is a function of the distance of said variant from the 5' end of the molecule in the case of "T" and from the 3' end of the molecule in the case of "A". (Item 42) 42. The method of claim 41, wherein a "T" variant located within a selected distance from the 5' end of a sequenced polynucleotide or an "A" variant located within a selected distance from the 3' end of a sequenced nucleic acid is not counted in the variant count. (Item 43) 42. The method of claim 41, wherein if the ratio of "T" variants located within a selected distance from the 5' end of the sequenced polynucleotide to "T" variants located outside said selected distance from the 5' end of the sequenced nucleic acid is greater than a predetermined ratio (e.g., greater than 50%), or if the ratio of "A" variants located within a selected distance from the 3' end of the sequenced nucleic acid to "A" variants located outside said selected distance from the 3' end of the sequenced nucleic acid is greater than a predetermined ratio (e.g., greater than 50%), then all "T" variants are discounted from the variant count. (Item 44) 42. The method of claim 41, wherein the number of variants is determined as the sum of the probabilities that each "T" variant or each "A" variant is a true variant. (Item 45) A method comprising administering to a subject determined to have a cancer marker by the method of any of the preceding items, a therapeutic intervention effective to treat a cancer characterized by said cancer marker. (Item 46) receiving data regarding the identity of one or more variant nucleotides in the cell-free nucleic acid of the subject by carrying out the method according to any one of items 1 to 45; determining the presence of a cancer marker from the one or more variant nucleotides; and administering a therapeutic intervention effective to treat a cancer characterized by said cancer markers. (Item 47) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; a computer in communication with the communications interface, the computer comprising: one or more computer processors; and when executed by the one or more computer processors, (a) receiving, via the communication network, the sequencing reads generated by the nucleic acid sequencer; (b) for each specified position in the reference sequence, (i) identifying a subset of sequencing reads that includes the specified position; (ii) identifying sequencing reads within said subset, wherein said designated position is occupied by a variant nucleotide; (c) calling the presence of a variant nucleotide at each designated position where the sequencing reads in the subset having the variation support the call, where the presence of a variant nucleotide at a designated position is supported if: (i) the variant is a C to T or a G to A variation compared to the reference nucleotide; and (ii) the variant nucleotide is (1) the nucleotide context surrounding the specified position; and / or (2) if a sequenced nucleic acid in said subset is classified as a deamination error based on the distance of said C to T transition at said designated position from the 5' end of the sequenced nucleic acid in said subset or the distance of said G to A transition at said designated position from the 3' end of the sequenced nucleic acid in said subset, then said sequenced nucleic acid in said subset is not called as a deamination error. and a computer readable medium including machine executable code for implementing the method comprising: Including, the system. (Item 48) 48. The system of claim 47, wherein step (c)(ii) identifies the number of nucleic acids in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in step (d)(i) and (ii), the presence of a variant nucleotide at each designated position is called when the number of sequenced nucleic acids in the subset having the variation meets a threshold. (Item 49) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; a computer in communication with the communications interface, the computer comprising: one or more computer processors; and when executed by the one or more computer processors, (a) receiving, via the communication network, the sequencing reads generated by the nucleic acid sequencer; (b) comparing the determined sequence to a reference sequence, wherein the determined sequence contains at least one C to T variation at at least one designated position within 20 nucleotides or less of the 5' end of the determined sequence, or at least one G to A variation within 20 nucleotides or less of the 3' end of the determined sequence; and (c) calling the sequence of the nucleic acid as the determined sequence, except that in at least one of said positions where a C to T variation is within 20 nucleotides or less of the 5' end of the determined sequence or a G to A variation is within 20 nucleotides or less of the 3' end of the determined sequence, the nucleotide occupying the reference sequence is called at the designated position. (Item 50) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; a computer in communication with the communications interface, the computer comprising: one or more computer processors; and when executed by the one or more computer processors, (a) receiving, via the communication network, the sequencing reads generated by the nucleic acid sequencer; (b) grouping sequences of the sequencing reads into families, members of a family having the same start and end points on the nucleic acid and the same barcode, and determining a consensus sequence for the family from the sequences of its respective members; (c) for each designated position in a reference sequence, determining a subset of families having consensus sequences that include the designated position, and identifying the consensus sequences in which the designated position is occupied by a variant nucleotide; (d) calling the presence of a variant nucleotide at each designated position, wherein the consensus sequences in the subset having the variant nucleotide support the call, but the presence of a variant nucleotide at the designated position is: (i) the variant nucleotide is a variation of C to T or G to A compared to the reference nucleotide; and (ii) the variant nucleotide is (1) the nucleotide context surrounding the specified position; and / or (2) if the error is classified as a deamination error based on the distance from the 5' end of the C to T transition at the specified position in a consensus sequence in the subset or the distance from the 3' end of the G to A transition at the specified position of a consensus sequence in the subset, then the error is not called; and a computer readable medium comprising machine executable code for implementing a method comprising: (Item 51) 51. The system of claim 50, wherein step (c) identifies a number of consensus sequences in the subset in which the designated position is occupied by a variant nucleotide, and, except as specified in (i) and (ii) of step (d), the presence of a variant nucleotide at each designated position is called if the number of consensus sequences in the subset having the variation meets a threshold. (Item 52) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; a computer in communication with the communications interface, the computer comprising: one or more computer processors; and when executed by the one or more computer processors, (a) receiving, via the communication network, the sequencing reads generated by the nucleic acid sequencer; (b) for each designated position in a reference sequence, identifying a subset of sequencing reads that includes the designated position, and identifying the number of sequenced nucleic acids in the subset in which the designated position is occupied by a reference nucleotide and the number of sequenced nucleic acids in the subset in which the designated position is occupied by a variant nucleotide; (c) calling the presence of a false positive variant nucleotide at each designated position, wherein the sequenced nucleic acid having a C to T or G to A variation at the designated position supports the call, wherein the variation is (1) the nucleotide context surrounding the specified position; and / or (2) classifying as a deamination error based on an over-occurrence of the C to T conversion in sequenced nucleic acids in a first proportion of the subset where the designated position is within a defined proximity of the 5' end, or an over-occurrence of the G to A conversion in sequenced nucleic acids in a second proportion of the subset where the designated position is within a defined proximity of the 3' end. (Item 53) a communication interface that receives sequencing reads generated by the nucleic acid sequencer via a communication network; a computer in communication with the communications interface, the computer comprising: one or more computer processors; and when executed by the one or more computer processors, (a) receiving, via the communication network, the sequencing reads generated by the nucleic acid sequencer; (b) adjusting the number of T or A variants in the sequencing reads based on the probability of deamination errors, where the probability of error is a function of the distance of the variant from the 5' end of the molecule in the case of "T" and from the 3' end of the molecule in the case of "A". (Item 54) 54. The system of any one of items 47 to 53, further comprising the nucleic acid sequencer. (Item 55) 54. The system of any one of items 47 to 53, wherein the nucleic acid sequencer sequences a sequencing library generated from cell-free DNA molecules derived from a subject, the sequencing library comprising the cell-free DNA molecules and adapters comprising barcodes. (Item 56) 54. The system of any one of items 47 to 53, wherein the nucleic acid sequencer performs sequencing by synthesis on the sequencing library to generate the sequencing reads. (Item 57) 54. The system of any one of items 47 to 53, wherein the nucleic acid sequencer performs pyrosequencing, single molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing by ligation, or sequencing by hybridization on the sequencing library to generate the sequencing reads. (Item 58) 54. The system of any one of items 47 to 53, wherein the nucleic acid sequencer generates the sequencing reads using a clonal single molecule array derived from the sequencing library. (Item 59) 54. The system of any one of items 47 to 53, wherein the nucleic acid sequencer comprises a chip having an array of microwells for sequencing the sequencing library to generate the sequencing reads. (Item 60) 54. The system of any one of items 47 to 53, wherein the computer-readable medium comprises a memory, a hard drive, or a computer server. (Item 61) 54. The system of any one of claims 47 to 53, wherein the communication network comprises a telecommunications network, the Internet, an extranet, or an intranet. (Item 62) 54. The system of any one of claims 47 to 53, wherein the communications network includes one or more computer servers capable of distributed computing. (Item 63) Item 65. The system of item 64, wherein the distributed computing is cloud computing. (Item 64) 54. The system of any one of items 47 to 53, wherein the computer is installed on a computer server that is remotely installed from the nucleic acid sequencer. (Item 65) 65. The system of claim 64, wherein the sequencing library further comprises a sample barcode that distinguishes the sample from one or more samples. (Item 66) 54. The system of any one of items 47 to 53, further comprising an electronic display in communication with the computer via a network, the electronic display including a user interface for displaying results upon performing (a) to (c). (Item 67) Item 67. The system of item 66, wherein the user interface is a graphical user interface (GUI) or a web-based user interface. (Item 68) Item 67. The system of item 66, wherein the electronic display is present in a personal computer. (Item 69) Item 67. The system of item 66, wherein the electronic display is present in an internet enabled computer. (Item 70) 70. The system of claim 69, wherein the Internet-enabled computer is located at a location remote from the computer.

Claims

[Claim 1] The invention as depicted in the drawings.

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