Synthetic nucleic acid incorporations

Abstract

The present disclosure provides methods for determining the relative abundance of one or more non-host species in a sample from a host. Methods involving adding a known concentration of synthetic nucleic acids to a sample and performing sequencing analysis to identify non-host species, such as pathogens, are also provided. Methods of tracking a sample, tracking reagents, and tracking loss of diversity in a sequencing analysis are also provided.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 313,668, filed March 25, 2016; U.S. Provisional Patent Application No. 62 / 397,873, filed September 21, 2016; and U.S. Provisional Patent Application No. 62 / 451,363, filed January 27, 2017, which are incorporated herein by reference in their entirety. Background of the Invention

[0004] Next-generation sequencing can be used to collect large amounts of data about the genetic content of a sample. It is particularly useful for analyzing nucleic acids in complex samples such as clinical samples, as well as for whole-genome sequencing. However, there is a need in the field for more efficient and accurate methods to detect and quantify nucleic acids, especially those in low-abundance samples or patient samples. Summary of the Invention

[0005] This article provides methods and compositions for improving nucleic acid identification or quantification in next-generation sequencing analyses and other assays using spike-in synthetic nucleic acids. In some cases, the spike-in synthetic nucleic acids have specific characteristics, such as a specific sequence, length, GC content, degeneracy, diversity, and / or a known starting concentration. The methods provided herein are particularly useful for detecting pathogen nucleic acids in clinical samples such as plasma, but can also be used to detect other types of targets.

[0006] In one aspect, this document provides a method for determining the abundance of nucleic acids in an initial sample containing a target nucleic acid, the method comprising: (a) adding an initial amount of at least 1000 synthetic nucleic acids to the sample, each of the at least 1000 synthetic nucleic acids containing a unique variable region; (b) performing sequencing analysis on a portion of the target nucleic acid and a portion of the at least 1000 synthetic nucleic acids in the sample to obtain target and synthetic nucleic acid sequence reads, wherein the synthetic nucleic acid sequence reads include unique variable region sequences; (c) detecting a loss of diversity of the at least 1000 synthetic nucleic acids by (i) quantifying a number of different variable region sequences within the synthetic nucleic acid sequence reads to obtain a unique sequencing value; and (ii) comparing the initial amount of the at least 1000 synthetic nucleic acids with the unique sequencing value to obtain a loss of diversity of the at least 1000 synthetic nucleic acids; and (d) using the loss of diversity of the at least 1000 synthetic nucleic acids to calculate the abundance of the target nucleic acid in the initial sample. In some cases, the initial amount compared is an initial concentration.

[0007] In some embodiments, the target nucleic acid comprises pathogen nucleic acid. In some cases, the target nucleic acid comprises pathogen nucleic acid from at least five different pathogens. In some cases, the target nucleic acid comprises pathogen nucleic acid from at least two different pathogens. In some cases, the target nucleic acid comprises pathogen nucleic acid from at least ten different pathogens.

[0008] In some cases, the at least 1,000 synthetic nucleic acids comprise DNA. In some cases, the at least 1,000 synthetic nucleic acids comprise RNA, ssRNA, dsDNA, ssDNA, or combinations thereof. In some cases, each of the at least 1,000 synthetic nucleic acids is less than 500 base pairs or nucleotides in length. In some cases, each of the at least 1,000 synthetic nucleic acids is less than 200 base pairs or nucleotides in length. In some cases, each of the at least 1,000 synthetic nucleic acids is less than 100 base pairs or nucleotides in length. In some cases, the sample is blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage fluid, urine, feces, saliva, or a nasal sample. In some cases, the sample is from a human subject. In some cases, the sample is a sample of isolated nucleic acids.

[0009] In some cases, the method further includes generating a sequencing library from the sample, wherein the at least 1,000 synthetic nucleic acids are added to the sample prior to generating the sequencing library. In some cases, the loss of diversity of the at least 1,000 synthetic nucleic acids indicates the loss of one or more nucleic acids during sample processing of the sample.

[0010] In some cases, each of the at least 1,000 synthetic nucleic acids contains a tag sequence. In some cases, quantifying the number of unique variable region sequences includes detecting sequences containing the tag sequence. In some cases, quantifying the at least 1,000 unique sequences within the first sequence read includes determining a read count of unique sequences within the first sequence read. In some cases, the at least 1,000 unique synthetic nucleic acids contain at least 10 4 A unique synthetic nucleic acid.

[0011] In some cases, the method further includes adding additional synthetic nucleic acids having at least three different lengths. In some cases, the method further includes adding a first set of additional synthetic nucleic acids having a first length, a second set of additional synthetic nucleic acids having a second length, and a third set of additional synthetic nucleic acids having a third length, wherein each of the first, second, and third sets of additional synthetic nucleic acids comprises synthetic nucleic acids having at least three different GC contents. In some cases, the method further includes using the additional synthetic nucleic acids to calculate the absolute abundance value of the target nucleic acid in the sample. In some cases, the method further includes using the additional synthetic nucleic acids to calculate the absolute or relative abundance of the target nucleic acid in the sample based on the length, GC content, or both length and GC content of the additional synthetic nucleic acids.

[0012] In some cases, the at least 1,000 synthetic nucleic acids are added to the sample in a first sample processing step. In some cases, the method further includes adding an additional pool of at least 1,000 unique synthetic nucleic acids to the sample in a second sample processing step, wherein the second sample processing step is different from the first sample processing step. In some cases, the method further includes calculating the diversity loss of the additional pool of at least 1,000 synthetic nucleic acids. In some cases, the method further includes identifying sample processing steps with relatively high diversity loss by comparing the diversity loss of the at least 1,000 synthetic nucleic acids with the diversity loss of the additional pool of at least 1,000 synthetic nucleic acids.

[0013] In some cases, each of the additional pool of at least 1,000 unique synthetic nucleic acids contains a domain that identifies the synthetic nucleic acid as a member of the additional pool of at least 1,000 synthetic nucleic acids. In some cases, the method further includes adding a sample-identifying nucleic acid to the sample. In some cases, (a) above further includes adding a non-unique synthetic nucleic acid to the sample.

[0014] In some implementations, the calculated abundance is a relative abundance. In some implementations, the calculated abundance is an absolute abundance.

[0015] In another aspect, this document provides a method for determining the relative abundance or initial abundance of pathogen nucleic acids in a sample, the method comprising: (a) obtaining a sample from a subject infected with or suspected of being infected with a pathogen, wherein the sample contains multiple pathogen nucleic acids; (b) adding multiple synthetic nucleic acids to the sample such that the sample contains the synthetic nucleic acids at a known initial abundance, wherein (i) the length of the synthetic nucleic acids is less than 500 base pairs; (ii) the synthetic nucleic acids comprise synthetic nucleic acids having a first length, synthetic nucleic acids having a second length, and synthetic nucleic acids having a third length, wherein the first, second, and third lengths are different; and (iii) the synthetic nucleic acids having the first length... The synthetic nucleic acids comprise at least three different GC contents; (c) the sample comprising the synthetic nucleic acids is sequenced to determine the final abundance of the synthetic nucleic acids and the final abundance of the pathogen nucleic acids; (d) the final abundance of the synthetic nucleic acids is compared with the known initial abundance to generate a recovery profile of the synthetic nucleic acids; and (e) the final abundance of the pathogen nucleic acids is normalized by comparing the pathogen nucleic acids with the synthetic nucleic acids having the closest GC contents and lengths using the recovery profile of the synthetic nucleic acids to determine the relative abundance or initial abundance of the pathogen nucleic acids.

[0016] In some cases, the at least three different GC contents include a first GC content between 10% and 40%, a second GC content between 40% and 60%, and a third GC content between 60% and 90%. In some cases, each of the at least three different GC contents is between 10% and 50%. In some cases, each of the at least three different GC contents is between 5% and 40%. In some cases, the length of the synthesized nucleic acid is less than 200 base pairs or nucleotides. In some cases, the length of the synthesized nucleic acid is less than 100 base pairs or nucleotides. In some cases, the at least three different GC contents are at least four different, at least five different, at least six different, at least seven different, or at least eight different GC contents. In some cases, the synthesized nucleic acid has at least a fourth length, at least a fifth length, at least a sixth length, at least a seventh length, at least a ninth length, at least a tenth length, at least a twelfth length, or at least a fifteenth length. In some implementations, each length contains synthetic nucleic acids having at least 3, 4, 5, 6, 7, 8, 9, or 10 different GC contents or no more than 50 different GC contents.

[0017] In some cases, the synthetic nucleic acid comprises double-stranded DNA. In some cases, the method further includes using the synthetic nucleic acid to monitor the denaturation of the pathogen nucleic acid. In some cases, the method further includes normalizing the relative abundance or initial abundance of the pathogen nucleic acid using a weighting factor. In some cases, the weighting factor is obtained by analyzing the raw measurements of the first synthetic nucleic acid and the second synthetic nucleic acid by comparing them with known concentrations of a first synthetic nucleic acid and a second synthetic nucleic acid.

[0018] In another aspect, this document provides a method for detecting nucleic acids from a pathogen, the method comprising: (a) obtaining a first sample containing a first pathogen nucleic acid, wherein the first sample is obtained from a first subject infected with the first pathogen; (b) obtaining a second sample from a second subject; (c) obtaining first and second sample markers, each marker containing a different synthetic nucleic acid that cannot hybridize with the first pathogen nucleic acid, and assigning the first sample marker to the first sample and the second sample marker to the second sample; (d) adding the first sample marker to the first sample and adding the second sample marker to the second sample; (e) performing sequencing analysis on the first sample containing the first sample marker and the second sample containing the second sample marker to obtain sequence results of the first and second samples; (f) detecting the presence of the first sample marker, the second sample marker, and the first pathogen nucleic acid in the sequence results of the first sample; and (g) determining that the detected first pathogen nucleic acid was initially present in the first sample when the sequencing analysis detected: (i) the first sample marker; (ii) the first pathogen nucleic acid; and (iii) the absence of the second sample marker or the second sample marker being below a threshold level.

[0019] In another aspect, this document provides a method for detecting nucleic acids, the method comprising: (a) obtaining a first nucleic acid sample containing a first nucleic acid; (b) obtaining a first control nucleic acid sample containing a first positive control nucleic acid; (c) adding a first sample marker to the first control nucleic acid, the first sample marker comprising a synthetic nucleic acid that cannot hybridize with the first nucleic acid; (d) performing sequencing analysis on the first nucleic acid sample and the first control nucleic acid sample containing the first sample marker to obtain sequence reads of the first nucleic acid sample and the control nucleic acid sample; (e) aligning the sequence reads of the first nucleic acid sample with a reference sequence to detect the presence of the first sample marker in the sequence reads of the first nucleic acid sample; and (f) determining, based on the alignment of the sequence reads, whether the first positive control nucleic acid is present in the first nucleic acid sample.

[0020] In some cases, the synthetic nucleic acid of the first sample marker is less than 150 base pairs or nucleotides in length. In some cases, the first positive control nucleic acid is a pathogen nucleic acid. In some cases, the first sample marker contains modified nucleic acid. In some cases, the first sample marker contains DNA. In some cases, the sample contains cell-free body fluids. In some cases, the sample is from a subject infected with a pathogen.

[0021] In another aspect, this document provides a method for detecting a reagent in a sample, the method comprising: (a) adding a first synthetic nucleic acid to the reagent, wherein the first synthetic nucleic acid contains a unique sequence; (b) adding a reagent containing the first synthetic nucleic acid to a nucleic acid sample; (c) preparing the nucleic acid sample for sequencing analysis; (d) performing sequencing analysis on the nucleic acid sample to obtain a sequence result of the nucleic acid sample; and (e) detecting the reagent in the sample by determining the presence of the first synthetic nucleic acid in the sample based on the sequence result of the nucleic acid sample.

[0022] In some cases, the length of the first synthetic nucleic acid is less than 150 base pairs or nucleotides. In some cases, the first synthetic nucleic acid is added to a first batch of reagents, and this further includes adding a second synthetic nucleic acid to a second batch of reagents. In some cases, the reagents for detecting the sample include reagents for detecting a specific batch. In some cases, the synthetic nucleic acid cannot be degraded by nucleases. In some cases, the reagents contain an aqueous buffer. In some cases, the reagents include extraction reagents, enzymes, ligases, polymerases, or dNTPs.

[0023] In another aspect, this paper provides a method for generating sequencing libraries, the method comprising: (a) obtaining a sample comprising: (i) a target nucleic acid; (ii) a sequencing adaptor; and (iii) at least one synthetic nucleic acid, wherein the at least one synthetic nucleic acid comprises DNA and is resistant to ligation with nucleic acids; and (b) performing a ligation reaction on the sample such that the sequencing adaptor is preferentially ligated to the target nucleic acid relative to the at least one synthetic nucleic acid.

[0024] In another aspect, this document provides a method for generating a sequencing library, the method comprising: (a) obtaining a sample containing a target nucleic acid and at least one synthetic nucleic acid; and (b) removing the at least one synthetic nucleic acid from the sample to obtain a sequencing sample containing the target nucleic acid but not the at least one synthetic nucleic acid; and (c) attaching a sequencing adaptor to the target nucleic acid within the sequencing sample.

[0025] In another aspect, this document provides a method for generating a sequencing library, the method comprising: (a) obtaining a sample containing a target nucleic acid and at least one synthetic nucleic acid, wherein the at least one synthetic nucleic acid comprises: (i) single-stranded DNA; (ii) a nucleotide modification that inhibits the amplification of the synthetic nucleic acid; (iii) an immobilized tag; (iv) a DNA-RNA hybrid; (v) a nucleic acid of a length greater than that of the target nucleic acid; or (vi) any combination thereof; and (b) generating a sequencing library from the sample for a sequencing reaction, wherein at least a portion of the synthetic nucleic acid is not sequenced in the sequencing reaction.

[0026] In another aspect, this paper provides a method for generating sequencing libraries, the method comprising: (a) obtaining a sample containing: (i) a target nucleic acid; (ii) a sequencing adaptor; and (iii) at least one synthetic nucleic acid, wherein the at least one synthetic nucleic acid contains DNA and is resistant to end repair; and (b) performing an end repair reaction on the sample such that the target nucleic acid is preferentially end-repaired relative to the at least one synthetic nucleic acid.

[0027] In another aspect, this article provides a kit for generating sequencing libraries, the kit comprising (a) a sequencing adaptor; and (b) at least one synthetic nucleic acid, wherein the at least one synthetic nucleic acid comprises DNA and is resistant to end repair of nucleic acids.

[0028] In one aspect, this document provides a method for determining the absolute or relative abundance of nucleic acids in an initial sample containing a target nucleic acid, the method comprising: (a) adding a starting amount of at least 1000 unique synthetic nucleic acids to the sample, each of said at least 1000 unique synthetic nucleic acids comprising (i) an identifier tag and (ii) a variable region; (b) performing sequencing analysis on a portion of the target nucleic acid and a portion of said at least 1000 synthetic nucleic acids in the sample to obtain target and synthetic nucleic acid sequence reads, wherein said synthetic nucleic acid sequence reads include identifier tag sequences and variable region sequences; (c) by ( (i) detecting sequence reads corresponding to at least a portion of the identified tag sequence to obtain a set of first sequence reads; (ii) quantifying a plurality of different variable region sequences within the first sequence reads to obtain unique sequencing values; and (iii) comparing the initial amount of the at least 1,000 unique synthetic nucleic acids with the unique sequencing values ​​to obtain a diversity loss of the at least 1,000 unique synthetic nucleic acids, to detect the diversity loss of the at least 1,000 synthetic nucleic acids; and (d) using the diversity loss of the at least 1,000 synthetic nucleic acids to calculate the abundance of the target nucleic acid in the initial sample. In some cases, the initial amount compared is the initial concentration.

[0029] In some cases, the target nucleic acid comprises pathogen nucleic acid. In some cases, the target nucleic acid comprises pathogen nucleic acid from at least five different pathogens. In some cases, the at least 1,000 unique synthetic nucleic acids comprise DNA.

[0030] In some cases, each of the at least 1,000 unique synthetic nucleic acids is less than 500 base pairs or nucleotides in length. In some cases, each of the at least 1,000 unique synthetic nucleic acids is less than 200 base pairs or nucleotides in length. In some cases, each of the at least 1,000 unique synthetic nucleic acids is less than 100 base pairs or nucleotides in length.

[0031] In some cases, the sample is blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage fluid, urine, feces, saliva, or a nasal sample. In some cases, the sample is an isolated nucleic acid sample. In some cases, the sample is from a human subject.

[0032] In some cases, the method further includes generating a sequencing library from the sample, wherein the at least 1,000 unique synthetic nucleic acids are added to the sample prior to generating the sequencing library. In some cases, the loss of diversity of the at least 1,000 unique synthetic nucleic acids indicates the loss of one or more nucleic acids during sample processing of the sample. In some cases, the identification tag contains a common sequence. In some cases, quantifying the at least 1,000 unique sequences within a first sequence read includes determining a read count of the unique sequences within the first sequence read.

[0033] In some cases, the at least 1,000 unique synthetic nucleic acids contain at least 10 4 Each unique synthetic nucleic acid. In some cases, the at least 1,000 unique synthetic nucleic acids comprise at least 10 5 Each unique synthetic nucleic acid. In some cases, the method further includes adding additional synthetic nucleic acids having at least three different lengths.

[0034] In some cases, the method further includes adding a first set of additional synthetic nucleic acids having a first length, a second set of additional synthetic nucleic acids having a second length, and a third set of additional synthetic nucleic acids having a third length, wherein each of the first, second, and third sets of additional synthetic nucleic acids comprises synthetic nucleic acids having at least three different GC contents. In some cases, the method further includes using the additional synthetic nucleic acids to calculate the absolute or relative abundance value of the target nucleic acid in the sample. In some cases, the method further includes using the additional synthetic nucleic acids to calculate the absolute or relative abundance value of the target nucleic acid in the sample based on the length, GC content, or both length and GC content of the additional synthetic nucleic acids.

[0035] In some cases, the at least 1,000 unique synthetic nucleic acids are added to the sample in a first sample processing step. In some cases, the method further includes adding an additional pool of at least 1,000 unique synthetic nucleic acids to the sample in a second sample processing step, wherein the second sample processing step is different from the first sample processing step. In some cases, the method further includes calculating the diversity loss of the additional pool of at least 1,000 unique synthetic nucleic acids. In some cases, the method further includes identifying sample processing steps with relatively high diversity loss by comparing the diversity loss of the at least 1,000 unique synthetic nucleic acids with the diversity loss of the additional pool of at least 1,000 unique synthetic nucleic acids.

[0036] In some cases, each of the additional pool of at least 1,000 unique synthetic nucleic acids contains a domain that identifies the synthetic nucleic acid as a member of the additional pool of at least 1,000 unique synthetic nucleic acids. In some cases, the method further includes adding a sample-identifying nucleic acid to the sample. In some cases, step (a) above further includes adding a non-unique synthetic nucleic acid to the sample. In some cases, the variable sequence reads are detected by alignment with a reference sequence. In some cases, the number of distinct variable sequence reads is quantified by aligning the variable sequence reads to each other and discarding duplicate reads.

[0037] This article provides a method for determining the relative abundance or concentration of pathogen nucleic acids in nucleic acid samples. In some cases, the method may include: obtaining a sample from a subject infected with or suspected of being infected with a pathogen, wherein the sample contains two or more pathogen nucleic acids, wherein the two or more pathogen nucleic acids contain first and second pathogen nucleic acids of different lengths; adding two or more synthetic nucleic acids of known concentration to the sample, wherein the two or more synthetic nucleic acids contain a first synthetic nucleic acid and a second synthetic nucleic acid, the length of the first synthetic nucleic acid being 65% to 135%, 75% to 125%, or 85% to 115% of the length of the first pathogen nucleic acid, and the length of the second synthetic nucleic acid being 65% to 135%, 75% to 125%, or 85% to 115% of the length of the second pathogen nucleic acid, and wherein the two or more synthetic nucleic acids do not hybridize with the first or second pathogen nucleic acid; performing sequencing analysis on the sample to obtain raw measurements of the two or more synthetic nucleic acids, the first pathogen nucleic acid, and the second pathogen nucleic acid; comparing the raw measurements of the first synthetic nucleic acid with a known concentration of the first synthetic nucleic acid to generate a recovery curve of the first synthetic nucleic acid; and normalizing the raw measurements of the first pathogen nucleic acid using the recovery curve of the first synthetic nucleic acid to determine the relative abundance or initial concentration of the first pathogen nucleic acid.

[0038] In some cases, the first and second pathogen nucleic acids originate from the same pathogen. In some cases, the first and second pathogen nucleic acids originate from different pathogens. In some cases, the method described herein further includes normalizing the relative abundance or initial concentration of the first pathogen nucleic acid using a weighting factor. In some cases, the weighting factor is obtained by analyzing the raw measurements of the first and second synthetic nucleic acids by comparing them with known concentrations of the first synthetic acid and the second synthetic nucleic acid.

[0039] This document provides a method for determining the relative abundance or initial concentration of nucleic acids in a sample, the method comprising: (a) obtaining a nucleic acid sample from a subject, wherein the nucleic acid sample comprises first and second nucleic acids of different lengths; adding two or more synthetic nucleic acids of known concentrations to the sample, wherein: (i) the two or more synthetic nucleic acids comprise a first synthetic nucleic acid and a second synthetic nucleic acid, the length of the first synthetic nucleic acid being 65% to 135%, 75% to 125%, or 85% to 115% of the length of the first nucleic acid, and the length of the second synthetic nucleic acid being 65% to 135%, 75% to 125%, or 85% to 115% of the length of the second nucleic acid; (ii) the first synthetic nucleic acid... The nucleic acid comprises a load domain and a marker domain of a specific length, the marker domain having a unique sequence encoding the specific length of the load domain; and (iii) the two or more synthetic nucleic acids cannot hybridize with the first nucleic acid or the second nucleic acid; (b) the sample is sequenced to obtain raw measurements of the two or more synthetic nucleic acids, the first nucleic acid, and the second nucleic acid; (c) the raw measurements of the first synthetic nucleic acid are compared with a known concentration of the first synthetic nucleic acid to generate a recovery curve; and (d) the raw measurements of the first nucleic acid are normalized using the recovery curve to determine the relative abundance or initial concentration of the first nucleic acid.

[0040] In some cases, the first nucleic acid is a pathogen nucleic acid. In some cases, the known concentrations of the two or more synthetic nucleic acids include 2 or more; 3 or more; 5 or more; 10 or more; 50 or more; 100 or more; or 1,000 or more different concentrations. In some cases, the known concentrations of the two or more synthetic nucleic acids are equimolar concentrations. In some cases, the two or more synthetic nucleic acids comprise DNA or modified DNA. In some cases, the two or more synthetic nucleic acids comprise RNA or modified RNA. In some cases, the two or more synthetic nucleic acids comprise 2 or more; 3 or more; 5 or more; 8 or more; 10 or more; 50 or more; 100 or more; or 1,000 or more different lengths. In some cases, the two or more synthetic nucleic acids comprise 2 or more; 3 or more; 5 or more; 8 or more; 10 or more; 50 or more; 100 or more; or 1,000 or more different sequences. In some cases, the length of the two or more synthetic nucleic acids is up to 50, 100, 200, 300, 350, 400, 450, 500, 750, or 1,000 nucleotides. In some cases, the length of the two or more synthetic nucleic acids is at least 10, 20, 30, 50, 100, or 150 nucleotides. In some cases, the two or more synthetic nucleic acids comprise a nucleic acid sequence that identifies the two or more synthetic nucleic acids as synthetic nucleic acids. In some cases, the length of the nucleic acid sequence that identifies the two or more synthetic nucleic acids as synthetic nucleic acids is up to 50, 100, 10, 20, 30, 40, 50, 100, 200, or 500 nucleotides. In some cases, the two or more synthetic nucleic acids comprise nucleic acid sequences that identify the length of the synthetic nucleic acids. In some cases, the length of the nucleic acid sequence identifying the length of the synthetic nucleic acids is up to 50, up to 100, up to 10, up to 20, up to 30, up to 40, up to 50, up to 100, up to 200, or up to 500 nucleotides.

[0041] In some cases, the sample is selected from blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage fluid, urine, feces, saliva, nasal swab, or any combination thereof. In some cases, the sample contains cell-free nucleic acids. In some cases, the sample contains circulating cell-free nucleic acids. In some cases, the subject is human. In some cases, the pathogen is a bacterium, virus, fungus, or parasite. In some cases, the subject has or is suspected of having sepsis. In some cases, the pathogen is associated with sepsis. In some cases, the two or more pathogen nucleic acids comprise 3 or more; 5 or more; 10 or more; 50 or more; 100 or more; 1,000 or more; 2,000 or more; 5,000 or more; 8,000 or more; 10,000 or more; 15,000 or more; or 20,000 or more pathogen nucleic acid sequences.

[0042] In some cases, determining the relative abundance of the first pathogen nucleic acid includes generating one or more genome copies. In some cases, generating one or more genome copies is expressed as genome copies per volume. In some cases, the method described herein further includes extracting nucleic acids from the sample. In some cases, nucleic acids are extracted from the sample using magnetic beads. In some cases, the method described herein further includes removing low-quality sequencing reads. In some cases, the method described herein further includes removing sequencing reads aligned to a reference sequence of the species of the subject or sequencing reads mapped to a reference sequence of the species of the subject. In some cases, the method described herein further includes determining the relative efficiency of recovering one or more nucleic acids of different lengths. In some cases, the method described herein further includes determining a measured concentration of one or more synthetic nucleic acids. In some cases, the method described herein further includes comparing the measured concentration of the one or more synthetic nucleic acids with a known concentration. In some cases, the methods described herein further include detecting one or more; two or more; three or more; five or more; ten or more; fifty or more; one hundred or more; one,000 or more; two,000 or more; five,000 or more; eight,000 or more; ten,000 or more; one,500 or more; or twenty,000 or more pathogen nucleic acids in a sequencing analysis. In some cases, the methods described herein further include detecting one or more; two or more; three or more; five or more; ten or more; fifty or more; one hundred or more; one thousand or more; two thousand or more; five thousand or more; eight thousand or more; ten thousand or more; fifteen thousand or more; or twenty thousand or more pathogen nucleic acids in a sequencing analysis, said pathogen nucleic acids indicating antimicrobial resistance, antibacterial resistance, antiviral resistance, or antifungal resistance. In some cases, the methods described herein further include identifying the co-occurrence of two or more; three or more; five or more; ten or more; fifty or more; one hundred or more pathogens in said sample.

[0043] In some cases, the two or more synthetic nucleic acids are added to the sample before or during nucleic acid extraction from the sample. In some cases, the two or more synthetic nucleic acids are added to the sample after nucleic acid extraction from the sample and before library preparation of the nucleic acids. In some cases, the two or more synthetic nucleic acids differ in length by at least about 20 base pairs. In some cases, the two or more synthetic nucleic acids include 3 or more, 5 or more, 8 or more, 10 or more, 20 or more, or 50 or more synthetic nucleic acids. In some cases, the two or more synthetic nucleic acids are selected from SEQ ID NO:111-SEQ ID NO:118 and any combination thereof. In some cases, the two or more synthetic nucleic acids share a common forward sequence. In some cases, the length of the common forward sequence is at most about 20 base pairs. In some cases, the two or more synthetic nucleic acids share a common reverse sequence. In some cases, the length of the common reverse sequence is at most about 20 base pairs.

[0044] In some cases, the method described herein further includes: comparing the raw measurement of the second synthetic nucleic acid with a known concentration of the second synthetic nucleic acid to generate a recovery curve of the second synthetic nucleic acid; and using the recovery curve of the second synthetic nucleic acid to normalize the raw measurement of the second pathogen nucleic acid to determine the relative abundance or initial concentration of the second pathogen nucleic acid.

[0045] In some cases, the two or more pathogen nucleic acids comprise five or more pathogen nucleic acids of different lengths; the two or more synthetic nucleic acids comprise one or more synthetic nucleic acids having a length of 65% to 135%, 75% to 125%, or 85% to 115% of the length of each of the five or more pathogen nucleic acids, and wherein the two or more synthetic nucleic acids do not hybridize with the five or more pathogen nucleic acids; sequencing analysis of the sample yields raw measurements of the two or more synthetic nucleic acids and the five or more pathogen nucleic acids; comparing the raw measurements includes comparing the raw measurements with a known concentration of each synthetic nucleic acid to generate a recovery curve for each synthetic nucleic acid; and / or using the recovery curves includes using the recovery curves of each synthetic nucleic acid to normalize the raw measurements of each of the five or more pathogen nucleic acids to determine the relative abundance or initial concentration of each of the five or more pathogen nucleic acids. In some cases, the five or more pathogen nucleic acids comprise 10 or more; 50 or more; 100 or more; 1,000 or more; 2,000 or more; 5,000 or more; 8,000 or more; 10,000 or more; 15,000 or more; or 20,000 or more pathogen nucleic acids. In some cases, the method described herein further comprises extracting or purifying the nucleic acid sample and the nucleic acids from the two or more synthetic nucleic acids. In some cases, extracting or purifying the nucleic acid sample and the nucleic acids from the two or more synthetic nucleic acids alters the relative concentration of the nucleic acid sample or the nucleic acids from the two or more synthetic nucleic acids. In some cases, the original measurement value is a read count.

[0046] This document provides a method for detecting nucleic acids from a pathogen, the method comprising: (a) obtaining a first nucleic acid sample containing nucleic acid of a first pathogen, wherein the first nucleic acid sample is obtained from a first subject infected with or suspected of being infected with the first pathogen; (b) obtaining a second nucleic acid sample containing nucleic acid of a second pathogen, wherein the second nucleic acid sample is obtained from a second subject infected with or suspected of being infected with the second pathogen; (c) obtaining first and second sample markers, each marker containing a different synthetic nucleic acid that cannot hybridize with the pathogen nucleic acid, and assigning the first sample marker to the first nucleic acid sample and the second sample marker to the second nucleic acid sample; (d) adding the first sample marker to the first nucleic acid sample and adding the second sample marker to the second nucleic acid sample; (e) performing sequencing analysis on the first nucleic acid sample containing the first sample marker and the second nucleic acid sample containing the second sample marker to obtain sequence results of the first and second samples; (f) detecting the presence of the first sample marker, the second sample marker, and the pathogen nucleic acid in the sequence results; and (g) determining that the target nucleic acid was initially present in the first sample when the sequencing analysis detected the first sample marker and the target nucleic acid but not the second sample marker.

[0047] In some cases, the synthetic nucleic acid is up to about 500 base pairs in length. In some cases, the synthetic nucleic acid is up to about 100 base pairs in length. In some cases, the synthetic nucleic acid is at least about 50 base pairs in length. In some cases, the synthetic nucleic acid is at least about 100 base pairs in length. In some cases, the synthetic nucleic acid comprises DNA or modified DNA. In some cases, the synthetic nucleic acid comprises RNA or modified RNA. In some cases, the synthetic nucleic acid is a modified nucleic acid. In some cases, the synthetic nucleic acid comprises a sequence selected from SEQ ID NO:111-SEQ ID NO:110 and any combination thereof. In some cases, the first sample comprises cell-free body fluid.

[0048] This document provides a method for detecting a reagent in a sample, the method comprising: adding a first synthetic nucleic acid to the reagent, wherein the first synthetic nucleic acid contains a unique sequence; adding a reagent containing the first synthetic nucleic acid to the nucleic acid sample; preparing the nucleic acid sample for sequencing analysis; performing sequencing analysis on the nucleic acid sample to obtain a sequence result of the nucleic acid sample; and detecting the reagent in the sample by determining the presence of the first synthetic nucleic acid in the sample based on the sequence result of the nucleic acid sample.

[0049] In some cases, adding the first synthetic nucleic acid to the reagent in step a includes adding the first synthetic nucleic acid to the specific batch of the reagent. In some cases, the method herein further includes detecting the specific batch of the reagent based on the sequence results of the nucleic acid sample. In some cases, the first synthetic nucleic acid does not hybridize with nucleic acids from pathogens. In some cases, the method herein further includes adding a second synthetic nucleic acid to a different batch of the reagent, wherein the second synthetic nucleic acid uniquely identifies the different batch of the reagent. In some cases, the method herein further includes detecting the target nucleic acid based on the results of sequencing analysis from the nucleic acid sample. In some cases, the method herein further includes: (i) using the specific batch of the reagent in a future sequencing analysis if the target nucleic acid is accurately detected; or (ii) not using the specific batch of the reagent in a future sequencing analysis if the target nucleic acid is not accurately detected. In some cases, the reagent comprises an aqueous solution. In some cases, the synthetic nucleic acid is about 50 to about 500 base pairs in length. In some cases, the synthetic nucleic acid comprises DNA or modified DNA. In some cases, the synthetic nucleic acid comprises RNA or modified RNA. In some cases, the synthetic nucleic acid is selected from SEQ ID NO:111-SEQ ID NO:110 and any combination thereof. In some cases, the synthetic nucleic acid cannot be degraded by DNases.

[0050] This article provides a method for determining the loss or abundance of nucleic acid diversity in a sample, the method comprising: adding 1,000 unique synthetic nucleic acids at a known concentration to a sample containing a target nucleic acid; performing sequencing analysis on the sample to obtain sequence read counts of the target nucleic acid and at least a portion of the 1,000 unique synthetic nucleic acids; detecting the loss of diversity of the 1,000 unique synthetic nucleic acids by aligning the sequence read counts of the at least a portion of the 1,000 unique synthetic nucleic acids to the sequences of the 1,000 unique nucleic acids added to the sample containing the target nucleic acid in step a, and comparing the diversity of the aligned sequence reads with the diversity of the 1,000 or more unique synthetic nucleic acids; and using the loss of diversity of the 1,000 unique synthetic nucleic acids to calculate the loss or abundance of the target nucleic acid in the sample.

[0051] In some cases, the 1,000 unique synthetic nucleic acids are at most about 500 base pairs in length or at most about 100 base pairs in length. In some cases, the 1,000 unique synthetic nucleic acids are added at an equimolar concentration. In some cases, the 1,000 unique synthetic nucleic acids have a content of at least about 1 × 10⁻⁶. 6 The diversity. In some cases, the 1,000 unique synthetic nucleic acids have at least about 1 × 10⁻⁶.7 The diversity. In some cases, the 1,000 unique synthetic nucleic acids have at least about 1 × 10⁻⁶. 8 The diversity of the 1,000 unique synthetic nucleic acids is as follows: In some cases, the 1,000 unique synthetic nucleic acids have a random portion. In some cases, the 1,000 unique synthetic nucleic acids comprise DNA, modified DNA, RNA, or modified RNA. In some cases, the 1,000 unique synthetic nucleic acids comprise the sequences identified in SEQ ID NO:119 and SEQ ID NO:120. In some cases, the 1,000 unique synthetic nucleic acids are added to the sample in a first sample processing step. In some cases, the method herein further comprises adding an additional pool of 1,000 unique synthetic nucleic acids to the sample in a second sample processing step, wherein the second sample processing step is different from the first sample processing step. In some cases, the diversity loss of the additional pool of 1,000 unique synthetic nucleic acids is calculated. In some cases, the method herein further comprises identifying sample processing steps with relatively high diversity loss by comparing the diversity loss of the 1,000 unique synthetic nucleic acids with the diversity loss of the additional pool of 1,000 unique synthetic nucleic acids. In some cases, the 1,000 unique synthetic nucleic acids include a domain that identifies the synthetic nucleic acid as a member of the additional pool of 1,000 unique synthetic nucleic acids. In some cases, the additional pool of 1,000 unique synthetic nucleic acids includes a domain that identifies the synthetic nucleic acid as a member of the additional pool of 1,000 unique synthetic nucleic acids. In some cases, the 1,000 unique synthetic nucleic acids are added to the sample prior to the extraction of the target nucleic acid. In some cases, the 1,000 unique synthetic nucleic acids are added to the sample prior to the preparation of a library of the target nucleic acid. In some cases, the method described herein further includes adding 5,000 unique synthetic nucleic acids at a known concentration to the sample containing the target nucleic acid.

[0052] This document further discloses methods and compositions for analyzing molecules. In one aspect, this document discloses a method for generating a sequencing library, the method comprising: a) obtaining a sample comprising: (i) a target nucleic acid; (ii) a sequencing adaptor; and (iii) at least one synthetic nucleic acid, wherein the at least one synthetic nucleic acid comprises DNA and is resistant to ligation with nucleic acids; and b) performing a ligation reaction on the sample such that the sequencing adaptor preferentially ligates to the target nucleic acid relative to the at least one synthetic nucleic acid.

[0053] In some cases, the at least one synthetic nucleic acid resists linkage to the nucleic acid via a phosphodiester bond. In some cases, the at least one synthetic nucleic acid resists linkage to the sequencing adaptor. In another aspect, this document discloses a method for generating a sequencing library, the method comprising: a) obtaining a sample containing a target nucleic acid and at least one synthetic nucleic acid; b) removing the at least one synthetic nucleic acid from the sample to obtain a sequencing sample containing the target nucleic acid but not the at least one synthetic nucleic acid; and c) attaching a sequencing adaptor to the target nucleic acid within the sequencing sample. In some cases, the removal of the at least one synthetic nucleic acid is not performed by endonuclease digestion. In some cases, the at least one synthetic nucleic acid removed from the sample does not attach to another synthetic nucleic acid. In some cases, the at least one synthetic nucleic acid resists end repair.

[0054] In another aspect, this document discloses a method for generating a sequencing library, the method comprising: a) obtaining a sample containing a target nucleic acid and at least one synthetic nucleic acid; b) attaching a sequencing adaptor to the target nucleic acid within the sample to obtain a sequencing sample; c) removing at least one synthetic nucleic acid from the sequencing sample by affinity-based consumption, RNA-directed DNase digestion, or a combination thereof, wherein removing the at least one synthetic nucleic acid from the sequencing sample comprises preferentially removing the at least one synthetic nucleic acid relative to the sequencing adaptor and relative to a multimer of the sequencing adaptor.

[0055] In some cases, the method further includes removing the at least one synthetic nucleic acid by endonuclease digestion, size-based consumption, or a combination thereof. In some cases, the sequencing adaptor is a nucleic acid. In some cases, the at least one synthetic acid is removed by affinity-based consumption, and the at least one synthetic nucleic acid contains an immobilized tag. In some cases, the at least one synthetic nucleic acid is removed by RNA-directed DNase digestion. In some cases, the RNA-directed DNase contains a CRISPR-related protein. In some cases, the at least one synthetic nucleic acid is removed by endonuclease digestion. In some cases, the at least one synthetic acid is removed by size-based consumption, and the length of the at least one synthetic nucleic acid is greater than the length of the target nucleic acid. In some cases, the at least one synthetic acid is removed using an RNase, and the at least one synthetic nucleic acid is a DNA-RNA hybrid. In some cases, attaching the sequencing adaptor to the target nucleic acid includes attaching the sequencing adaptor to the target nucleic acid. In some cases, attaching the sequencing adaptor to the target nucleic acid includes attaching the sequencing adaptor to the target nucleic acid.

[0056] In another aspect, this document discloses a method for generating a sequencing library, the method comprising: a) obtaining a sample containing a target nucleic acid and at least one synthetic nucleic acid, wherein the at least one synthetic nucleic acid comprises: (i) single-stranded DNA; (ii) a nucleotide modification that inhibits the amplification of the synthetic nucleic acid; (iii) an immobilized tag; (iv) a DNA-RNA hybrid; (v) a nucleic acid of a length greater than that of the target nucleic acid; or (vi) any combination thereof; and b) generating a sequencing library from the sample for a sequencing reaction, wherein at least a portion of the synthetic nucleic acid is not sequenced in the sequencing reaction.

[0057] In some cases, the at least one synthetic nucleic acid further comprises an endonuclease recognition site. In some cases, obtaining the sample includes extracting the target nucleic acid from a test sample, and further includes adding the at least one synthetic nucleic acid to the test sample after extracting the target nucleic acid from the test sample. In some cases, obtaining the sample includes extracting the target nucleic acid from a test sample, and further includes adding the at least one synthetic nucleic acid to the test sample before extracting the target nucleic acid from the test sample. In some cases, the at least one synthetic nucleic acid comprises a blocking group that inhibits the ligation reaction, and the blocking group comprises a modified nucleotide. In some cases, the modified nucleotide comprises a reverse deoxyglucose. In some cases, the reverse deoxyglucose comprises a 3' reverse deoxyglucose. In some cases, the modified nucleotide comprises reverse thymidine, reverse adenosine, reverse guanosine, or reverse cytidine. In some cases, the modified nucleotide comprises a reverse dideoxyglucose. In some cases, the reverse dideoxyglucose comprises a 5' reverse dideoxyglucose. In some cases, the modified nucleotide comprises reverse dideoxythymidine, reverse dideoxyadenosine, reverse dideoxyguanosine, or reverse dideoxycytidine. In some cases, the modified nucleotide is dideoxycytidine. In some cases, the at least one synthetic nucleic acid comprises a blocking group that inhibits the ligation reaction, and the blocking group comprises a spacer region. In some cases, the spacer region comprises a C3 spacer region or a spacer region 18. In some cases, the at least one synthetic nucleic acid comprises a blocking group that inhibits the ligation reaction, and the blocking group comprises a hairpin structure. In some cases, the synthetic nucleic acid comprises a nucleotide modification that inhibits the amplification of the at least one synthetic nucleic acid, and the nucleotide modification comprises at least one abase-free site. In some cases, the at least one abase-free site is at least one internal abase-free site. In some cases, the nucleotide modification comprises 8 to 10 abase-free sites. In some cases, the at least one abase-free site is a single abase-free site. In some cases, the at least one abase-free site is on the modified ribose. In some cases, the at least one base-free site comprises 1',2'-dideoxyribose, locked nucleic acid, bridging nucleic acid, or twisted insert nucleic acid. In some cases, the at least one synthetic nucleic acid comprises an immobilized tag, and the immobilized tag comprises biotin, digitoxin, polyhistidine, or Ni-nitrotriacetic acid. In some cases, the at least one synthetic nucleic acid comprises DNA and is internally tagged with uracil. In some cases, the at least one synthetic nucleic acid is removed from the sequencing sample using a uracil-specific excision enzyme.

[0058] In some cases, the test sample is a biological sample. In some cases, the biological sample is whole blood, plasma, serum, or urine. In some embodiments, the target nucleic acid is a cell-free nucleic acid. In some cases, the cell-free nucleic acid is cell-free DNA. In some cases, the cell-free nucleic acid is pathogen nucleic acid. In some cases, the cell-free nucleic acid is circulating cell-free nucleic acid. In some cases, the at least one synthetic nucleic acid comprises a double-stranded nucleic acid. In some cases, the at least one synthetic nucleic acid comprises a single-stranded nucleic acid. In some cases, the at least one synthetic nucleic acid comprises DNA, RNA, a DNA-RNA hybrid, or any analogue thereof.

[0059] In some cases, the method further includes one or more of the following: (a) extracting the target nucleic acid from the sample; (b) purifying the target nucleic acid from the sample; (c) end-repairing the target nucleic acid; (d) fragmenting the target nucleic acid; (e) amplifying the target nucleic acid; (f) attaching a sequencing adaptor to the target nucleic acid; and (g) sequencing the target nucleic acid. In some cases, the method includes attaching the sequencing adaptor to the target nucleic acid, and further includes treating the sequencing sample with an endonuclease before attaching the sequencing adaptor to the target nucleic acid. In some cases, the method includes attaching the sequencing adaptor to the target nucleic acid, and further includes treating the sequencing sample with an endonuclease after attaching the sequencing adaptor to the target nucleic acid. In some cases, the method includes end-repairing the target nucleic acid, wherein the at least one synthetic nucleic acid is added to the sample before end-repairing the target nucleic acid. In some cases, the method includes end-repairing the target nucleic acid, wherein the at least one synthetic nucleic acid is added to the sample after end-repairing the target nucleic acid. In some cases, the method includes attaching the sequencing adaptor to the target nucleic acid and adding the at least one synthetic nucleic acid to the sample prior to attaching the sequencing adaptor to the target nucleic acid. In some cases, the ratio of the concentration of the at least one synthetic nucleic acid in the sample to the concentration of the target nucleic acid in the sample is from 1:1 to 1000:1.

[0060] In some cases, the difference between the size of the at least one synthetic nucleic acid and the size of the target nucleic acid allows for size-based separation of the at least one synthetic nucleic acid from the target nucleic acid. In some cases, the synthetic nucleic acid contains a blocking group that inhibits the ligation reaction and a nucleotide modification that inhibits the amplification reaction. In some cases, the blocking group that inhibits the ligation reaction contains a 3' reverse deoxy-T, and the nucleotide modification that inhibits the amplification reaction contains an internal base-free site. In some cases, the blocking group further contains a 5' reverse dideoxy-T. In some cases, the method further includes incubating the sample with endonuclease VIII. In some cases, the sample is incubated with endonuclease VIII for no more than 1 hour. In some cases, the method includes extracting the target nucleic acid from the sample, and the extraction of the target nucleic acid has a higher yield compared to extraction from a sample that does not contain the at least one synthetic nucleic acid. In some cases, the method includes end repair of the target nucleic acid, and the end repair of the target nucleic acid has a higher efficiency compared to end repair of the target nucleic acid in a sample that does not contain the at least one synthetic nucleic acid. In some cases, the target nucleic acid contains a naturally occurring nucleic acid or a copy thereof. In some cases, the method further includes using a computer to obtain sequence information of at least one of the target nucleic acids.

[0061] In another aspect, this document discloses a method for generating sequencing libraries, the method comprising: (a) obtaining a sample comprising: (i) a target nucleic acid; (ii) a sequencing adaptor; and (iii) at least one synthetic nucleic acid, wherein the at least one synthetic nucleic acid comprises DNA and is resistant to end repair; and (b) performing an end repair reaction on the sample such that the target nucleic acid is preferentially end-repaired relative to the at least one synthetic nucleic acid.

[0062] In some implementations, any of the foregoing methods may include reporting the results of the methods to the patient, caregiver, or other person.

[0063] In another aspect, this document discloses a kit for generating sequencing libraries, the kit comprising: a) a sequencing adaptor; and b) at least one synthetic nucleic acid, wherein the at least one synthetic nucleic acid comprises DNA and is resistant to nucleic acid end repair. In some cases, the ratio of the amount of the at least one synthetic nucleic acid to the amount of the sequencing adaptor is not greater than 1:1. Attached Figure Description

[0064] The novel features of the disclosed subject matter are set forth in the appended claims. A better understanding of the features and advantages of the disclosed subject matter will be gained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of the disclosed subject matter; in these drawings:

[0065] Figure 1 A schematic diagram illustrating the basic method of this disclosure is shown.

[0066] Figure 2 A schematic diagram of an exemplary infection is shown.

[0067] Figure 3 The general schemes of some of the methods presented in this paper are shown.

[0068] Figure 4 Eight exemplary Spark large group incorporation designs are shown.

[0069] Figure 5 This paper presents a general scheme for determining diversity loss.

[0070] Figure 6 An exemplary design incorporating Spank is shown.

[0071] Figure 7 A computer control system is shown, which is programmed or otherwise configured to implement the methods provided herein.

[0072] Figure 8 The steps in preparing a sequencing library are shown, in which vector nucleic acids may be added.

[0073] Figure 9 The normalized read counts for 110 exemplary IDs Spike are shown.

[0074] Figure 10 The normalized read count of an exemplary ID Spike with 110 signal normalizations is shown.

[0075] Figure 11 Results are shown from a method used to identify cross-contamination of a positive control for Shigella flexneri using pathogen tracers.

[0076] Figure 12 Results are shown from a method used to identify cross-contamination of positive controls for Salmonella enterica using pathogen tracers.

[0077] Figure 13 Results are shown from a method used to identify cross-contamination in a Staphylococcus aureus positive control using pathogen tracers.

[0078] Figure 14Results are shown from a method used to identify cross-contamination in a positive control of Pseudomonas aeruginosa using pathogen tracers.

[0079] Figure 15 Results are shown from a method used to identify cross-contamination of a positive control for Clostridium sporogenes using pathogen tracers.

[0080] Figure 16 The effect of size-selective library processing methods on the relative yield of exemplary Spark size incorporation is shown.

[0081] Figure 17 Results are shown from a method for preparing sequencing libraries containing an enzyme heating-killing step using nucleic acids with different GC contents.

[0082] Figure 18 Results are shown from a method for preparing sequencing libraries lacking an enzyme heat-kill step using nucleic acids with different GC contents.

[0083] Figure 19 An exemplary method for preparing sequencing libraries using a vector synthesizing nucleic acid has a large size to prevent ligation at both ends.

[0084] Figure 20A An exemplary method for preparing sequencing libraries using a vector to synthesize nucleic acids having base-free sites and modifications is shown.

[0085] Figure 20B Results from sequencing libraries generated using vector-synthesized nucleic acids that have not undergone endonuclease VIII digestion are shown. Lane A1: TapeStation ladder. Lane B1: No CNA, 1st replication. Lane C1: No CNA, 2nd replication. Lane D1: No CNA, 3rd replication. Lane E1: 10 ng CNA, 1st replication. Lane F1: 10 ng CNA, 2nd replication. Lane G1: 10 ng CNA, 3rd replication.

[0086] Figure 20CResults from sequencing libraries generated using vector-synthesized nucleic acids digested with reninase VIII are shown. Lane A1: TapeStation ladder band. Lane B1: No CNA, 1st replication. Lane C1: No CNA, 2nd replication. Lane D1: No CNA, 3rd replication. Lane E1: 10 ng CNA, 1st replication. Lane F1: 10 ng CNA, 2nd replication. Lane G1: 10 ng CNA, 3rd replication.

[0087] Figure 21A The results of using vector-synthesized nucleic acids to prepare sequencing libraries are shown. The vector-synthesized nucleic acids have base-free sites that are not digested by endonuclease VIII.

[0088] Figure 21B The results of preparing sequencing libraries using vector-synthesized nucleic acids having base-free sites digested by endonuclease VIII are shown.

[0089] Figure 22 An exemplary sequence of nucleic acid synthesized by a vector having a DNA-RNA hybrid is shown. The letter "rX" (e.g., rG, rC, rA) represents an RNA sequence.

[0090] Figure 23A An exemplary method for preparing sequencing libraries using a vector to synthesize nucleic acids having a DNA-RNA hybrid is shown.

[0091] Figure 23B The results are shown from sequencing libraries generated using vector-synthesized nucleic acids that have DNA-RNA hybrids.

[0092] Incorporation

[0093] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent or patent application were specifically and individually incorporated by reference. Detailed Implementation

[0094] Overview

[0095] This disclosure provides a variety of methods and approaches for improved identification or quantification of nucleic acids in next-generation sequencing analyses and other assays. Typically, the methods described herein involve the use of incorporated synthetic nucleic acids with specific characteristics such as a particular sequence, length, GC content, degeneracy, diversity level, and / or known initial concentration. Using such incorporated synthetic nucleic acids enables and improves absolute abundance assays, relative abundance assays, abundance normalization, universal quantification, bias control, sample identification, cross-contamination detection, information transfer efficiency, reagent traceability, diversity loss normalization, absolute or relative loss assays, quality control, and many other applications. The incorporated synthetic nucleic acids provided herein also include specially designed vector nucleic acids that increase the total concentration of nucleic acids in a sample but have the ability to evade detection by sequencing or other assays.

[0096] In a preferred embodiment, this disclosure provides a group of incorporated synthetic nucleic acid species, wherein the length and / or GC content of each species is designed to match or closely approximate the expected or observable length and / or GC content of the target nucleic acid group to be analyzed. For example, the length of the incorporated synthetic nucleic acid may be close to the length of disease-specific or pathogen-specific cell-free nucleic acids in samples (e.g., plasma) obtained from human patients infected with pathogens. In other preferred embodiments, this disclosure provides incorporated synthetic nucleic acids containing sequences that uniquely identify a sample, reagent, or batch of reagent. In other preferred embodiments, this disclosure provides pools containing a large number of incorporated synthetic nucleic acids having unique sequences (e.g., 10) that can be used to track absolute nucleic acid loss in a sample by reducing the diversity of unique incorporated sequences during high-throughput sequencing analysis, particularly during sample processing steps such as nucleic acid extraction and / or library preparation. 4 10 5 10 6 10 7 10 8 10 9 Or 10 10 A unique incorporated synthetic nucleic acid).

[0097] The ability to track absolute nucleic acid loss allows for the determination of the absolute abundance of target nucleic acids in an initial sample. For example, the absolute amount of a pathogen in a clinical sample can be determined based on the number of sequencing reads attributed to the pathogen. Drug treatment can be monitored or adjusted, such as before, during, and after treatment with antibiotics or drug combinations, by determining the absolute abundance of pathogens in clinical samples collected over time. In addition to determining the presence of a specific pathogen, the extent or stage of infection or disease can also be determined.

[0098] The method may include adding synthetic nucleic acids to a variety of different samples, including but not limited to clinical samples, processed samples (e.g., extracted nucleic acids, extracted cell-free DNA, extracted cell-free RNA, plasma, serum), unprocessed samples (e.g., whole blood), and any other type of sample, particularly samples containing nucleic acids. In some cases, the method may involve adding synthetic nucleic acids to reagents, particularly laboratory reagents (or specific reagent batches) used at any stage of sample analysis by sequencing (e.g., next-generation sequencing). In a preferred embodiment, the method may include introducing synthetic nucleic acids of known concentration into reagents and samples. The method may particularly help improve the accuracy and efficiency of assays designed to detect, identify, monitor, or quantify low-abundance pathogens or pathogen-derived nucleic acids in clinical samples. The method may also reduce undesirable results associated with errors in sample tracking; undesirable results caused by unequal loss of nucleic acid sequences during sample preparation, nucleic acid purification, or sequencing library preparation; or undesirable results caused by the lack of internal normalization criteria when comparing analyses of different target nucleic acids or different samples.

[0099] Figure 1 A general overview of the steps of many of the methods provided herein is given, particularly where they involve abundance normalization. The methods may include obtaining samples from a subject 110, such as a human patient. In some specific embodiments, the subject has an infectious disease or is otherwise suspected of being infected with a pathogen. As shown, the sample may be a blood sample 120 or a plasma sample 130, or any other type of biological sample, especially biological samples containing body fluids, tissues, and / or cells, or cell-free biological samples.

[0100] Nucleic acids (e.g., cell-free nucleic acids) can be extracted from sample 140 and used for assays such as sequencing analysis (e.g., next-generation sequencing analysis). One or more types of synthetic nucleic acids 150 can be added (or incorporated) to, for example, blood sample 120, plasma sample 130, or sample nucleic acid 140 in one or more steps of the method. This synthetic nucleic acid may have a length designed to approximate the length of the target nucleic acid set to be analyzed and / or a GC content designed to approximate the GC content of the target nucleic acid set to be analyzed. Typically, the synthetic nucleic acid also has a known starting concentration. The sample containing the synthetic nucleic acid can then be analyzed by sequencing analysis 160, such as next-generation sequencing analysis. In some cases, the amount of synthetic nucleic acid identified by sequencing analysis is compared to the known starting concentration of the synthetic nucleic acid so that the read count is correlated with the known starting concentration. Thus, target nucleic acids within sample nucleic acids can be identified or quantified, particularly by comparing the abundance of the detected target nucleic acid with the abundance of synthetic nucleic acids whose length and / or GC content are closest to that of such target nucleic acids 170. By using such methods and other methods provided herein, the condition of a subject can be identified with a higher level of accuracy and certainty. In some specific implementations, sequencing analysis (e.g., next-generation sequencing analysis) detects pathogen nucleic acids in cell-free nucleic acid (e.g., DNA) samples derived from human patients.

[0101] The steps can be performed in any order and in any combination. In some cases, certain steps are repeated several times. In some cases, certain steps are not performed. In some cases, new steps are added to the described steps or scattered between the described steps.

[0102] Figure 2 A schematic diagram of an exemplary infection is provided. The source of pathogen infection can be, for example, the lungs. Cell-free nucleic acids derived from the pathogen, such as cell-free DNA, can cross the bloodstream and be collected in plasma samples for analysis. This can then be achieved through methods such as... Figure 1 The sequencing analysis shown is used to analyze nucleic acids in the sample.

[0103] Figure 3The general scheme of some of the methods provided herein is described. The methods may include obtaining samples containing host (e.g., human) and non-host (e.g., pathogen) nucleic acids. Samples may be obtained from subjects such as patients. In some specific embodiments, the subject has an infectious disease or is otherwise suspected of being infected with a pathogen. Samples may be blood or plasma samples, or any other type of biological sample, especially biological samples containing body fluids, tissues, and / or cells. Nucleic acids from the sample (e.g., cell-free nucleic acids) may be combined with a known amount of synthetic nucleic acid. The sample containing the synthetic nucleic acid can then be analyzed by sequencing analysis such as next-generation sequencing analysis. Sequencing results may be mapped to known host and non-host reference sequences. In some cases, the amount of synthetic nucleic acid identified by sequencing analysis is compared with a known starting concentration of the synthetic nucleic acid to correlate read counts with the known starting concentration. Thus, the relative abundance of non-host sequences can be determined. The steps may be performed in any order and in any combination. In some cases, certain steps are repeated several times. In some cases, certain steps are not performed. In some cases, new steps are added to the described steps or scattered between the described steps.

[0104] The method presented in this paper enables improved identification or quantification of target nucleic acids via next-generation sequencing, particularly when the target nucleic acid is present in low abundance in a sample or when comparing or tracking multiple samples or multiple target nucleic acids. For example, accurate detection and quantification of target pathogens, tumor cells, or cancer genomic markers in clinical samples via next-generation sequencing can be compromised or negatively impacted if samples are not tracked correctly or if target nucleic acids are not accurately normalized or quantified. Therefore, the method presented in this paper helps avoid defects caused by errors in sample tracking or identification, nucleic acid quantification, or other cloud analysis of sequencing data.

[0105] The methods and compositions provided herein can be used to add and / or remove synthetic nucleic acids during sequencing library preparation to improve the yield, quality, or efficiency of the sequencing library, particularly when the starting sample has relatively low levels of nucleic acids. Typically, in some cases, synthetic nucleic acids can act as carrier nucleic acids in these applications to increase the total nucleic acid concentration during sample preparation. Adding synthetic nucleic acids to a sample can improve the yield and / or efficiency of one or more steps in sequencing library generation. These steps can be sensitive to nucleic acid concentration. For example, the yield and / or efficiency of these steps may depend on the nucleic acid concentration in the sample. Such steps may include nucleic acid extraction, purification, ligation, and end repair. In some cases, synthetic nucleic acids can be removed from the sequencing library. Synthetic nucleic acids may contain certain features that prevent them from participating in one or more steps in sequencing library generation. Therefore, synthetic nucleic acids may not be sequenced in the sequencing steps.

[0106] The methods and compositions described herein can be used to analyze samples from multiple subjects (e.g., to generate sequencing libraries from target nucleic acids in the samples). The concentration of target nucleic acids in these samples may vary among subjects. Adding the synthetic nucleic acids described herein to these samples reduces concentration variations between samples, thereby improving the accuracy of the analysis.

[0107] The methods and compositions described herein can be used to prepare sequencing libraries from a sample by adding at least one synthetic nucleic acid. The synthetic nucleic acids may have one or more properties that prevent them from being sequenced in the sequencing reaction. In some cases, the synthetic nucleic acids contain modifications that inhibit one or more reactions in the generation of the sequencing library, such as adaptor ligation and nucleic acid amplification. For example, the nucleic acid may contain inverse sugars at one or both ends, and / or one or more abase sites.

[0108] In some cases, synthetic nucleic acids can be removed from the sequencing library prior to sequencing. In some cases, synthetic nucleic acids can be removed by enzymatic digestion. For example, synthetic nucleic acids may contain restriction enzyme recognition sites and can be degraded by restriction enzymes. In some cases, synthetic nucleic acids can be removed by affinity-based consumption. For example, synthetic nucleic acids may contain one or more immobilized tags and can be removed by affinity-based consumption. In some cases, synthetic nucleic acids can be removed by size-based consumption. Synthetic nucleic acids may also have a different size than other molecules in the sequencing library, allowing for size-based removal. In some cases, synthetic nucleic acids may contain a combination of properties and / or modifications described herein that prevent them from participating in one or more steps of sequence library generation and can also be removed prior to sequencing.

[0109] sample

[0110] The methods presented in this paper enable improved analysis of a wide range of samples. The synthetic nucleic acids described herein can be used to analyze such samples, which may involve adding synthetic nucleic acids directly to the sample or to a processed form, such as cell-free nucleic acids extracted from clinical plasma samples.

[0111] The samples analyzed in the methods provided herein are preferably any type of clinical sample. In some cases, the sample contains cells, tissues, or body fluids. In a preferred embodiment, the sample is a liquid or fluid sample. In some cases, the sample contains body fluids such as whole blood, plasma, serum, urine, feces, saliva, lymph, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage fluid, nasal swabs, respiratory secretions, vaginal fluid, amniotic fluid, semen, or menstrual blood. In some cases, the sample consists entirely or partially of cells or tissues. In some cases, cells, cell debris, or foreign bodies are removed from the sample, such as by centrifugation or filtration. The samples described herein may be biological samples.

[0112] Samples may contain any concentration of nucleic acids. The compositions and methods described herein can be used for samples with low concentrations of total nucleic acids. In some cases, samples may have a total nucleic acid concentration of up to 100 ng / μL, 50 ng / μL, 10 ng / μL, 5 ng / μL, 2 ng / μL, 1.5 ng / μL, 1.2 ng / μL, 1 ng / μL, 0.8 ng / μL, 0.4 ng / μL, 0.2 ng / μL, 0.1 ng / μL, 0.05 ng / μL, 0.01 ng / μL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, 0.8 ng / mL, 0.6 ng / mL, 0.5 ng / mL, or 0.1 ng / mL. In some cases, the sample contains a total nucleic acid concentration of at least 0.1 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 0.01 ng / μL, 0.05 ng / μL, 0.1 ng / μL, 0.2 ng / μL, 0.4 ng / μL, 0.8 ng / μL, 1 ng / μL, 1.2 ng / μL, 1.5 ng / μL, 2 ng / μL, 5 ng / μL, 10 ng / μL, 50 ng / μL, or 100 ng / μL. In some cases, the sample has a total nucleic acid concentration in the range of about 0.1 ng / mL to about 10,000 ng / mL (i.e., about 0.1 ng / mL to about 10 ng / μL).

[0113] Samples may include one or more controls. In some cases, samples include one or more negative controls. Exemplary negative controls include samples prepared for identifying contaminants (e.g., samples minus plasma), plasma from healthy subjects, and low-diversity samples (e.g., samples collected from seemingly healthy subjects). In some cases, samples include one or more positive controls. Exemplary positive controls include samples from healthy subjects (e.g., plasma samples) containing genomic DNA from a known pathogen. The genomic DNA from the known pathogen may be intact genomic DNA. In some cases, the genomic DNA from the known pathogen may be cut, for example, cut to various average lengths. Cutting may be performed by mechanical shearing (e.g., ultrasound, hydrodynamic shearing forces), enzymatic shearing (e.g., endonucleases), thermal fragmentation (e.g., incubation at high temperatures), or chemical fragmentation (e.g., alkaline solutions, divalent ions).

[0114] The sample may contain a target nucleic acid. A target nucleic acid can refer to the nucleic acid to be analyzed in the sample. For example, the target nucleic acid may be originally present in the sample, such as a natural nucleic acid. The sample may further contain one or more synthetic nucleic acids disclosed herein. In some cases, the target nucleic acid is a cell-free nucleic acid as described herein. For example, the target nucleic acid may be cell-free DNA, cell-free RNA (e.g., cell-free mRNA, cell-free miRNA, cell-free siRNA), or any combination thereof. In some cases, the cell-free nucleic acid is a pathogen nucleic acid, such as nucleic acid derived from a pathogen. Cell-free nucleic acid may be a circulating nucleic acid, such as circulating tumor DNA or circulating fetal DNA. The sample may contain nucleic acids derived from pathogens such as viruses, bacteria, fungi, and / or eukaryotic parasites.

[0115] In some cases, the sample also contains an adaptor. The adaptor can be a nucleic acid with a known or unknown sequence. The adaptor can attach to the 3' end, 5' end, or both ends of the nucleic acid. The adaptor can contain known and / or unknown sequences. The adaptor can be double-stranded or single-stranded. In some cases, the adaptor is a sequencing adaptor. A sequencing adaptor attaches to the target nucleic acid and aids in the sequencing of the target nucleic acid. For example, a sequencing adaptor may contain one or more of the following: a sequencing primer binding site, a unique marker sequence, a non-unique marker sequence, and a sequence for immobilizing the target nucleic acid on a solid support. The target nucleic acid attached to the sequencing adaptor can be immobilized on a solid support on a sequencer. Sequencing primers can hybridize with the adaptor and use the target nucleic acid as a template for extension in the sequencing reaction. In some cases, markers in the adaptor are used to label sequence reads of different target sequences, thereby allowing high-throughput sequencing of multiple target nucleic acids.

[0116] The term "attachment" and its grammatical equivalents can refer to joining two molecules using any attachment pattern. For example, attachment can refer to joining two molecules by chemical bonds or other methods to create a new molecule. Attaching an adaptor to a nucleic acid can refer to forming a chemical bond between the adaptor and the nucleic acid. In some cases, attachment is performed via ligation, such as using ligases. For example, a nucleic acid adaptor can be attached to a target nucleic acid via ligation through the formation of a phosphodiester bond catalyzed by a ligase.

[0117] Sequencing libraries can be generated from samples using the methods and compositions provided herein. Sequencing libraries may contain a variety of nucleic acids that are compatible with the sequencing system to be used. For example, the nucleic acids in a sequencing library may contain target nucleic acids attached to one or more adaptors. The steps for preparing a sequencing library may include one or more of the following: extracting target nucleic acids from a sample, fragmenting the target nucleic acids, attaching adaptors to the target nucleic acids, amplifying the target nucleic acid-adaptor complex, and sequencing the amplified target nucleic acid-adaptor complex.

[0118] Samples (particularly cell samples or tissue biopsies) can come from any part or region of the body. Exemplary samples may be obtained from, for example, blood, the central nervous system, brain, spinal cord, bone marrow, pancreas, thyroid gland, gallbladder, liver, heart, spleen, colon, rectum, lungs, respiratory system, larynx, nasal cavity, stomach, esophagus, ear, eye, skin, limbs, uterus, prostate, reproductive organs, or any other organ or region of the body.

[0119] Typically, samples are taken from human subjects, particularly human patients. However, samples can also be taken from any other type of subject, including any mammal, non-human mammal, non-human primate, domesticated animal (e.g., laboratory animal, domestic pet, or livestock), or undomesticated animal (e.g., wild animal). In some specific implementations, the subject may be a dog, cat, rodent, mouse, hamster, cow, bird, chicken, pig, horse, goat, sheep, rabbit, ape, monkey, or chimpanzee.

[0120] In a preferred embodiment, the subject is a host organism (e.g., a human) that is infected with a pathogen, at risk of infection with a pathogen, or suspected of having a pathogen infection. In some cases, the subject is suspected of having a specific infection, such as suspected tuberculosis. In other cases, the subject is suspected of having an infection of unknown origin. In some cases, the host or subject is infected (e.g., infected by one or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites). In some cases, the host or subject has been diagnosed with one or more cancers or is at risk of developing one or more cancers. In some cases, the host or subject is not infected (e.g., not infected by one or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites). In some cases, the host or subject is healthy. In some cases, the host or subject is susceptible to infection or at risk of infection.

[0121] In some cases, the subject may have been treated or could be treated with antimicrobial, antibacterial, antiviral, or antiparasitic drugs. The subject may have an actual infection (e.g., infection by one or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites). In some cases, the subject is not infected (e.g., not infected by one or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites). In some cases, the subject is healthy. In some cases, the subject is susceptible to infection or at risk of infection (e.g., the patient is immunocompromised). The subject may have another disease or condition or be at risk of developing another disease or condition. For example, the subject may have a disease, be at risk of developing a disease, or be suspected of having a disease such as cancer (e.g., breast cancer, lung cancer, pancreatic cancer, blood cancer, etc.).

[0122] The sample may be a nucleic acid sample; in some cases, the sample contains a certain amount of nucleic acid. Nucleic acids in the sample may include double-stranded (ds) nucleic acids, single-stranded (ss) nucleic acids, DNA, RNA, cDNA, mRNA, cRNA, tRNA, ribosomal RNA, dsDNA, ssDNA, miRNA, siRNA, circulating nucleic acids, circulating cell-free nucleic acids, circulating DNA, circulating RNA, cell-free nucleic acids, cell-free DNA, cell-free RNA, circulating cell-free DNA, cell-free dsDNA, cell-free ssDNA, circulating cell-free RNA, genomic DNA, foreign bodies, cell-free pathogen nucleic acids, circulating pathogen nucleic acids, mitochondrial nucleic acids, non-mitochondrial nucleic acids, nuclear DNA, nuclear RNA, chromosomal DNA, circulating tumor DNA, circulating tumor RNA, circular nucleic acids, circular DNA, circular RNA, circular single-stranded DNA, circular double-stranded DNA, plasmids, or any combination thereof. In some cases, the sample nucleic acids may include synthetic nucleic acids. In some cases, synthetic nucleic acids include any type of nucleic acid disclosed herein, such as DNA, RNA, and DNA-RNA hybrids. For example, synthetic nucleic acids may be DNA.

[0123] In some cases, different types of nucleic acids may be present in a sample. For example, a sample may contain cell-free RNA and cell-free DNA. Similarly, the methods described herein may include methods for analyzing both RNA and DNA present in a sample, either alone or in combination.

[0124] As used herein, the term "cell-free" refers to the state of nucleic acids present in the body before a sample is obtained from the body. For example, circulating cell-free nucleic acids in a sample may originate from cell-free nucleic acids circulating in the human bloodstream. Conversely, nucleic acids extracted from solid tissues such as biopsies are generally not considered "cell-free".

[0125] In some cases, the sample may be an untreated sample (e.g., whole blood) containing cell-free or cell-associated nucleic acids, or a treated sample (e.g., serum, plasma). In some cases, the sample has been enriched for a specific type of nucleic acid, such as DNA, RNA, cell-free DNA, cell-free RNA, cell-free circulating DNA, cell-free circulating RNA, etc. In some cases, the sample has been treated in a manner to isolate nucleic acids or to separate nucleic acids from other components within the sample. In some cases, the sample has been enriched for pathogen-specific nucleic acids.

[0126] Typically, the sample is a fresh sample. In some cases, the sample is a frozen sample. In other cases, the sample is fixed, for example with chemical fixatives, such as formalin fixation or paraffin-embedded tissue.

[0127] target nucleic acid

[0128] The methods provided herein can be used to detect any number of target nucleic acids. Target nucleic acids include, but are not limited to: whole or partial genomes, exomes, gene loci, genes, exons, introns, modified nucleic acids (e.g., methylated nucleic acids), and / or mitochondrial nucleic acids. Typically, the methods provided herein can be used to detect pathogen target nucleic acids; in some cases, pathogen target nucleic acids are present in complex clinical samples containing nucleic acids from subjects. Pathogen target nucleic acids may be associated with infectious diseases such as influenza, tuberculosis, or any other known infectious disease or condition, including those further described herein. In some cases, the target nucleic acids described herein may be target nucleic acids.

[0129] In some cases, the pathogen target nucleic acid is present in tissue samples, such as tissue samples from sites of infection. In other cases, the pathogen target nucleic acid has migrated from the site of infection; for example, it can be obtained from samples containing circulating cell-free nucleic acids (e.g., DNA).

[0130] In some cases, the target nucleic acid is derived from cancerous tissue. Target nucleic acids can be obtained directly from tissue or tumor. In other cases, target cancer nucleic acids are obtained from circulating cell-free nucleic acids or from circulating tumor cells (CTCs).

[0131] In some cases, target nucleic acids may constitute only a very small portion of the entire sample, for example, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, less than 0.0001%, less than 0.00001%, less than 0.000001%, or less than 0.0000001% of the total nucleic acids in the sample. In other cases, target nucleic acids may constitute about 0.00001% to about 0.5% of the total nucleic acids in the sample. Typically, the total nucleic acids in the original sample can vary. For example, the total cell-free nucleic acids (e.g., DNA, mRNA, RNA) may be in the range of 1-100 ng / ml (e.g., about 1, 5, 10, 20, 30, 40, 50, 80, 100 ng / ml). In some cases, the total concentration of cell-free nucleic acids in the sample is outside this range (e.g., less than 1 ng / ml; in other cases, the total concentration is greater than 100 ng / ml). Cell-free nucleic acid (e.g., DNA) samples, primarily composed of human DNA and / or RNA, may be in this state. In such samples, pathogen target nucleic acids or cancer target nucleic acids may be present in very small amounts compared to human nucleic acids or healthy nucleic acids, for example, in the case of samples from subjects undergoing chemotherapy. For instance, pathogen target nucleic acids may constitute less than 0.001% of the total nucleic acids in the sample, and cancer target nucleic acids may constitute less than 1% of the total nucleic acids in the sample.

[0132] The length of the target nucleic acid can vary. In some cases, the length of the target nucleic acid can be approximately or at least approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 10000, 15000, 20000, 25000, or 50000 nucleotides (or base pairs). In some cases, the target nucleic acid can be up to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 10000, 15000, 20000, 25000, or 50000 nucleotides (or base pairs). In some specific embodiments, the target nucleic acid is relatively short, for example, less than 500 base pairs (or nucleotides) or less than 1000 base pairs (or nucleotides). In some cases, the target nucleic acid is relatively long, for example, greater than 1000, 1500, 2000, 2500, 3000, or 5000 base pairs (or nucleotides). In other cases, the target nucleic acid can be in the range of about 20 to about 120 base pairs. In still other cases, the target nucleic acid can be in the range of about 40 to about 100 base pairs.

[0133] Similar to the sample nucleic acid, the target nucleic acid can be any type of nucleic acid, including: double-stranded (ds) nucleic acid, single-stranded (ss) nucleic acid, DNA, RNA, cDNA, mRNA, cRNA, tRNA, ribosomal RNA, dsDNA, ssDNA, miRNA, siRNA, circulating nucleic acid, circulating cell-free nucleic acid, circulating DNA, circulating RNA, cell-free nucleic acid, cell-free DNA, cell-free RNA, circulating cell-free DNA, cell-free dsDNA, cell-free ssDNA, circulating cell-free RNA, genomic DNA, foreign bodies, cell-free pathogen nucleic acid, circulating pathogen nucleic acid, mitochondrial nucleic acid, non-mitochondrial nucleic acid, nuclear DNA, nuclear RNA, chromosomal DNA, circulating tumor DNA, circulating tumor RNA, circular nucleic acid, circular DNA, circular RNA, circular single-stranded DNA, circular double-stranded DNA, plasmids, or any combination thereof. The target nucleic acid is preferably derived from a pathogen, including but not limited to viruses, bacteria, fungi, parasites, and any other microorganisms, especially infectious microorganisms. The target nucleic acid can be derived from a specific organ or tissue. In some cases, the target nucleic acid is directly derived from the subject, rather than from a pathogen.

[0134] Incorporation into synthetic nucleic acids

[0135] This disclosure describes single synthetic nucleic acids and synthetic nucleic acid assemblies for use in a variety of applications, particularly relating to high-throughput or next-generation sequencing analyses. In some cases, when used in the described methods, incorporation of synthetic nucleic acids allows for efficient normalization of nucleic acids (e.g., disease-specific nucleic acids, pathogen nucleic acids) across samples, independent of the individual from which the sample originated, preanalytical sample processing conditions, nucleic acid extraction methods, manipulation of nucleic acids using molecular biology tools and methods, nucleic acid purification methods, the behavior of the measurement itself, storage conditions, and time progression. In some cases, this disclosure provides pools or sets of synthetic nucleic acids with specific characteristics such as a large number of unique sequences. Synthetic nucleic acid assemblies can be used to monitor for diversity loss during sample analysis, which can then be used to determine the abundance of the starting nucleic acid. The synthetic nucleic acids provided herein can also be used for sample tracking, monitoring for cross-contamination between samples, reagent tracking, reagent batch tracking, and many other applications. Typically, the design, length, quantity, concentration, diversity level, and sequence of synthetic nucleic acids can be tailored to a specific application. In some cases, incorporation of synthetic nucleic acids includes vector synthetic nucleic acids (e.g., vector synthetic nucleic acids) as described herein.

[0136] The collection (or group) of synthetic nucleic acids provided in this article may contain several species of synthetic nucleic acids. In some cases, the length, concentration, and / or sequence of the species may be the same or similar. In other cases, the length, concentration, and / or sequence of the species may be different.

[0137] In preferred embodiments, the synthetic nucleic acid species have different lengths. For example, the set of synthetic nucleic acid species may collectively span the length of an observable range of certain target nucleic acids in the sample, or at least a portion of such an observable range. For example, the species may collectively span the length of disease-specific or pathogen-specific nucleic acids in the sample (particularly from samples obtained from subjects infected with or suspected of being infected with pathogens). In some cases, the length of disease-specific or pathogen-specific nucleic acids in the sample may be in the range of about 40 to about 100 base pairs. In some cases, the species may collectively span the lengths of various different disease-specific or pathogen-specific nucleic acids in the sample. In some cases, the species may collectively span the length of specific pathogen-specific nucleic acids, such as nucleic acids within a specific pathogen genome. In some cases, the nucleic acid may be a specific nucleic acid within a pathogen genome, such as a virulence region of the pathogen, an antibiotic resistance region of the pathogen, or other regions or specific nucleic acids or nucleic acids within a gene. In some cases, the length or nucleic acid may be specific to a specific type of infection (e.g., acute, chronic, active, or latent). In other instances, the species may collectively span the lengths of certain subject nucleic acids and / or pathogen nucleic acids in the sample (e.g., from an infected subject).

[0138] The length of the synthetic nucleic acid species within the ensemble may exactly match the length of a specific target nucleic acid (e.g., pathogen-specific or disease-specific nucleic acids within an observable range in a sample). In other cases, the length of the synthetic nucleic acid species within the ensemble may closely match, or substantially match, the length of the target nucleic acid. For example, the length of the synthesized nucleic acid can be within 50%-150% of the target nucleic acid length, 55%-145% of the target nucleic acid length, 60%-140% of the target nucleic acid length, 65%-135% of the target nucleic acid length, 70%-130% of the target nucleic acid length, 75%-125% of the target nucleic acid length, 80%-120% of the target nucleic acid length, 85%-115% of the target nucleic acid length, 90%-110% of the target nucleic acid length, 95%-105% of the target nucleic acid length, 96%-104% of the target nucleic acid length, 99%-101% of the target nucleic acid length, or 99.5%-100.5% of the target nucleic acid length. In some cases, the length of the synthesized nucleic acid can be within 50%-150% of the target nucleic acid length. In some cases, the length of the synthesized nucleic acid can be up to two, three, four, or five times the target nucleic acid length. In some cases, the length of the synthesized nucleic acid species can be within 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, or 200 nucleotides of the target nucleic acid length. In other cases, the synthesized nucleic acid species in the set are greater than 65%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, or 99% of the length of the most closely matched target nucleic acid.

[0139] Each or most nucleic acid “species” within the collection (or pool) of synthetic nucleic acids disclosed herein may contain one or more domains or regions of interest. In some cases, the domain or region of interest is a length marker sequence. The length marker sequence may contain a predetermined code to indicate or represent a specific length; typically, such length markers may be short sequences, such as 10 base pairs (bp), 9 bp, 8 bp, 7 bp, 6 bp, 5 bp, 4 bp, or 3 bp; less than 9 bp, less than 8 bp, less than 7 bp, or less than 6 bp; or 6–15 bp, 5–10 bp, 4–8 bp, or 6–9 bp. A species may contain one, two, or more length marker sequences. In some cases, the length marker exists as a forward and / or reverse sequence.

[0140] In some cases, the domain within a nucleic acid species in a synthetic nucleic acid assembly can be a load sequence of a specific length, typically corresponding to the length encoded by a length-discriminating sequence (if present) in the synthetic nucleic acid. The length of the incorporated nucleic acid or load can vary. In some cases, the total length of the incorporated nucleic acid can be about or at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 nucleotides. In some cases, the length of the incorporated nucleic acid can be up to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 nucleotides. In other cases, the incorporated nucleic acid can be in the range of about 20 to about 200 base pairs, such as about 20 to about 120 base pairs. In some cases, the length of the load sequence domain incorporated into the nucleic acid can be about or at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 nucleotides. In some cases, the length of the load sequence domain incorporated into nucleic acid can range from 0 to approximately 200 bp.

[0141] The domains within a nucleic acid species within a synthetic nucleic acid set can be synthetic nucleic acid identification sequences (e.g., Spark identification sequences, Spank identification sequences), which contain unique codes indicating that the nucleic acid is incorporated rather than part of the original sample. Typically, these unique codes are codes not present in the original sample or the target nucleic acid pool. Synthetic nucleic acid identification sequences can contain specific bp numbers, such as 25bp, 20bp, 19bp, 18bp, 16bp, 15bp, 12bp, 10bp, or other lengths. A species can contain one, two, or more synthetic nucleic acid identification sequences or domains. In some cases, synthetic nucleic acid identification sequences exist as forward and / or reverse sequences.

[0142] In some cases, the domains within nucleic acid species within a synthetic nucleic acid pool can be "diversity codes" associated with the overall pool or set of synthetic nucleic acids. These diversity-coding domains can be unique codes representing the amount of diversity within the synthetic nucleic acid pool. In such cases, sequences representing the degree of diversity within the pool (e.g., 10) can be used. 8 Each synthetic nucleic acid within a diversity pool is encoded by a unique sequence. In some cases, such as when two or more diversity pools are used on the same sample, diversity encoding can be used to identify diversity loss in two or more pools.

[0143] In some cases, depending on the application, the domains within a nucleic acid species within a synthetic nucleic acid set can be characteristic domains associated with one or more characteristics of a sample or reagent. For example, characteristic domains may include sequences encoding a specific reagent; a specific reagent batch; or a specific sample (e.g., sample number, patient number, patient name, patient age, patient sex, patient ethnicity, location from which the sample was obtained).

[0144] The domains or regions of interest can exist in any combination and number. For example, a synthetic nucleic acid may contain: one or more length marker sequences, one or more load sequences, one or more synthetic nucleic acid-marker sequences, one or more diversity-coding and / or one or more characteristic domains, in any combination or ratio. For example, in some cases, the synthetic nucleic acid contains both a length marker sequence and a load sequence. In some cases, the synthetic nucleic acid contains both a synthetic nucleic acid-marker sequence and a characteristic domain sequence. In some cases, the synthetic nucleic acid contains a synthetic nucleic acid marker sequence, while in others it does not.

[0145] In some cases, synthetic nucleic acids may contain domains with overlapping purposes. For example, in some cases, synthetic nucleic acids contain one or more length marker sequences, which also serve as load sequences. In some cases, length marker sequences and / or load sequences are also used as marker sequences for synthetic nucleic acids.

[0146] Nucleic acids can be selectively synthesized or incorporated to match nucleic acid libraries. In some cases, the synthesized nucleic acids or incorporates may contain adaptors, common sequences, random sequences, poly-A tails, blunt or non-blunt ends, or any combination thereof. In some cases, the synthesized nucleic acids or incorporates are designed to mimic nucleic acids in samples with one or more of these or other properties.

[0147] The synthetic nucleic acids provided herein (e.g., incorporated synthetic nucleic acids) may contain any type of nucleic acid or combination of nucleic acid types. In a preferred embodiment, the synthetic or incorporated nucleic acid is DNA. In some cases, the synthetic or incorporated nucleic acid is single-stranded DNA. In some cases, the synthetic or incorporated nucleic acid is double-stranded DNA. In some cases, the synthetic or incorporated nucleic acid is RNA. In some cases, the synthetic or incorporated nucleic acid may contain modified bases or artificial bases. Double-stranded synthetic or incorporated nucleic acids may have blunt or concave ends. Synthetic or incorporated nucleic acids may have phosphorylated or dephosphorylated ends. In some cases, synthetic nucleic acids may contain double-stranded (ds) nucleic acids, single-stranded (ss) nucleic acids, DNA, RNA, cDNA, mRNA, cRNA, tRNA, ribosomal RNA, dsDNA, ssDNA, snRNA, genomic DNA, oligonucleotides, double-stranded oligonucleotides, longer assembled double-stranded DNA (e.g., gBlock from Integrated DNA Technologies), plasmids, PCR products, in vitro synthesized transcripts, viral particles, fragmented or unfragmented genomic DNA, circular nucleic acids, circular DNA, circular RNA, circular single-stranded DNA, circular double-stranded DNA, plasmids, or any combination thereof. Synthetic nucleic acids may typically contain nucleobases such as adenine (A), cytosine (C), guanine (G), thymine (T), and / or uracil (U).

[0148] Synthetic nucleic acids can be or may contain any synthetic nucleic acid or nucleic acid analogue. Synthetic nucleic acids may contain a modified or altered phosphate backbone; a modified pentose sugar (e.g., modified ribose or deoxyribose); or a modified or altered nucleobase (e.g., modified adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)). In some cases, synthetic nucleic acids may include one or more modified bases, such as 5-methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and / or 7-methylguanosine (m7G). In some cases, synthetic nucleic acids may include peptide nucleic acids (PNA), bridging nucleic acids (BNA), nucleic acid mimics, glycerol nucleic acids (GNA), threonine nucleic acids (TNA), locked nucleic acids (LNA), 2'-O-methyl-substituted RNA, morpholino- or other synthetic polymers having nucleotide side chains. In some cases, synthetic nucleic acids may include DNA, RNA, PNA, LNA, BNA, or any combination thereof. In some cases, synthesized nucleic acids may include double helices, triple helices, or other structures.

[0149] Synthetic nucleic acids can include any combination of any nucleotides. Nucleotides can be naturally occurring or synthetic. In some cases, nucleotides may be oxidized or methylated. Nucleotides may include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), and deoxyadenosine diphosphate (cAMP). dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate, and 5-hydroxymethyl-2'-deoxycytidine triphosphate.

[0150] Synthetic or incorporated nucleic acids can refer to any molecule added to a sample, and is not limited to chemically synthesized (e.g., column-synthesized) molecules. In some cases, synthetic or incorporated nucleic acids can be synthesized, for example, by PCR amplification, in vitro transcription, or other template-based replication. In some cases, the synthetic or incorporated nucleic acid is a cleaved or otherwise fragmented nucleic acid or contains such a nucleic acid. Cleaved or fragmented nucleic acids can include genomic nucleic acids, such as human or pathogen genomic nucleic acids. In some cases, the synthetic nucleic acid does not contain human nucleic acids. In some cases, the synthetic nucleic acid does not contain nucleic acids that can be found in nature. In some cases, the synthetic nucleic acid does not contain sample nucleic acids.

[0151] The guanine-cytosine content (GC content) of the incorporated or synthesized nucleic acid can vary. In some cases, the GC content of the incorporated or synthesized nucleic acid can be about or at least about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In other cases, the GC content can be at most about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some cases, the GC content of the incorporated or synthesized nucleic acid can range from about 15% to about 85%, such as from about 20% to about 80%. The GC content of the synthesized nucleic acid species within the ensemble can exactly match the GC content of a specific target nucleic acid (e.g., pathogen-specific or disease-specific nucleic acid within an observable range in the sample). In other cases, the GC content of the synthesized nucleic acid species within the ensemble can closely match, or substantially match, the GC content of the target nucleic acid. For example, the GC content of the synthesized nucleic acid species can be within 75%–125% of the GC content of the target nucleic acid, within 80%–120% of the GC content of the target nucleic acid, within 85%–115% of the GC content of the target nucleic acid, within 90%–110% of the GC content of the target nucleic acid, within 95%–105% of the GC content of the target nucleic acid, within 96%–104% of the GC content of the target nucleic acid, within 99%–101% of the GC content of the target nucleic acid, or within 99.5%–100.5% of the GC content of the target nucleic acid.

[0152] Nucleic acid incorporation can attach, link, or conjugate to various molecules, such as beads, fluorophores, and polymers. Examples of fluorophores include, but are not limited to, fluorescent proteins, green fluorescent protein (GFP), Alexa dyes, luciferin, red fluorescent protein (RFP), and yellow fluorescent protein (YFP). Nucleic acid incorporation can be associated with proteins (e.g., histones, nucleic acid-binding proteins, DNA-binding proteins, RNA-binding proteins). In other cases, the incorporation is not associated with a protein. Nucleic acid incorporation can be particle-protected (e.g., similar to nucleic acids in viral particles). In some cases, the incorporation is encapsulated within or associated with particles. In some cases, particles include proteins, lipids, metals, metal oxides, plastics, polymers, biopolymers, ceramics, or composite materials.

[0153] Incorporated nucleic acids may have sequences different from those found in the sample or host. In some cases, the incorporated nucleic acid sequence is naturally occurring. In some cases, the incorporated nucleic acid sequence is not naturally occurring. In some cases, the incorporated nucleic acid sequence is derived from the host. In some cases, the incorporated nucleic acid sequence is not derived from the host. In some cases, the incorporated or synthesized nucleic acid cannot hybridize (or is not complementary) with one or more target nucleic acids (e.g., pathogen nucleic acids, disease-specific nucleic acids) and / or one or more sample nucleic acids.

[0154] The concentration of nucleic acid incorporated into a sample can vary. Incorporations can be added at various concentrations, which is useful for determining sensitivity and sample loss. In some cases, approximately or at least approximately 100,000, 500,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000, 20,000,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100 million, 500 million, or 1 billion molecules of each nucleic acid are added per mL of plasma or sample. In some cases, approximately 10 million to approximately 100 million molecules of each nucleic acid are added per mL of plasma or sample. In some cases, synthetic nucleic acids are incorporated at equimolar concentrations. In other cases, synthetic nucleic acids are incorporated into the sample at different concentrations.

[0155] The number of different incorporating nucleic acids added to a sample may vary. Multiple incorporating nucleic acids may be added to a sample or reagent. In some cases, about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 incorporating nucleic acids are added to the sample or reagent. In some cases, up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 incorporating nucleic acids are added to the sample or reagent. In some cases, the incorporating nucleic acids added to the sample or reagent are of the same length. In some cases, the incorporating nucleic acids added to the sample or reagent are of different lengths. In some cases, the incorporating nucleic acids are selected from SEQ ID NO 1-120 and any combination thereof.

[0156] The level of uniqueness of the incorporated nucleic acid can vary. A virtually unlimited number of incorporations can be designed or used (e.g., ID Spike).

[0157] The steps involved in adding incorporated nucleic acids can vary. For sample tracking, adding incorporated nucleic acids earlier may be preferable and reduces the likelihood of subsequent operator or system errors. In some cases, the tube to which the sample (e.g., blood) is first added may already contain incorporated nucleic acids. This allows for more systematic control and testing of the manufacture of these tubes compared to adding incorporated nucleic acids to samples in a clinical or laboratory setting, thus reducing the possibility of sample contamination. In some cases, ID Spikes can replace all external labels (“white labels”).

[0158] In some cases, identifying nucleic acid tags can be added to each nucleic acid fragment in a sample, so that each sequence read contains the identifying tag. This method will achieve differentiation between initial and downstream cross-contamination. If the tagging of the fragments is sufficiently complete, it can also allow for intentional multiplexing of the sample once the barcode is added to the sample fragments. Methods for incorporating tags include, but are not limited to, transposons, terminal transferases, cleavage at methylation sites, and cleavage at demethylation sites.

[0159] For other applications, including but not limited to those involving process quality control or development work, incorporated nucleic acids can be added at different steps of the process. For example, for RNA analysis, multiple RNA incorporations can be added at the beginning of sample preparation, each with different concentrations, lengths, sequences, and / or GC contents, and DNA incorporations can be added after RNA has been converted into DNA. For DNA libraries, different forms of DNA can be added at different steps of the library generation process. For example, to test the end repair step, DNA incorporations with non-blunt ends, + / - 5'-phosphate, and + / - 3'-adenine extensions can be used. To test the step of ligating adaptors to end repair fragments, + / - pre-ligated incorporations can be used. Sequencing or qPCR can quantify sample loss at each step. Incorporated qPCR can also be used in conjunction with other library quantification methods for final library evaluation before sequencing.

[0160] The terms “incorporation,” “incorporated synthetic nucleic acid,” “incorporation,” and “synthetic nucleic acid” are used interchangeably in this document and should be interpreted equally unless the context indicates otherwise. The term “IDSpike” or “traceable” is generally used herein to refer to an identification incorporation that can be used for, for example, sample identification tracking, cross-contamination detection, reagent tracking, or reagent batch tracking. The term “Spark” is generally used herein to refer to a nucleic acid as a size or length marker that can be used for abundance normalization, development and / or analytical purposes, and other purposes. The term “Spank” is generally used herein to refer to a degenerate pool or nucleic acid pool with different sequences and is generally used for diversity assessment and abundance calculation.

[0161] Universal normalization of nucleic acid measurement values

[0162] This disclosure describes a synthetic nucleic acid genome that, when used in the methods described, allows for efficient and improved normalization of the amount of disease-specific nucleic acids, pathogen-specific nucleic acids, or other target nucleic acids in a sample. The incorporated nucleic acid genome may contain several nucleic acid “types” of different lengths, such that the set of incorporated nucleic acid types collectively spans the observable length range of pathogen nucleic acids, disease-specific nucleic acids, or other target nucleic acids to be tested.

[0163] Incorporation of synthetic nucleic acids can be used to normalize samples in a variety of ways. Typically, normalization can be performed across samples, independently of the subject from which the sample originated, preanalytical sample processing, nucleic acid extraction methods, manipulation of nucleic acids using molecular biology tools and methods, nucleic acid purification methods, the behavior of the measurement itself, storage conditions, and / or time progression.

[0164] In some preferred embodiments, nucleic acid incorporation can normalize across all samples and all methods for measuring disease-specific nucleic acids, pathogen-specific nucleic acids, or other target nucleic acids. In some cases, incorporation can be used to determine the relative abundance of pathogen nucleic acids (or disease-specific nucleic acids or target nucleic acids) in a sample relative to other pathogen nucleic acids.

[0165] Typically, the methods described herein involve incorporating or introducing one or more sets of synthetic nucleic acids into a sample. This incorporation step can occur at any point throughout the process, including early, midway, or near the end of the procedure. For example, synthetic nucleic acids can be introduced instantaneously upon or immediately after sample collection from a subject, before or during sample storage, before sample transfer, before or during nucleic acid extraction, before or during library preparation, before sequencing analysis, or before any other step of the method. In some cases, the method may include incorporating a known amount of a unique nucleic acid molecule, measured by the same method, but readily distinguishable from pathogen-specific or disease-specific nucleic acids or other sample nucleic acids, early in the process. In some cases, synthetic nucleic acids are incorporated into the biological sample in a single step of the process, for example, upon sample collection from a subject, upon obtaining the sample for analysis, during sample storage, before or during nucleic acid extraction, before or during library preparation, or instantaneously before sequencing analysis. In other cases, the same or different incorporated synthetic nucleic acids are introduced at different steps of the process. For example, unique synthetic nucleic acids can be introduced early in the process, such as during sample collection, and different sets of unique synthetic nucleic acids can be introduced later in the process, such as before or after extraction, purification, or library preparation. The same set of incorporating nucleic acids or sets that differ in some respects can also be used to repeatedly add steps in different steps of the method.

[0166] Typically, synthetic nucleic acid species at known concentrations (or multiple concentrations) are incorporated into each sample. In many cases, the synthetic nucleic acid species are incorporated at equimolar concentrations. In some cases, the concentrations of the synthetic nucleic acid species vary.

[0167] When samples are processed and ultimately measured, the relative abundance of nucleic acid species may change due to inherent biases in sample processing, preparation, and measurement. After measurement, the efficiency of recovering nucleic acids of each length can be determined by comparing the measured abundance of each "species" incorporated with the initial amount incorporated. This produces a "length-based recovery profile".

[0168] "Length-based recovery curves" can be used to normalize the abundance of all (or most or some) disease-specific nucleic acids, pathogen nucleic acids, or other target nucleic acids by normalizing the abundance of disease-specific nucleic acids (or pathogen nucleic acids or other target nucleic acids) to a function fitted to the closest length of incorporating molecules or to incorporating molecules of different lengths. This process can be applied to disease-specific nucleic acids and produces an estimate of the "raw length distribution of all disease-specific nucleic acids" at the time of incorporation. Similarly, this process can be applied to other target nucleic acids, such as pathogen-specific nucleic acids, and produces an estimate of the "raw length distribution of all pathogen-specific nucleic acids" at the time of incorporation. The "raw length distribution of all target nucleic acids" can be displayed as a length distribution curve of the target nucleic acids (e.g., disease-specific nucleic acids, pathogen-specific nucleic acids) at the time of incorporation. Incorporated nucleic acids can seek to generalize this length distribution to achieve perfect or near-perfect abundance normalization.

[0169] Because it may be impossible to incorporate a mixture of known nucleic acids that accurately summarize the relative abundance curves of disease-specific nucleic acids, pathogen nucleic acids, or other target nucleic acids in a particular sample—partly because the sample may have been exhausted or the relative abundance curves may have changed over time—each incorporated "species" can be weighted proportionally to its relative abundance within the "original length distribution of all disease-specific nucleic acids." The sum of all "weighting factors" can equal 1.0.

[0170] Normalization can involve a single step or a series of steps. In some cases, the abundance of disease-specific nucleic acids (or pathogen nucleic acids or other target nucleic acids) can be normalized using the raw measurement of the incorporation nucleic acid abundance closest to the size, to produce a “normalized disease-specific nucleic acid (or pathogen nucleic acid or other target nucleic acid) abundance”. This “normalized disease-specific nucleic acid abundance” (or pathogen nucleic acid or other target nucleic acid abundance) can then be multiplied by a “weighting factor” to adjust for the relative importance of recovering that length, resulting in a “weighted normalized disease-specific (or pathogen-specific or other target) nucleic acid abundance”. One advantage of this normalization method is that it allows for comparable measurements of target nucleic acid (e.g., disease-specific nucleic acid, pathogen nucleic acid) abundance across all (or most) methods for measuring disease-specific nucleic acid abundance, regardless of the method.

[0171] Measurements of target nucleic acid abundance or relative abundance are particularly useful for detection, prediction, monitoring, and diagnostic assays. Such assays may involve measuring the amount of target nucleic acids (e.g., disease-specific nucleic acids) in biological samples (e.g., plasma) to detect the presence of pathogens or identify disease states. The methods described herein enable comparability of these measurements across samples, measurement times, nucleic acid extraction methods, nucleic acid manipulation methods, nucleic acid measurement methods, and / or various sample handling conditions.

[0172] The exact sequence of the incorporated molecules, the exact number of "types," the length range of the "types," the concentration of the incorporated molecules, the relative amount of each molecule, the actual amount of each incorporated molecule, and the stage of molecule incorporation can be optimized or customized based on the sample. GC content, nucleic acid structure, DNA damage, or DNA modification status can be used instead of or analyzed for length.

[0173] In some cases, the methods presented herein may include the use of incorporating nucleic acids containing a single-length nucleic acid, typically with a largely fixed sequence composition (except for some short randomized portions in some methods). This method works well when disease-specific nucleic acids, pathogen-specific nucleic acids, or other target nucleic acids are nearly the same length as the incorporating nucleic acid.

[0174] Single-length nucleic acids can be used alone, or this method can be combined with another method involving the use of multi-length nucleic acids. For example, multi-length nucleic acid pools can be incorporated into the sample at the time of sample acquisition or before nucleic acid extraction, and single-length nucleic acid pools can be incorporated into the sample at different points in the process, such as after nucleic acid extraction and before library preparation. When using single-length and / or multi-length nucleic acids, the amount of disease-specific nucleic acids, pathogen nucleic acids, or other target nucleic acids can be normalized relative to the amount of incorporated nucleic acids measured at the end of the method.

[0175] In many cases, as described herein, using synthetic nucleic acids of varying lengths is superior to methods involving synthetic nucleic acids of a single length. The methods presented herein are particularly useful when the target nucleic acid has multiple lengths. For example, the lengths of disease-specific (or pathogen-specific) nucleic acids can vary widely. Therefore, using incorporated nucleic acids of observable lengths spanning disease-specific nucleic acids can be particularly useful. Furthermore, the length of the measured disease-specific nucleic acid can be significantly influenced by numerous factors, including the metabolism of the individual from which it is derived, preanalytical sample processing conditions, nucleic acid extraction methods, manipulation of the nucleic acid using molecular biology tools and methods, nucleic acid purification methods, the behavior of the measurement itself, storage conditions, and the passage of time. These factors have different effects on nucleic acids of different lengths; therefore, a single incorporated nucleic acid may not be sufficient to reflect the overall efficiency of the process involving nucleic acids of mixed lengths.

[0176] Calculation of "genome copies per volume"

[0177] The methods and synthetic nucleic acids presented in this article can be used to aid in certain calculations, including determining the number of microbial or pathogen genome copies per volume in a sample from next-generation sequencing results. Typically, genome copies per volume can refer to an absolute measure of the amount of target nucleic acids (e.g., target nucleic acids derived from a specific pathogen) per 1 ml of fluid (e.g., plasma, urine, buffer, etc.), and is often used as an expression of the abundance or relative abundance of an individual pathogen. The total number of reads and / or the magnitude of pathogen abundance can vary from sample to sample. It may be necessary to report values ​​corresponding to the biological level of infection, and this value may be useful for sample-to-sample comparisons.

[0178] In specific instances, the method can be used to determine the genomic copies of pathogen nucleic acids per volume in a sample, particularly from samples obtained from subjects infected with or suspected of being infected with the pathogen. A statistical framework can be used to determine or estimate the genomic copies per volume. This statistical framework can be used to estimate the relative abundance of one or more genomes that produce a set of non-human reads (e.g., pathogen reads) in the sequencing results of a sample.

[0179] Using the incorporated synthetic nucleic acids provided herein, estimates of the "genome copies per volume" of one or more pathogens / organisms in a sample can be calculated. Typically, nucleic acids of various lengths can be incorporated into a sample at known concentrations. In some cases, the portion of information from the sample actually observed in the sequencing data for each incorporation length can be observed (e.g., by comparing observed reads with reads associated with the incorporated nucleic acids, or by dividing the observed reads by the incorporated reads). The original number of non-host or pathogen molecules of each length can also be inversely calculated (e.g., inferred in part from the number of incorporated reads of each length). When the genome length of each pathogen is known, this load can be converted into a "genome copies per volume" measure.

[0180] In many cases, methods for detecting per-volume genome copies (and others provided herein) may involve removing or isolating low-quality reads. Removing low-quality reads can improve the accuracy and reliability of the methods provided herein. In some cases, the methods may include removing or isolating the following reads (in any combination): unmappable reads, reads generated by PCR replication, low-quality reads, adaptor dimer reads, sequencing adaptor reads, reads that are not uniquely mapped, and / or reads mapped to non-informative sequences.

[0181] In some cases, sequence reads are mapped to a reference genome, and reads not mapped to such a reference genome are mapped to a target or pathogen genome. In some cases, reads may be mapped to a human reference genome (e.g., hg19), while the remaining reads are mapped to a curated reference database of viruses, bacteria, fungi, and other eukaryotic pathogens (e.g., fungi, protozoa, parasites).

[0182] In some specific instances, the method may include incorporating a known concentration of synthetic nucleic acid (e.g., DNA) into a sample (e.g., a plasma sample) before DNA extraction (e.g., cell-free DNA extraction, cell-free RNA extraction) or at different stages of the assay (e.g., after extraction, before library preparation, before sequencing, during sample storage). The synthetic nucleic acid may also be added to negative and / or positive control samples. In some cases, control samples may be processed together with the sample. The method may further include generating a sequencing library of the sample (e.g., a plasma sample, a positive control, a negative control). The library may be multiplexed and sequenced on sequencing devices known in the art, particularly those capable of next-generation sequencing. The method may further include discarding low-quality reads and removing human reads by aligning them with a human reference sequence (e.g., hg19). The remaining reads may then be aligned with a database of pathogen sequences. In some cases, reads from the NGS read set correspond to the target sequence of interest (e.g., a pathogen sequence). Based on this information, the relative abundance of the target nucleic acid (e.g., pathogen nucleic acid) may be expressed as genomic copies per volume. The genome copy value per volume can be determined, for example, by determining the number of sequences present in each organism (e.g., pathogen) normalized to a known number of oligonucleotides in an incorporated sample (e.g., plasma). The calculation of the genome per volume may also take into account the relative length of the individual pathogen genome. In some cases, the genome copy value per volume can be determined by quantifying the number of sequences present in each organism (e.g., pathogen) normalized to a known number of synthetic nucleic acids in an incorporated sample, where the normalization of pathogen sequences considers the synthetic nucleic acid with the closest length to the pathogen sequence. Similarly, normalization may involve using a set of incorporated synthetic nucleic acids of different lengths (e.g., 2, 3, 4, 5, 6, 10, 15, 20 or more different lengths), where the pathogen nucleic acid is normalized relative to the incorporating nucleic acid of the corresponding length within the incorporation set.

[0183] Incorporation for sample tracking and / or analysis

[0184] Molecules can be incorporated into samples to provide unique identifiers and tracers. These molecules can become part of the sample and can be read by appropriate measuring devices; the concept is similar to a 1D or 2D barcode read on the outside of a sample tube by a laser scanner. Optical, radiometric, and other tracers are possible, but nucleic acid tracers are likely the most suitable option for analyzing nucleic acid samples because the incorporation's identity can be revealed in the same process used to assess the sample's nucleic acids (e.g., DNA or RNA sequencing).

[0185] Exogenous nucleic acids may include, but are not limited to, oligonucleotides, double-stranded oligonucleotides, longer assembled double-stranded DNA (e.g., gBlock from Integrated DNA Technologies), plasmids, PCR products, in vitro synthesized transcripts, viral particles, and fragmented or unfragmented genomic DNA, which can be added to samples, such as bodily fluids from a subject. The advantages of using incorporation include, but are not limited to, the ability to customize the nucleic acid sequence, length, diversity, and concentration for a sample or application.

[0186] Applications include, but are not limited to, sample tracking (e.g., ID Spikes can be used in addition to or potentially replace traditional label barcodes), sample cross-contamination (e.g., if ID Spikes are not naturally present in any sample, and if different ID Spikes are added to different samples, sample mixing can be determined), reagent tracking (e.g., ID Spikes can also be added to reagents. For example, each batch of reagents used can be tracked for each sample, providing an error-resistant reagent tracking molecular laboratory information management system (LIMS)), quality control or development efforts (e.g., different incorporations can be added at various times during sample processing to monitor library complexity (e.g., PCR replicates), sample loss, or sensitivity), normalization or yield (e.g., comparing known inputs of incorporation with measurement outputs enables inferences about unknown inputs (e.g., in the sample) from the measurement outputs. These measurements and calculations can inform, for example, the pathogen load of the sample), and increasing nucleic acid concentration (e.g., if the barcodes are nucleic acids, they can be used at high concentrations for samples with limited nucleic acid concentrations, which can improve sample recovery).

[0187] In some preferred embodiments, incorporation can be used to estimate the likelihood that a particular nucleic acid sequence of interest originated from the observed sample, or whether its presence in the observed sample might be a result of cross-contamination or legacy from different samples. By introducing unique incorporating molecules into each sample at concentrations higher than reasonably expected concentrations of molecules from a particular pathogen (or other sequence class of interest), any pathogen sequence (or other sequence class of interest) that might have been accidentally introduced by cross-contamination or legacy will be accompanied by a greater number of incorporating molecules from the source of the contamination or legacy sequence. Therefore, the ratio of pathogen sequence count (or other class of sequences) to cross-contamination or legacy incorporating molecule count can be used to identify any pathogen sequence that might be a result of sample-to-sample cross-contamination or legacy. In some cases, the absence of cross-contamination or legacy incorporating molecules, or their presence at levels below a threshold, indicates that the sample is not contaminated.

[0188] For some applications, the genotype of the subject from whom the sample is derived can be used, particularly for sample tracking. In some cases, the genotype can be determined during the analysis procedure or by removing aliquots and performing a separate genotyping process. In some cases, the genotype of the sample is known. The sequencing output of the subject's DNA can be compared with the genotype from an independently sourced source. The advantage of using the genotype is that it is already part of the sample and is inherent to the sample. An exemplary orthogonal genotyping method is short tandem repeat (STR) analysis; see, for example, ATCC's testing services.

[0189] In some cases, phenotypic characteristics can help identify samples. For example, a subject's eye color, blood type, sex, ethnicity, and other traits can provide clues about their genotype.

[0190] ID Spike

[0191] The unique sample identifiers can be completely disrupted (e.g., a randomer for DNA with A, C, G, and T, or for RNA with A, C, G, and U), or they may have some regions of shared sequence. For example, shared regions at each end can reduce sequence deviation in ligation events. In some cases, the shared region is about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 common base pairs. In other cases, the shared region is at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 common base pairs. Exemplary sequences are shown in Table 1.

[0192] Combinations of ID Spikes can be added to increase diversity without using a large number of ID Spikes. For example, an ID Spike can be used as an identifier for the well location in a microtiter plate (e.g., 96 different ID Spikes for a 96-well plate), and another ID Spike can be used as an identifier for the plate number (e.g., 24 different ID Spikes for 24 different plates), giving 96 × 24 = 2,304 combinations using only 96 + 24 = 120 sequences. Using 3 or more ID Spikes per sample can significantly increase the achievable diversity.

[0193] Table 1: Example ID Spike Sequences

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] Spark bias control incorporation

[0205] Nucleic acid sequence groups spanning multiple lengths (“Spark”) can serve as size markers. These sequences can be incorporated into a sample and processed along with the sample nucleic acids (e.g., extraction, purification, sequencing). Certain processes can affect nucleic acids of different lengths differentially. For example, nucleic acid purification using silica membrane columns may favor longer sequences or be optimized to retain sequences of specific lengths. Since nucleic acid sequencing typically occurs after nucleic acid extraction from a sample, the prevalence or distribution of lengths in the sequencing results may not be representative of the original sample. By incorporating Spark sequences of known quantity and length, the effects of processing and sequencing on sample nucleic acids of different lengths can be monitored and quantified. Furthermore, the relative and / or absolute quantities of sample nucleic acids of different lengths in the original sample can be estimated by measuring the final number of sequencing reads of the sample nucleic acids and Spark macronuclei and normalizing them to the known quantity of Spark macronuclei incorporated into the original sample.

[0206] In some cases, Spark groups may include approximately or at least approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 250, 300, 350, 400, 500, 600, 700, 800, 1000 or more nucleic acids. In some cases, Spark groups may include up to approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100 or 200 nucleic acids. In some cases, Spark groups include approximately 3 to approximately 50 nucleic acids, such as approximately 3 to approximately 30 nucleic acids. In some cases, the nucleic acids in Spark groups have one or more different properties, such as different lengths, different GC contents and / or different sequences.

[0207] Spark nucleic acids may contain any of the characteristics of synthetically incorporated nucleic acids described herein, including length-identifying sequences, loaded sequences, synthetic nucleic acid identification sequences (which are Spark identification sequences in this context), and characteristic structural domains. In some cases, nucleic acids in a Spark ensemble contain fixed forward and / or fixed reverse sequences. The fixed forward and / or fixed reverse sequences may be common to all nucleic acids in the Spark ensemble and identify the sequence as Spark. In some cases, the length of the fixed forward and / or fixed reverse sequences is about or at least about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 32, 40, 50, 60, 70, 80, 90, or 100 base pairs. In some cases, the length of the fixed forward sequence and / or fixed reverse sequence is at most about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 32, 40, 50, 60, 70, 80, 90, or 100 base pairs. In some cases, the fixed forward sequence and / or fixed reverse sequence is in the range of about 8 bp to about 50 bp, such as about 8 bp to about 20 bp or about 16 bp to about 40 bp. In some cases, the Spark identification sequence is not naturally occurring or is not present in the sample. In some cases, the fixed forward sequence differs from the fixed reverse sequence.

[0208] In some cases, nucleic acids within a Spark group contain unique forward and / or unique reverse sequences. These unique forward and / or unique reverse sequences distinguish Sparks within the group from one another. In some cases, the length of the unique forward and / or unique reverse sequences is approximately or at least approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 32, 40, 50, 60, 70, 80, 90, or 100 base pairs. In some cases, the length of the unique forward and / or unique reverse sequences is at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 32, 40, 50, 60, 70, 80, 90, 100, 200, 300, 306, 400, or 500 base pairs. In some cases, the length of the unique forward and / or unique reverse sequences ranges from about 4 to about 10 base pairs. In some cases, each nucleic acid in the Spark group has a different unique forward and / or unique reverse sequence. In some cases, each nucleic acid in the Spark group has a unique forward and / or unique reverse sequence of the same length. In some cases, each nucleic acid in the Spark group has a unique forward and / or unique reverse sequence of different lengths.

[0209] In some cases, nucleic acids within a Spark group contain padding sequences. In some cases, the padding sequences distinguish Sparks within the group from one another. In some cases, the length of the padding sequences is approximately or at least approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 32, 40, 50, 60, 70, 80, 90, or 100 base pairs. In some cases, the length of the filler sequence is at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 32, 40, 50, 60, 70, 80, 90, 100, 200, 300, 306, 400, or 500 base pairs. In some cases, the filler sequence ranges from 0 to about 350 bp. In some cases, each nucleic acid in the Spark group has a different length of filler sequence. In some cases, the filler sequence length is selected from 0, 8, 31, 56, 81, 106, 131, and 306 bp.

[0210] In some cases, the length of nucleic acids in Spark groups is about or at least about 10, 20, 30, 32, 40, 50, 60, 70, 80, 90, or 100 base pairs. In some cases, the length of nucleic acids in Spark groups is at most about 10, 200, 300, 350, 400, 500, 600, 700, 800, 900, or 1,000 base pairs. In some cases, the length of nucleic acids in Spark groups ranges from about 20 to about 500 base pairs, from about 20 to about 400 base pairs, or from about 20 to about 200 base pairs.

[0211] For example, a set of eight double-stranded DNA sequences can be designed (SEQ ID NO: 111-118 in Table 2). Figure 4 It has the following characteristics: a size range of 32-350bp (e.g., fragments of 32, 52, 75, 100, 125, 150, 175 and 350bp with padding sequence lengths of 0, 8, 31, 56, 81, 106, 131 and 306bp respectively), a fixed 16bp forward sequence, a fixed 16bp reverse sequence different from the forward sequence, and unique 6bp forward and reverse sequences.

[0212] Table 2: Exemplary Spark Large and Small Group Sequences

[0213]

[0214]

[0215]

[0216] GC content incorporation group

[0217] Incorporating nucleic acids (e.g., DNA) at known concentrations into a sample, followed by measurement after processing, provides yield and other information about the process, which can be used to infer yield and other properties of the sample itself. For example, a range of nucleic acid incorporations can be added to a sample (e.g., plasma), then extracted, and subsequently sequenced using next-generation sequencing (NGS). The yield of each incorporation size can vary depending on many factors during processing, including intentional size selection, temperature and other denaturing factors, and PCR bias. This information can be used to develop new procedures designed to maximize recovery within a desired size range or for monitoring existing processes (e.g., quality control).

[0218] For double-stranded DNA library preparation, the relatively low melting temperature (T0) of the DNA double strand is crucial. m The denaturation of ) reduces the yield of these duplexes, compared to T m Inversely proportional. For given conditions (e.g., salt concentration, temperature, pH, etc.), the effect on the double strand T... m Contributing factors include length and GC content. A range of bistrands, each size represented by a single species with a single GC content, can only provide information about T under different conditions. m Partial information from the response.

[0219] How do nucleic acid length and / or GC content affect nucleic acid T? m The information processed can be important, for example, when using incorporation as a surrogate to infer the recovery of short, cell-free fragments from different pathogens in the blood. The GC content of pathogen nucleic acids can vary significantly, thus the short fragment lengths can have very different Tg values. m For example, given the short length of many cfDNA fragments (e.g., 30, 40, 50 bp), they may be prone to denaturation during NGS processing. This is useful for tracking large T cells. m A finer incorporation group within the scope of recovery allows for better inference of the initial amount of unknown samples.

[0220] Spanning a series of T mA set of incorporated nucleic acids of GC and / or length can be used for absolute abundance determination and / or for detailed monitoring of denaturation. For example, a set of 28 different nucleic acids (e.g., duplexes) containing four different lengths (e.g., 32, 42, 52, and 75 bp) and seven different GC contents (approximately 20, 30, 40, 50, 60, 70, or 80% GC) for each length can be used, as shown in Table 3. For each size, the set can provide more spacing overall than a set with a single GC content. In some cases, a set of synthetic nucleic acids (dsDNA, ssDNA, dsRNA, ssRNA) may contain at least three different lengths, each with at least two, three, four, five, seven, or ten different GC contents. In some cases, a group of synthetic nucleic acids (dsDNA, ssDNA, dsRNA, ssRNA) may contain at least 5 different lengths, each with at least 2 different GC contents, at least 3 different GC contents, at least 4 different GC contents, at least 5 different GC contents, at least 7 different GC contents, or at least 10 different GC contents.

[0221] In some cases, the incorporation group contains at least 3, 5, 10, 15, 20, 25, or 30 unique nucleic acids. In other cases, the incorporation group contains up to 15, 20, 25, 30, 35, 40, 45, 50, or 100 unique nucleic acids.

[0222] Incorporated nucleic acids with varying GC contents can be used. In some cases, the incorporated group contains nucleic acids with GC contents ranging from approximately 40-60%, 45-65%, 30-70%, 25-75%, or 20-80%. In some cases, the incorporated group contains nucleic acids with at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different GC contents. In some cases, the incorporated group contains nucleic acids with up to 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 different GC contents. In some cases, the incorporated group contains nucleic acids with different GC contents differing by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20%. The percentage of GC can be calculated by dividing the sum of the number of G and C nucleotides by the total number of nucleotides in the sequence. For example, for the sequence ACTG, %GC would be calculated as (1+1) / 4 = 50% GC.

[0223] Incorporated nucleic acids of varying lengths can be used. In some cases, the incorporated group contains nucleic acids of at least 3, 4, 5, 6, 7, 8, 9, 10, or 15 different lengths. In some cases, the incorporated group contains nucleic acids of up to 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or 100 different lengths. In some cases, the incorporated group contains nucleic acids of lengths spanning approximately 40-50 bp, approximately 35-55 bp, approximately 30-60 bp, approximately 35-60 bp, approximately 35-65 bp, approximately 35-70 bp, approximately 35-75 bp, approximately 30-70 bp, approximately 30-80 bp, approximately 30-90 bp, approximately 30-100 bp, approximately 25-150 bp, approximately 20-300 bp, or approximately 20-500 bp. In some cases, the incorporation group contains nucleic acids of varying lengths, differing by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 bp. In other cases, the incorporation group contains nucleic acids of lengths of 32, 42, 52, and 75 bp, or 27, 37, 47, 57, 62, and 67 bp.

[0224] Incorporated nucleic acids of length and GC content selected from a set of values ​​can be used. For example, synthetic nucleic acid sets can be selected from two or more lengths and two or more GC contents. The 28 synthetic nucleic acid sets (SEQ ID NO:125-SEQ ID NO:152) in Table 3 are formed from four different lengths (e.g., 32, 42, 52, and 75 bp) and seven different GC contents (e.g., approximately 20, 30, 40, 50, 60, 70, and 80% GC). Similar synthetic nucleic acid sets can be generated using different lengths (e.g., 20, 30, 40, 50, 60, 70, and 67 bp) and different GC contents (e.g., approximately 15, 25, 35, 45, 55, 65, and 75% GC).

[0225] Different melting temperatures (T) can be used m Incorporated nucleic acids. In some cases, the incorporated group contains melting temperatures (T0) spanning approximately 40-50°C, approximately 35-55°C, approximately 30-60°C, approximately 35-60°C, approximately 35-65°C, approximately 35-70°C, approximately 35-75°C, or approximately 30-70°C. m Nucleic acids. In some cases, the incorporation group contains nucleic acids with different melting temperatures (T0) differing by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 °C. m ) nucleic acid.

[0226] In some cases, T can be calculated based on one or more parameters other than the double-strand length and GC content. mFactors such as duplex concentration, nearest neighbor effect of nucleotide sequence, higher-order DNA structure, concentration of monovalent and / or divalent cations, and nucleotide concentration can be considered. In some cases, T can be calculated empirically for given conditions. m For example, using double-stranded DNA-specific dyes and gradually increasing the temperature, as well as detecting the dye signal.

[0227] Incorporated nucleic acids with different sequences can be used. Preferably, non-natural or unnatural sequences, or sequences that cannot hybridize with the sample nucleic acids, are used. In some cases, the incorporation set contains nucleic acids having at least 3, 4, 5, 6, 7, 8, 9, 10, or 15 different sequences. In some cases, the incorporation set contains nucleic acids having up to 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or 100 different sequences.

[0228] Different numbers of incorporated nucleic acids can be used. In some cases, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 nucleic acids are used. For example, a subset of the 28 sequences listed in Table 3 can be used, such as 32 / 42 / 52 / 75bp × 20 / 50 / 80% GC.

[0229] RNA omes can be used for RNA applications. As discussed herein, RNA omes can include the same or different molecules that differ in length, GC content, and / or other properties.

[0230] The group of 8 DNA sequences (SEQ ID NO: 111-118 in Table 2, each approximately 50% GC) provides partial coverage of the 28 member GC groups listed in Table 3.

[0231] Table 3: Exemplary GC Incorporation Sequences

[0232]

[0233]

[0234]

[0235] Degeneracy and incorporation: Spank

[0236] Incorporation into synthetic nucleic acids can be a degenerate pool of nucleic acids or a pool of nucleic acids with high diversity (sometimes referred to as "Spank" in this paper). Typically, Spank can be used to determine the absolute or relative loss of nucleic acids or diversity that may occur during sample processing steps that lead to and / or include sequencing reactions. For a unique Spank sequence pool, the loss of sequence diversity within the pool should directly correspond to the loss of nucleic acid abundance, without considering the effects of amplification or PCR bias. For example, if 10 8A unique spank sequence was incorporated into the sample, and only 10 were recovered after sequencing. 4 Each unique spank sequence reduces both nucleic acid abundance and nucleic acid diversity by 10%. 4 In some cases, spanks can be used to determine the degree of recovery of replicated molecules. For example, after extraction and library processing (which may include PCR and potentially uneven amplification of various input molecules), sequencing and alignment of individual spanks can reveal the degree of recovery of replicated molecules.

[0237] The determined diversity loss can then be used to determine the absolute abundance of nucleic acids (e.g., target nucleic acids) in the initial sample prior to one or more sample processing or sequencing steps. In some cases, the determined diversity loss is used to determine the relative abundance of nucleic acids in the initial sample. Figure 5 As shown, prior to one or more sample processing steps, the sample nucleic acids (S1, S2, ..., S...) are... m It can be incorporated into synthetic nucleic acids (SP1, SP2, ..., SP) with Spank. n ) combination. For example, approximately 10 8 Each unique spank is added to the sample. During sample processing (e.g., nucleic acid extraction, purification, ligation, and / or end repair), a portion of the sample nucleic acid and a portion of the synthetic nucleic acid may be lost. After sample processing, the initial 10% can be retained. 8 Approximately 10 of the unique sequences 6 Each sequence is unique. A portion of these sequences can then be sequenced, for example, 10... 4 Each unique sequence. The absolute loss of diversity can be calculated as the initial number of unique sequences divided by the number of unique sequences sequenced or recovered (e.g., 10). 8 / 10 4 =10 4 Similarly, the recovery value can be calculated as the number of unique sequences sequenced or recovered divided by the initial number of unique sequences (e.g., 10). 4 / 10 8 =10 -4 The calculated diversity loss can be used to determine the absolute abundance of nucleic acids in the initial sample. For example, sequencing read counts of the spank and sample sequences can be determined from sequencing analysis, and the initial concentration or amount of spank sequences incorporated into the sample can be known. The determined diversity loss can be used to determine the initial concentration or amount of nucleic acids (e.g., nucleic acids from a specific organism, pathogen, tumor, or organ) in the initial sample. The absolute amount of sample nucleic acids in the original sample can be estimated by measuring the final number of sequencing reads of the sample and spank nucleic acids and / or the final diversity of the spank nucleic acids and normalizing it to the known quantity or diversity of the spank group of nucleic acids incorporated into the original sample.

[0238] The number of unique sequence reads can be determined using various methods. For example, sequence reads with tagged identifiers can be identified. The number of unique sequences within a tagged sequence read can then be determined by deduplication (“duplicate removal”) or by removing duplicate sequences. For example, sequences can be aligned to a reference database of possible sequences or to each other to determine which sequences are duplicates and which are unique or different. Since identifiers are generally conserved between sequences, randomized sequence regions embedded within each incorporated molecule can be analyzed. In some cases, spank nucleic acids do not include identifiers; in such cases, spanks can be identified using other methods, such as by referencing or aligning to databases containing known sequences.

[0239] Spank sequences can be used to detect relative and / or absolute losses. In some cases, if the diversity of spank sequences is high enough, it can be assumed that the spank sequences incorporated into the sample are substantially all unique. Therefore, any repetitive spank sequences sequenced are likely due to PCR amplification rather than multiple copies of the same spank sequence added to the sample and can be removed from the analysis. Alternatively, if each spank sequence is unique, the total number of spank sequences initially added to the sample can be determined based on the concentration and volume of nucleic acids added to the sample, and the total number of unique spank sequencing reads after sequencing can also be determined; these values ​​can be used together to calculate the diversity loss value or recovery value.

[0240] The methods presented in this paper include approaches to identifying steps during sample processing that are associated with population bottlenecks or diversity loss. In some cases, when a population bottleneck is identified, a correction factor can be applied to other initially unknown molecules in the starting population. For example, if the input Spank molecules are substantially all unique, but the recovered Spanks are only 50% unique, this suggests that the bottleneck and diversity loss can inform the interpretation of the diversity of other molecules from the sample.

[0241] A set of spanks can be incorporated into the sample at any step during sample processing to identify the step where the bottleneck occurs. For example, a first set of spanks may be introduced when collecting the sample from the subject (e.g., body fluids), a second set of spanks may be introduced into the sample before or during subsequent processing of the collected sample (e.g., removal of residual cells, storage), and / or a third set of spanks may be introduced before library preparation. In some cases, the spank sets incorporated into the sample at different steps in the sample processing procedure have the same or similar composition. In some cases, different spank sets are incorporated into the sample at different steps in the sample processing procedure.

[0242] In some cases, spank nucleic acids may each contain a randomized portion with a unique sequence. Spanks may contain one or more distinct domains. In some cases, spanks may contain one or more process codes, one or more diversity codes, one or more length marker sequences, one or more load sequences, one or more synthetic nucleic acid marker sequences (or spank marker sequences), and / or one or more characteristic domains. In some cases, spanks may contain a tag and a unique nucleic acid sequence.

[0243] When using different spank sets, each set can be coded using "process coding" to identify spank sets that introduce samples in a specific step (e.g., sample collection, extraction, library processing). In such cases, spanks with the same process code can be grouped bioinformatically and diversity loss analyzed. The degree of diversity loss associated with a specific step can then be determined and compared between each sample processing step.

[0244] Spanks may contain "diversity coding" associated with the synthetic nucleic acid pool or the overall pool or set of spanks. The diversity coding domain can be a unique code representing the amount of diversity within the synthetic nucleic acid pool. In such cases, a value representing the degree of diversity within the pool (e.g., 10) can be used. 8 A unique sequence (each synthetic nucleic acid in a diversity pool) encodes each synthetic nucleic acid within the diversity pool. In some cases, such as when two or more diversity pools are used on the same sample, diversity encoding can be used to identify diversity loss in two or more pools.

[0245] In some cases, a spank may contain one or more codes (e.g., process codes) that identify the spank as a member of a particular spank pool or set. In other cases, a spank may contain one or more spank-identifying domains that recognize the spank as a spank, rather than a nucleic acid initially present in the sample. A spank may also contain characteristic domains, length marker domains, and load domains, as further described herein.

[0246] Spank can be used alone or in combination with other synthetic nucleic acids to calculate nucleic acid abundance or for other applications. In some cases, Spank can be used together with other synthetic nucleic acids. For example, in some cases, a set of Spank and a set of Spark can be added to a sample. In some cases, sample identification nucleic acids can also be added to the sample.

[0247] Spank pools preferably contain diverse mixtures of nucleic acid sequences; therefore, spank pools can be designed to maximize diversity. In some cases, spank pools are derived from larger spank pools. For example, in some cases, a 75-bp oligonucleotide can be synthesized using two 8-bp N strings (e.g., A / C / G / T in equal proportions). The spank can be a synthetic nucleic acid containing (i) one or more identifying tags and (ii) a unique nucleic acid sequence. In some cases, the unique nucleic acid sequence can be multiple degenerate or random positions, such as two sets of 8-bp strings of degenerate positions separated by one or more nucleotides, as shown in the example. Figure 6 As shown in the table. Table 4 lists two exemplary sequences. The design incorporates two 8-bp N-string oligonucleotides containing a total of 16 Ns, corresponding to 4... 16 =4.3×10 9 A pool of different oligonucleotides. For example, if the pool contains 1×10 8 If a single molecule is incorporated into 1 mL of plasma and IDSpike and Spark are treated as described above, then almost all Spanks will be unique. For example, in this case, more than 90%, 95%, or 99% of Spanks can be unique.

[0248] In some cases, the length of a spank nucleic acid can be approximately or at least approximately 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides. In some cases, the length of a spank nucleic acid can be up to about 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides. In some cases, a spank nucleic acid can have a length ranging from about 20 to about 175 base pairs. In some cases, the nucleic acids in a spank group have the same length. In some cases, the nucleic acids in a spank group have two or more lengths (e.g., 2, 3, 4, 5, or more lengths).

[0249] In some cases, a spank nucleic acid may have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 degenerate positions. In some cases, a spank nucleic acid may have up to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 degenerate positions. In some cases, a spank nucleic acid may have a number of degenerate positions ranging from about 5 to about 25. In some cases, the degenerate positions may be consecutive, discrete, or divided into two or more groups, such as two, three, four, or five groups. In some cases, when degenerate positions are grouped, the degenerate positions may be evenly distributed between groups (e.g., two groups of 8-bp degenerate position strings, totaling 16 degenerate positions) or unevenly distributed between different groups (e.g., one group of 10 degenerate positions and another group of 6 degenerate positions, totaling 16 degenerate positions). In some cases, when degenerate positions are grouped, the groups may be separated by one or more nucleotides. In some cases, the groups may be separated by about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 nucleotides. In some cases, the groups may be separated by at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 nucleotides.

[0250] In some cases, Spank nucleic acids may have approximately or at least approximately 1 × 10⁻⁶. 3 1×10 4 1×10 5 1×10 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 1×10 7 2×10 7 3×10 7 4×10 7 5×10 7 6×10 7 7×10 7 8×10 7 9×10 7 1×10 8 2×10 8 3×108 4×10 8 5×10 8 6×10 8 7×10 8 8×10 8 9×10 8 1×10 9 2×10 9 3×10 9 4×10 9 5×10 9 6×10 9 7×10 9 8×10 9 9×10 9 1×10 10 Or 1×10 11 The diversity of unique sequences. In some cases, spank nucleic acids can have up to about 1 × 10⁻⁶ sequences. 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 1×10 7 2×10 7 3×10 7 4×10 7 5×10 7 6×10 7 7×10 7 8×10 7 9×10 7 1×10 8 2×10 8 3×10 8 4×10 8 5×10 8 6×10 8 7×10 8 8×10 8 9×10 8 1×10 9 2×10 9 3×10 9 4×10 9 5×10 9 6×10 9 7×10 9 8×10 9 9×10 9 1×1010 Or 1×10 11 The diversity of unique sequences. In some cases, spank nucleic acids can have a diversity of approximately 1 × 10⁻⁶. 4 To approximately 1×10 11 Diversity within a unique sequence range.

[0251] Table 4: Exemplary Spank Diversity Sequences

[0252]

[0253] Tracking sequence

[0254] Laboratory-derived nucleic acids (e.g., pathogen genomic DNA) can serve as standards for the development, validation, verification, and assay control of diagnostic tests for infectious diseases. However, because these same organisms can be present in clinical samples (e.g., samples infected with pathogens), laboratory-derived materials can cross-contaminate clinical samples during testing, leading to false positives. This can not only provide incorrect information to patients and physicians but may also trigger required reporting to health authorities for certain pathogen species. While real reference nucleic acids (e.g., real pathogen genomic DNA, cancer nucleic acids, tumor nucleic acids, or other disease-related nucleic acids) are useful and even necessary as positive controls, routine or even extreme caution in handling them may not be sufficient to prevent cross-contamination, especially for sensitive assays such as next-generation sequencing (NGS).

[0255] Synthetic tracer nucleic acids, which are not found in nature or cannot hybridize with sample nucleic acids, can be added to the original positive control nucleic acid at an effective concentration at least as high as that of the positive control nucleic acid. The tracer and positive control nucleic acids are in a form that allows them to be treated and detected in the same way. Therefore, the endpoint (e.g., sequence alignment in the case of NGS) is the same for both the tracer and the positive control nucleic acid, and due to the higher effective concentration of the tracer, it is at least as easily detectable as the positive control nucleic acid. In some cases, the positive control nucleic acid is pathogen genomic DNA. In some cases, the positive control nucleic acid contains disease-related nucleic acids, such as oncogenes.

[0256] The tracer sequence can vary in one or more properties, such as sequence, length, concentration, GC content, etc. The sequences shown in Table 5 and used in Example 6 have approximately 50% GC content, but the tracer sequence can be varied to match the composition of the positive control or their paired genome, for example, 30% GC content, 35% GC content, 40% GC content, 45% GC content, 50% GC content, 55% GC content, 60% GC content, 65% GC content, or 70% GC content.

[0257] In some cases, the tracer sequence can be added to the positive control nucleic acid or genomic DNA after fragmentation, as described in Example 6, for example. In other cases, the tracer sequence can be added to the positive control nucleic acid or genomic DNA before fragmentation to better represent the complete processing of the positive control nucleic acid or sample nucleic acid. Positive control nucleic acids that are scarce and present in low concentrations in clinical samples (e.g., pathogen DNA) can be labeled with the tracer sequence as early as possible to minimize cross-contamination with unlabeled nucleic acids.

[0258] In some cases, more than one tracer sequence is added to each positive control nucleic acid. In other cases, two or more, three or more, four or more, or five or more tracer sequences are added at the same or different concentrations.

[0259] Different forms of tracking sequences can be used for different applications. For example, the length of a tracking sequence can be matched with the length of a control sequence, such as the mean or median length. In some cases, the length of a tracking sequence can be within 5%, 10%, or 20% of the mean or median length of the control sequence.

[0260] RNA tracer sequences can be used for RNA applications.

[0261] Table 5: Exemplary Tracking Sequences

[0262]

[0263]

[0264] Molecular LIMS

[0265] A Laboratory Information Management System (LIMS) is a way to track the consumption and use of consumables. In some cases, it is used to ensure that only the chemicals or reagents necessary for a given experiment are used in that experiment. LIMS can also help track the batch number of chemicals used in each repeated experiment. All these functions (e.g., batch tracking) can help troubleshoot failed experiments, such as when the quality of a single chemical deteriorates or when incorrect reagents are used in the experiment.

[0266] LIMS systems can be designed as electronic or networked applications, where laboratory personnel enter the catalog and batch number of each consumable used in the process. Barcodes are typically used to speed up the process and improve its accuracy. However, for a given repeatable reaction, human error can still lead to incomplete recording.

[0267] This article provides methods for labeling reagents at the molecular level, particularly reagents, reagent batches, aliquots, or shipments. In some cases, the methods involve using incorporated synthetic nucleic acids to molecularly barcode various reagents in different containers. For example, adding an incorporated nucleic acid or a short nucleic acid oligomer (e.g., 50 to 100 bp) with a unique sequence (e.g., non-human, non-pathogen) to each reagent, reagent batch, reagent aliquot, or reagent shipment can help track reagent inventory used to prepare a specific library. In some cases, one or more ID Spike, Spark, or Spank sequences can be used for molecular LIMS. The batch number and reagents used in each sample processing can then be automatically detected by sequencing and can be used to troubleshoot problematic runs, for example, by comparing with the batch number used in successful runs or identifying missed or added reagents in processing that sample.

[0268] Similarly, the detection of incorporated nucleic acids associated with specific reagents, reagent batches, aliquots, or shipments can be used to identify the batches, aliquots, or shipments of reagents used in successful sequencing runs. In some cases, nucleic acids or incorporations can be detected by methods other than sequencing; for example, fluorescent detection of general polymers labeled with one or more fluorescent probes can be used.

[0269] While DNA oligomers can be used in many aqueous solutions, nucleic acid oligomers (e.g., RNA, DNA oligomers with modified backbones) resistant to DNase can be designed for solutions containing DNase. Similarly, synthetic nucleic acids (e.g., DNA) resistant to RNase can be used to track RNase-containing solutions.

[0270] Nucleic acid enrichment and library preparation

[0271] In the methods provided herein, nucleic acids can be isolated from samples using any means known in the art. For example, nucleic acids can be extracted using liquid extraction techniques (e.g., Trizol, DNAzol). Commercially available kits (e.g., QIAamp Cyclic Nucleic Acid Kit, Qiagen DNeasy Kit, QIAamp Kit, Qiagen Midi Kit, QIAprep Rotation Kit) can also be used to extract nucleic acids.

[0272] Nucleic acids can be enriched or precipitated using known methods, including, to name only, centrifugation. For purification purposes, nucleic acids can be bound to selective membranes (e.g., silica). Nucleic acids can also be enriched for desired lengths, such as fragments less than 1000, 500, 400, 300, 200, or 100 base pairs. Such size-based enrichment can be performed using, for example, PEG-induced precipitation, electrophoretic gels or chromatographic materials (Huber et al. (1993) Nucleic Acids Res. 21:1061-6), gel filtration chromatography or TSKgel (Kato et al. (1984) J. Biochem, 95:83-86), all of which are incorporated herein by reference in their entirety for all purposes.

[0273] Nucleic acid samples can be enriched for target polynucleotides, particularly those associated with inflammation or infection. In some preferred cases, the target nucleic acid is pathogen nucleic acid (e.g., cell-free pathogen nucleic acid). In some preferred cases, the target nucleic acid is cell-free RNA associated with a specific organ or tissue (including, but not limited to, uterus, heart, lung, kidney, fetal brain, liver, or cervical tissue).

[0274] Target enrichment can be performed by any method known in the art. For example, nucleic acid samples can be enriched by amplifying the target sequence using target-specific primers (e.g., primers specific to pathogen nucleic acids). Target amplification can occur in the form of digital PCR using any method or system known in the art. Nucleic acid samples can be enriched by capturing the target sequence onto an array on which target-selective oligonucleotides are immobilized. Nucleic acid samples can be enriched by hybridizing with target-selective oligonucleotides free in solution or on a solid support. The oligonucleotides may contain a capture portion that can be captured by a capture reagent. In some embodiments, the nucleic acid sample is not enriched for a target polynucleotide, for example, representing the entire genome.

[0275] In some cases, target (e.g., pathogen, organ) nucleic acids can be enriched relative to background (e.g., subject, healthy tissue) nucleic acids in the sample, for example by pull-down assays (e.g., by hybridizing the target nucleic acid with complementary oligonucleotides conjugated to a label such as a biotin tag and using, for example, avidin or streptoavidin attached to a solid support, preferentially pulling down the target nucleic acid in a pull-down assay), targeted PCR, or other methods. Examples of enrichment techniques include, but are not limited to: (a) self-hybridization techniques, in which the major population in the nucleic acid sample self-hybridizes faster than the minor population in the sample; (b) depletion of nucleosome-associated DNA from cell-free DNA; (c) removal and / or isolation of DNA in specific length ranges; (d) foreign body depletion or enrichment; and (e) strategic capture of regions of interest.

[0276] In some cases, the enrichment step includes (a) providing a nucleic acid sample from the host, wherein the nucleic acid sample from the host is a single-stranded nucleic acid sample from the host and contains both host nucleic acids and non-host nucleic acids; (b) renaturing at least a portion of the single-stranded nucleic acids from the host to generate a population of double-stranded nucleic acids within the sample; and (c) using a nuclease to remove at least a portion of the double-stranded nucleic acids within the sample, thereby enriching the non-host sequences in the nucleic acid sample from the host. In some cases, the enrichment step includes (a) providing a nucleic acid sample from the host, wherein the nucleic acid sample from the host contains both nucleosome-associated host nucleic acids and non-host nucleic acids; and (b) removing at least a portion of the nucleosome-associated host nucleic acids, thereby enriching the non-host nucleic acids in the nucleic acid sample from the host. In some cases, the enrichment step includes (a) providing a nucleic acid sample from the host, wherein the nucleic acid sample from said host contains both host nucleic acids and non-host nucleic acids; and (b) removing or isolating DNA having one or more length ranges, thereby enriching the non-host nucleic acids in the nucleic acid sample from said host. In some cases, the enrichment step includes (a) providing a nucleic acid sample from a host, wherein the nucleic acid sample from said host comprises host nucleic acids, non-host nucleic acids, and foreign bodies; and (b) removing or isolating at least a portion of the foreign bodies, thereby enriching the non-host sequences in the nucleic acid sample from said host. In some cases, the enrichment step includes preferentially removing nucleic acids longer than about 300 bases from the sample. In some cases, the enrichment step includes preferentially amplifying or capturing non-host nucleic acids from the sample.

[0277] The enrichment step may include preferentially removing nucleic acids of length greater than about 120, about 150, about 200, or about 250 bases from the sample. In some cases, the enrichment step includes preferentially enriching nucleic acids of length from about 10 to about 60 bases, from about 10 to about 120 bases, from about 10 to about 150 bases, from about 10 to about 300 bases, from about 30 to about 60 bases, from about 30 to about 120 bases, from about 30 to about 150 bases, from about 30 to about 200 bases, or from about 30 to about 300 bases. In some cases, the enrichment step includes preferentially digesting nucleic acids derived from the host (e.g., a subject). In some cases, the enrichment step includes preferentially replicating non-host nucleic acids.

[0278] In some cases, the enrichment step increases the ratio of non-host nucleic acids to host (e.g., subject) nucleic acids by at least 2X, at least 3X, at least 4X, at least 5X, at least 6X, at least 7X, at least 8X, at least 9X, at least 10X, at least 11X, at least 12X, at least 13X, at least 14X, at least 15X, at least 16X, at least 17X, at least 18X, at least 19X, at least 20X, at least 30X, at least 40X, at least 50X, at least 60X, at least 70X, at least 80X, at least 90X, at least 100X, at least 1000X, at least 5000X, or at least 10,000X. In some cases, the enrichment step increases the ratio of non-host nucleic acids to host (e.g., subject) nucleic acids by at least 10X. In some cases, the enrichment step increases the ratio of non-host nucleic acids to host (e.g., subject) nucleic acids by a range from about 10X to about 100X.

[0279] In some cases, nucleic acid libraries are prepared. Nucleic acid libraries can be single-stranded or double-stranded. In some cases, single-stranded nucleic acid libraries can be single-stranded DNA libraries (ssDNA libraries) or RNA libraries. In some cases, double-stranded nucleic acid libraries are double-stranded DNA libraries (dsDNA libraries). Methods for preparing ssDNA libraries may include denaturing a double-stranded DNA fragment into an ssDNA fragment, ligating a primer docking sequence to one end of the ssDNA fragment, and hybridizing the primers to the primer docking sequence. The primers may contain at least a portion of an adaptor sequence coupled to a next-generation sequencing platform. The method may further include extending the hybridized primers to produce a double-stranded structure, wherein the double-stranded structure comprises the original ssDNA fragment and an extended primer strand. The extended primer strand may be separated from the original ssDNA fragment. The extended primer strand may be collected, wherein the extended primer strand is a member of the ssDNA library. Methods for preparing RNA libraries may include ligating a primer docking sequence to one end of an RNA fragment and hybridizing the primers to the primer docking sequence. The primers may contain at least a portion of an adaptor sequence conjugated to a next-generation sequencing platform. The method may further include extending the hybridization primers to produce a double-stranded structure, wherein the double-stranded structure comprises an original RNA fragment and an extended primer strand. The extended primer strand may be separated from the original RNA fragment. The extended primer strand may be collected, wherein the extended primer strand is a member of the RNA library. Methods for preparing a dsDNA library may include ligating an adaptor sequence to one or both ends of a dsDNA fragment.

[0280] In all respects, dsDNA can be fragmented in any manner known in the art or as described herein. In some cases, dsDNA can be fragmented by physical means (e.g., mechanical shearing, atomization, or sonication), by enzymatic means, or by chemical means.

[0281] In some implementations, cDNA is generated from RNA. For example, cDNA can be generated by reverse transcription (RNase H+) using random primers to produce cDNA of random size.

[0282] The length of nucleic acids can vary. Nucleic acids or nucleic acid fragments (e.g., dsDNA fragments, RNA, or randomly sized cDNA) can be less than 1000 bp, less than 800 bp, less than 700 bp, less than 600 bp, less than 500 bp, less than 400 bp, less than 300 bp, less than 200 bp, or less than 100 bp. DNA fragments can be approximately 40 to approximately 100 bp, approximately 50 to approximately 125 bp, approximately 100 to approximately 200 bp, approximately 150 to approximately 400 bp, approximately 300 to approximately 500 bp, approximately 100 to approximately 500 bp, approximately 400 to approximately 700 bp, approximately 500 to approximately 800 bp, approximately 700 to approximately 900 bp, approximately 800 to approximately 1000 bp, or approximately 100 to approximately 1000 bp. In some cases, nucleic acids or nucleic acid fragments (e.g., dsDNA fragments, RNA, or randomly sized cDNA) can be in the range of approximately 20 to approximately 200 bp, such as in the range of approximately 40 to approximately 100 bp.

[0283] The ends of dsDNA fragments can be flattened (e.g., made blunt-ended). This can be achieved by treating the DNA fragment with a polymerase. Flattening may include removing 3' overhangs, filling 5' overhangs, or a combination thereof. The polymerase may be a correction polymerase (e.g., containing 3' to 5' exonuclease activity). Correction polymerases may be, for example, T4 DNA polymerase, Pol 1Klenow fragment polymerase, or Pfu polymerase. Flattening may include removing damaged nucleotides (e.g., abase sites) using any means known in the art.

[0284] Linking an adaptor to the 3' end of a nucleic acid fragment may include forming a bond between the 3' OH group of the fragment and the 5' phosphate group of the adaptor. Therefore, removing the 5' phosphate group from the nucleic acid fragment minimizes aberrant linkages between two library members. Thus, in some embodiments, the 5' phosphate group is removed from the nucleic acid fragment. In some embodiments, the 5' phosphate group is removed from at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95% of the nucleic acid fragment in the sample. In some embodiments, substantially all phosphate groups are removed from the nucleic acid fragment. In some embodiments, substantially all phosphate groups are removed from at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95% of the nucleic acid fragment in the sample. Phosphate groups can be removed from a nucleic acid sample by any means known in the art. Phosphate group removal may include treating the sample with a heat-labile phosphatase. In some embodiments, phosphate groups are not removed from the nucleic acid sample. In some implementations, the adaptor is ligated to the 5' end of the nucleic acid fragment.

[0285] sequencing

[0286] This disclosure provides methods for analyzing nucleic acids. Such analytical methods include sequencing the nucleic acids and performing bioinformatics analysis on the sequencing results. Nucleic acids produced according to this method can be analyzed to obtain various types of information, including genome, epigenetics (e.g., methylation), and RNA expression. Methylation analysis can be performed, for example, by converting methylated bases followed by DNA sequencing. RNA expression analysis can be performed, for example, by multinucleotide array hybridization, RNA sequencing technology, or sequencing of cDNA derived from RNA.

[0287] In a preferred embodiment, sequencing is performed using next-generation sequencing analysis. As used herein, the term "next-generation" is well understood in the art and generally refers to any high-throughput sequencing method, including but not limited to one or more of the following: massively parallel signature sequencing, pyrosequencing (e.g., using a Roche 454 sequencing device), Illumina (Solexa) sequencing, sequencing synthesis (Illumina), Ion torrent sequencing, ligation sequencing (e.g., SOLiD sequencing), single-molecule real-time (SMRT) sequencing (e.g., Pacific Biosciences), polymerase cloning sequencing, DNA nanosphere sequencing, Heliscope single-molecule sequencing (Helicos Biosciences), and nanopore sequencing (e.g., Oxford Nanopore). In some cases, sequencing analysis uses nanopore sequencing. In some cases, sequencing analysis includes some form of Sanger sequencing. In some cases, sequencing involves shotgun sequencing; in some cases, sequencing includes bridge PCR. In some cases, sequencing is broad-spectrum. In some cases, sequencing is targeted.

[0288] In some cases, sequencing analysis includes the Gilbert sequencing method. In such methods, nucleic acids (e.g., DNA) are chemically modified and then cleaved at specific bases. In other cases, sequencing analysis includes dideoxynucleotide chain termination or Sanger sequencing.

[0289] The method described herein utilizes a synthetic sequencing approach. In some cases, fluorescently labeled reversible terminator nucleotides are introduced into a clone-amplified DNA template immobilized on a glass flow cell surface. During each sequencing cycle, a single labeled deoxynucleoside triphosphate (dNTP) can be added to the nucleic acid strand. The labeled terminator nucleotide can be imaged upon addition to identify bases, and then enzymatically cleaved to allow incorporation into the next nucleotide. Since all four reversibly terminator-bound dNTPs (A, C, T, G) typically exist as single molecules, natural competition minimizes incorporation bias.

[0290] In some cases, a method called single-molecule real-time (SMRT) is used. In such an approach, nucleic acids (e.g., DNA) are synthesized in a zero-mode waveguide (ZMW), a small, porous container with a trapping tool located at the bottom of the pore. Sequencing is performed using an unmodified polymerase (attached to the bottom of the ZMW) and fluorescently labeled nucleotides flowing freely in solution. The fluorescent label detaches from the nucleotides as it is incorporated into the DNA strand, leaving the unmodified DNA strand. The light emission can then be detected using a detector such as a camera; and the data can be analyzed bioinformatically to obtain sequence information.

[0291] In some cases, sequencing via ligation pathways is used to sequence nucleic acids in a sample. One example is SOLiD (Sequencing by Oligonucleotide Ligation and Detection) sequencing (Life Technologies), a next-generation sequencing method. This next-generation technology can generate hundreds of millions to billions of small sequence reads simultaneously. The sequencing method may include preparing a library of DNA fragments from the sample to be sequenced. In some cases, this library is used to prepare a population of cloning beads, where only one fragment is present on the surface of each bead (e.g., magnetic beads). The fragments attached to the magnetic beads may have a universal P1 adaptor sequence for ligation, such that the starting sequence of each fragment is known and identical. In some cases, the method may further involve PCR or emulsion PCR. For example, emulsion PCR may involve using a microreactor containing reagents for PCR. The resulting PCR products attached to the beads can then be covalently bound to a glass slide. Sequencing analysis (such as SOLiD sequencing analysis or other sequencing analyses via ligation) may include steps involving the use of primers. Primers may hybridize with the P1 adaptor sequence or other sequences within the library template. The method may further involve introducing four fluorescently labeled dibase probes that competitively ligate to sequencing primers. Specificity of the dibase probes can be achieved by querying each first and second base in each ligation reaction. Multiple cycles of ligation, detection, and cleavage can be performed, where the number of cycles determines the final read length. In some cases, after a series of ligation cycles, the extension product is removed, and the template is reset with a primer complementary to the n-1 position for a second ligation cycle. Multiple rounds (e.g., 5 rounds) of primer reset can be performed for each sequence tag. Through the primer reset process, each base can be queried in two separate ligation reactions using two different primers. For example, the base at read position 5 can be determined in ligation cycle 2 using primer number 2 and in ligation cycle 1 using primer number 3.

[0292] In any implementation, the detection or quantification of oligonucleotides can be performed via sequencing. Subunits or complete synthetic oligonucleotides can be detected by complete sequencing of all oligonucleotides using any suitable method known in the art, such as the Illumina HiSeq 2500, including the sequencing methods described herein.

[0293] Sequencing can be performed using the classic Sanger sequencing method, well-known in the art. High-throughput systems can also be used, some of which allow detection of the sequenced nucleotides immediately or simultaneously after incorporation into the growing strand, for example, real-time or near-real-time sequence detection. In some cases, high-throughput sequencing generates at least 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 100,000, or 500,000 sequence reads per hour. In some cases, each read is at least 50, 60, 70, 80, 90, 100, 120, or 150 bases. In some cases, each read is up to 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 bases long. Long-read sequencing may include sequencing consecutive sequence reads that provide, for example, more than 500, 800, 1000, 1500, 2000, 3000, or 4500 bases long.

[0294] In some cases, high-throughput sequencing involves the use of techniques available from Illumina's Genome Analyzer II, MiSeq personal sequencers, or HiSeq systems (such as the HiSeq 2500, HiSeq 1500, HiSeq 2000, or HiSeq 1000). These machines use sequencing based on reversible terminators via synthetic chemistry. These machines can complete 200 billion DNA reads or more in 8 days. Smaller systems can be used to run in 3 days, 2 days, or 1 day or less. Shorter synthesis cycles can be used to minimize the time spent obtaining sequencing results.

[0295] In some cases, high-throughput sequencing involves the use of technologies available from the ABI Solid System. This genetic analysis platform enables massively parallel sequencing of bead-linked cloned amplified DNA fragments. The sequencing method is based on the sequential ligation of dye-labeled oligonucleotides.

[0296] Next-generation sequencing can include ion semiconductor sequencing (e.g., using technology from Life Technologies (IonTorrent)). Ion semiconductor sequencing leverages the fact that ions are released when nucleotides are incorporated into a DNA strand. For ion semiconductor sequencing, a high-density array of micromechanical pores can be formed. Each pore can hold a single DNA template. Below the pores can be an ion-sensitive layer, and below that, an ion sensor. When nucleotides are added to the DNA, H+ ions are released, which can be measured as a change in pH. The H+ ions are converted into a voltage and recorded by the semiconductor sensor. The array chip can be sequentially filled with successive nucleotides. No scanning, light, or camera is required. In some cases, IONPROTON is used. TM Sequencing instruments are used to sequence nucleic acids. In some cases, IONPGM is used. TM Sequencing instrument. The Ion Torrent Personal Genome Machine (PGM) can complete 10 million reads in two hours.

[0297] In some cases, high-throughput sequencing involves the use of technologies available from Helicos BioSciences Corporation (Cambridge, MA), such as synthetic single-molecule sequencing (SMSS) methods. SMSS allows sequencing of the entire human genome in up to 24 hours. Similar to MIP technology, SMSS may not require a pre-amplification step before hybridization. SMSS may not require any amplification. Parts of the SMSS description are found in U.S. Publication Nos. 20060024711; 20060024678; 20060012793; 20060012784; and 20050100932.

[0298] In some cases, high-throughput sequencing involves the use of technologies available from 454 Lifesciences, Inc. (Branford, Connecticut), such as the Pico titration plate device, which includes an optical fiber plate that transmits the chemiluminescent signal generated by the sequencing reaction to be recorded by a CCD camera in the instrument. The use of this optical fiber allows for the detection of at least 20 million base pairs in 4.5 hours.

[0299] The method of using bead amplification followed by fiber optic detection is described in Marguiles, M. et al., "Genomesequencing in microfabricated high-density picolitre reactors", Nature, doi:10.1038 / nature03959; and U.S. Publication Nos. 20020012930; 20030058629; 20030100102; 20030148344; 20040248161; 20050079510; 20050124022; and 20060078909.

[0300] In some cases, high-throughput sequencing is performed using cloned single-molecule arrays (Solexa, Inc.) or sequencing-by-synthesis (SBS) utilizing reversible terminator chemistry. These techniques are described in part in U.S. Patent Nos. 6,969,488; 6,897,023; 6,833,246; 6,787,308; and U.S. Publication Nos. 20040106110; 20030064398; 20030022207; and Constans, A., The Scientist 2003, 17(13):36.

[0301] In some cases, next-generation sequencing is nanopore sequencing (see, for example, Soni GV and Meller A. (2007) Clin Chem 53:1996-2001). Nanopores can be small pores, for example, with a diameter of about 1 nanometer. Immersing a nanopore in a conductive fluid and applying a potential therebetween results in a slight current generated by the conduction of ions through the nanopore. The amount of current flowing can be sensitive to the size of the nanopore. As a DNA molecule passes through the nanopore, each nucleotide on the DNA molecule can block the nanopore to varying degrees. Therefore, the change in the current flowing through the nanopore as a DNA molecule passes through can indicate a readout of the DNA sequence. Nanopore sequencing technology can be derived from Oxford Nanopore Technologies; for example, the GridION system. Individual nanopores can be inserted into a polymer membrane that passes through the top of a micropore. Each micropore can have an electrode for individual sensing. Micropores can be fabricated into array chips, each chip having 100,000 or more micropores (e.g., greater than 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000). Instruments (or nodes) can be used to analyze the chips. Data can be analyzed in real time. One or more instruments can be operated simultaneously. Nanopores can be protein nanopores, such as protein α-hemolysin or heptameric protein pores. Nanopores can be solid-state nanopores made from nanoscale pores, for example, formed in synthetic membranes (e.g., SiNx or SiO2). Nanopores can be hybrid pores (e.g., protein pores integrated into solid membranes). Nanopores can be nanopores with integrated sensors (e.g., tunneling electrode detectors, capacitive detectors, or graphene-based nanogap or edge state detectors (see, for example, Garaj et al. (2010) Nature vol.67, doi:10.1038 / nature09379)). Nanopores can be functionalized to analyze specific types of molecules (e.g., DNA, RNA, or proteins). Nanopore sequencing can include “strand sequencing,” where a complete DNA polymer can pass through a protein nanopore, and sequencing is performed in real time as the DNA translocates within the pore. Enzymes can separate the strands of double-stranded DNA and advance the strands through the nanopore. One end of the DNA may have a hairpin, and the system can read both strands. In some cases, nanopore sequencing is “exonuclease sequencing,” where individual nucleotides can be processed by an exonuclease to cleave the DNA strand, and the nucleotides can pass through the protein nanopore. Nucleotides can transiently bind to molecules in the pore (e.g., cyclodextrins). Characteristic disruption by an electric current can be used to identify bases.

[0302] Nanopore sequencing technology from GENIA can be used. Engineered protein pores can be embedded in lipid bilayer membranes. “Active control” technology can be used to achieve efficient nanopore-membrane assembly and control the movement of DNA through the channels. In some cases, nanopore sequencing technology comes from NABsys. Genomic DNA can be fragmented into strands with an average length of approximately 100 kb. 100 kb fragments can be made into single strands and subsequently hybridized with hexameric probes. Genomic fragments with probes can be driven through nanopores, which generates current-time tracking. Current tracking provides the position of the probe on each genomic fragment. Genomic fragments can be aligned to generate probe maps against the genome. This process can be performed in parallel against probe libraries. Genomic-length probe maps for each probe can be generated. Errors can be corrected through a process called “moving window hybridization sequencing (mwSBH)”. In some cases, nanopore sequencing technology comes from IBM / Roche. Electron beams can be used to create nanopore-sized openings in microchips. DNA can be pulled or passed through nanopores using an electric field. DNA transistor devices in nanopores can include alternating nanoscale metal and dielectric layers. The discrete charges in the DNA backbone can be captured by an electric field within the DNA nanopores. The gate voltages, which are then turned off and on, allow the DNA sequence to be read.

[0303] Next-generation sequencing may include DNA nanosphere sequencing (e.g., via Complete Genomics; see, for example, Drmanac et al. (2010) Science 327:78-81). DNA can be isolated, fragmented, and size-selected. For example, DNA can be fragmented (e.g., by sonication) to an average length of approximately 500 bp. An adaptor (Adl) can be attached to the end of the fragment. The adaptor can be used to hybridize with an anchor for a sequencing reaction. The DNA bound to each end by the adaptor can be amplified by PCR. The adaptor sequence can be modified so that complementary single-stranded ends bind together to form circular DNA. DNA can be methylated to protect it from cleavage by IIS-type restriction enzymes used in subsequent steps. The adaptor (e.g., the right adaptor) may have a restriction recognition site, and the restriction recognition site may remain unmethylated. The unmethylated restriction recognition site in the adaptor can be recognized by a restriction enzyme (e.g., Acul), and Acul can cleave the DNA 13 bp to the right of the right adaptor to form linear double-stranded DNA. The second round of right and left adaptors (Ad2) can be ligated to either end of the linear DNA, and all DNA bound by the two adaptors can be amplified by PCR (e.g., via PCR). The Ad2 sequence can be modified to allow them to bind to each other and form circular DNA. The DNA can be methylated, but the restriction enzyme recognition site on the left Ad1 adaptor remains unmethylated. A restriction enzyme (e.g., Acul) can be applied, and the DNA can be cleaved 13 bp to the left of Ad1 to form a linear DNA fragment. The third round of right and left adaptors (Ad3) can be ligated to the right and left sides of the linear DNA, and the resulting fragment can be amplified by PCR. The adaptors can be modified so that they can bind to each other and form circular DNA. A type III restriction enzyme (e.g., EcoP15) can be added; EcoP15 cleaves the DNA 26 bp to the left of Ad3 and 26 bp to the right of Ad2. This cleavage removes a large segment of DNA and linearizes the DNA again. The fourth round of right and left adaptors (Ad4) can be linked to DNA, which can be amplified (e.g., by PCR) and modified so that they bind together to form a complete circular DNA template.

[0304] Small fragments of DNA can be amplified using rolling circle replication (e.g., using Phi 29 DNA polymerase). The four adaptor sequences may contain palindromic sequences capable of hybridization, and the single strands can fold onto themselves to form DNA nanospheres (DNBs). TMThe average diameter of the DNA nanospheres can be approximately 200-300 nanometers. DNA nanospheres can be attached (e.g., by adsorption) to a microarray (sequencing flow cell). The flow cell can be a silicon wafer coated with silica, titanium, and hexamethyldisilazane (HMDS) and a photoresist material. Sequencing can be performed via non-stranded sequencing by attaching fluorescent probes to the DNA. The fluorescence color at the query location can be visualized using a high-resolution camera. The identity of the nucleotide sequences between adaptor sequences can be determined.

[0305] The methods provided herein may include using a system, such as one containing a nucleic acid sequencer (e.g., a DNA sequencer, an RNA sequencer), to generate DNA or RNA sequence information. The system may include a computer containing software for performing bioinformatics analysis of the DNA or RNA sequence information. Bioinformatics analysis may include, but is not limited to, compiling sequence data, detecting and quantifying genetic variants in a sample, including germline variants and somatic variants (e.g., genetic variants associated with cancer or precancerous conditions, genetic variants associated with infection).

[0306] Sequencing data can be used to determine genetic sequence information, ploidy status, the identity of one or more genetic variants, and quantitative measures of variants (including relative and absolute relative measures).

[0307] In some cases, genome sequencing involves whole-genome sequencing or partial genome sequencing. Sequencing can be unbiased and can involve sequencing all or substantially all (e.g., greater than 70%, 80%, 90%) of the nucleic acids in a sample. Genome sequencing can be selective, for example, targeting a portion of the genome of interest. For example, many genes (and mutated forms of these genes) are known to be associated with various cancers. Sequencing of selected genes or gene portions can satisfy the desired analysis. Polynucleotides mapped to specific loci in the genome of the subject of interest can be isolated for sequencing via, for example, sequence capture or site-specific amplification.

[0308] application

[0309] The methods described herein can be used for a variety of purposes, such as diagnosing or detecting conditions (e.g., infection), predicting the occurrence or recurrence of conditions, monitoring treatment, selecting or modifying treatment regimens, or optimizing treatment. Using this approach, treatment and / or diagnostic protocols can be individualized and tailored based on data obtained at different times during treatment, thereby providing an individualized approach.

[0310] Detection / Diagnosis / Prediction of Disease

[0311] The methods described herein can be used to detect, diagnose, or predict infections or diseases in patient samples, such as human blood samples. These methods can be used to detect rare fragments of microbial nucleic acids in samples primarily composed of human nucleic acids. For example, cell-free DNA (cfDNA) in blood consists mainly of DNA fragments derived from the host, but also contains small fragments from in vivo microorganisms. Extracting cfDNA and then performing deep sequencing (e.g., next-generation sequencing or NGS) can produce millions or billions of sequence reads that can be mapped against host and non-host genome databases. Similarly, these methods can be used to detect rare circulating or cell-free RNA populations from specific organs. For samples where non-host reads constitute a very small percentage of the total, the methods described herein improve the sensitivity and specificity of the assay (otherwise hampered by a lack of internal normalization criteria) to compare different target nucleic acids (e.g., derived from different microorganisms or organisms) or to track different samples or reagents. Furthermore, these methods can be used in cases where the target nucleic acids constitute a large portion of the total nucleic acid population.

[0312] The methods provided herein can be used to detect, monitor, diagnose, prognose, treat, or prevent a variety of diseases and conditions. In particular, the methods can be used to detect one or more target nucleic acids derived from pathogens associated with infectious diseases or conditions. Exemplary diseases and conditions include any infection-related disease or condition, such as sepsis, pneumonia, tuberculosis, HIV infection, hepatitis infection (e.g., hepatitis A, hepatitis B, or hepatitis C), human papillomavirus (HPV) infection, chlamydia infection, syphilis infection, Ebola infection, Staphylococcus aureus infection, or influenza. The methods provided herein are particularly suitable for detecting infections caused by drug-resistant microorganisms, including multidrug-resistant microorganisms, or microorganisms that are difficult to culture or commonly tested.Some non-limiting examples of diseases and conditions that can be detected using this method include: cancer, dilated cardiomyopathy, Guillain-Barré syndrome, multiple sclerosis, tuberculosis, anthrax poisoning, sleeping sickness, dysentery, toxoplasmosis, tinea, candidiasis, histoplasmosis, Ebola, Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired Immunodeficiency Syndrome), HIV infection, amoebiasis, anaplasmosis, Cryptococcus hemolyticus infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, and bacterial vaginosis (BV). Bacteroides infection, giardiasis, ascariasis, BK virus infection, melanoma, blastomycosis, Bolivian hemorrhagic fever, Treponema pallidum infection, botulism (including infant botulism), Brazilian hemorrhagic fever, brucellosis, bubonic plague, Burkholderia infection, Buruli ulcer, calicivirus infection (norovirus and Sapporo virus), Campylobacteriosis, candidiasis (candidiasis; thrush), cat scratch disease, cellulitis, Chagas disease (American trypanosomiasis), chancroid, varicella, Chekungunya fever, Chlamydia, Chlamydia pneumoniae infection (Taiwan acute respiratory pathogens). (agent) or TWAR), cholera, chromoblastomycosis, clonorchiasis, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever (CTF), common cold (acute viral nasopharyngitis; acute rhinitis), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), cryptococcosis, cryptosporidiosis, cutaneous larval migration syndrome (CLM), protococcosis, cysticercosis, cytomegalovirus infection, dengue fever, Binuclear amoebiasis, diphtheria, sparganosis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, pinworm infection, enterococcal infection, enterovirus infection, epidemic typhus, erythema infectiosum (fifth disease), roseola infantum (sixth disease), fascioliasis, liver fluke infection, filariasis, food poisoning caused by Clostridium perfringens, infection with living amoeba, fusobacterium infection, gas gangrene (clostridial myonecrosis), geomycosis, Gerstmann's disease. - Scheinker syndrome (GSS), Giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma inguinale (the fifth sexually transmitted disease), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand-foot-mouth disease (HFMD), Hantavirus pulmonary syndrome (HPS), Heartland virus disease, Helicobacter pylori infection, hemolytic uremic syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), hepatitis A, hepatitis B, hepatitis C Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human Ehrlich's disease, Human granulocytic anaplasmosis (HGA), Human metapneumovirus infection, Human monocytic erythrocyte disease, Human papillomavirus (HPV) infection, Human parainfluenza virus infection, Hymenolepis hymenodelasia, Epstein-Barr virus infectious mononucleosis (Mono), Influenza (flu), Coccidioidomycosis, Kawasaki disease, Keratitis, Laryngotracheitis kinens infection, Kuru Lassa fever, Legionnaires' disease (Legionis), Legionnaires' disease (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listeria infection, Lyme disease (Lyme Borrelia), Lymphatic filariasis (elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever (MHF), Measles, Middle East Respiratory Syndrome (MERS), Whitmore's disease, Meningitis, Meningococcal infection, Paragonimiasis, Microsporidiosis, Molluscum contagiosum (… MC), monkeypox, mumps, murine typhus (endemic typhus), mycoplasma pneumonia, Madura mycobacterium infection, myiasis, neonatal conjunctivitis (neonatal ophthalmia), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiasis, onchocerciasis (river blindness), paracoccidioidomycosis (South American bud fungicide), paragonimiasis, pastoral infection, head lice, body lice, pubic liceCrab lice), pelvic inflammatory disease (PID), pertussis, plague, pneumococcal infection, Pneumocystis pneumonia (PCP), pneumonia, poliomyelitis, Prevotella infection, primary amoebic meningoencephalitis (PAM), progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsial pox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), rotavirus infection, rubella, salmonellosis, severe acute respiratory syndrome (SARS), scabies, schistosomiasis, sepsis, Shigella infection (bacterial dysentery), herpes zoster, smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute sclerosis. Panencephalitis, syphilis, tapeworm infection, tetanus (trismus), tinea barbae (facial tinea), tinea capitis (scalp tinea), tinea corporis (body tinea), tinea cruris (jock itch), tinea manuum (hand tinea), palmar tinea, tinea pedis (athlete's foot), onychomycosis (nail fungus), tinea versicolor (pityriasis versicolor), toxocariasis (ocular larval migration (OLM)), toxocariasis (visceral larval migration (VLM)), trachoma, trichomonas, trichinosis, trichomonas, whipworm infection, tuberculosis, tularemia, typhoid fever, Ureaplasma urealyticum infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, trichomonaditis (white spot disease), pseudotuberculosis Yersinia infection, Yersinia infection, yellow fever, Zika virus, and zygomycosis.

[0313] In some cases, the methods described herein involve determining whether an infection is active or latent. In some cases, gene expression quantification can provide a method for detecting, predicting, diagnosing, or monitoring active infection. In some cases, the methods described herein involve detecting active infection. In some cases, gene expression can be quantified by detecting or sequencing one or more target nucleic acids of interest. In some cases, gene expression quantification can provide a method for detecting, predicting, diagnosing, or monitoring latent infection. In some cases, the methods described herein involve detecting latent infection.

[0314] The methods described in this article can be used to detect cancer, particularly in subjects who have, are at risk of, or are suspected of having such cancer. Examples of cancers include, but are not limited to: brain cancer, head and neck cancer, laryngeal cancer, oral cancer, breast cancer, bone cancer, blood cancer, leukemia, lymphoma, lung cancer, kidney cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, skin cancer, reproductive tract cancer, prostate cancer, etc. In some cases, the methods described in this article can be particularly useful for detecting non-hematologic cancers, such as cancers of solid organs (e.g., lung cancer, breast cancer, pancreatic cancer, etc.).

[0315] The methods described can also be used to detect any other type of disease or condition in the subject. Typically, they can be used to detect rare genetic variations; or nucleic acid sequences that constitute only a very small portion of the total nucleic acid population in a sample.

[0316] Detection of pathogen or organ nucleic acids may involve comparing the level of pathogen or organ nucleic acids to a control or reference value to determine the presence or absence of pathogen or organ nucleic acids and / or the quantity of pathogen or organ nucleic acids. This level can be qualitative or quantitative. In some cases, the control or reference value is a predetermined absolute value indicating the presence or absence of cell-free pathogen nucleic acids or cell-free organ-derived nucleic acids. For example, detecting a cell-free pathogen nucleic acid level above the control value may indicate the presence of a pathogen or infection, while a level below the control value may indicate the absence of a pathogen or infection. The control value may be a value obtained by analyzing the cell-free nucleic acid levels of uninfected subjects; in some cases, the control value may be a positive control and may be obtained by analyzing cell-free nucleic acids from subjects with a specific infection or a specific infection in a specific organ.

[0317] In some cases, to determine the presence of an infection—and often to obtain precise results—one or more of the following methods may be applied: (i) as described in patent WO 2015070086 A1, all reads obtained by sequencing may be compared with a targeted host genome reference database, which may be from humans, dogs, cats, primates or any other host, including, for example, the GenBank hg19 human reference sequence; (ii) a data processor for bioinformatics analysis may subtract or isolate host sequences so that only non-host sequences, including pathogen-related sequences, can be further analyzed; (iii) a data processor may determine the presence of one or more pathogens by comparing non-host sequences with a targeted microbial reference sequence database (including, for example, reference sequences from GenBank and Refseq); (iv) a statistical analysis framework may be applied to determine whether the presence of one or more pathogens is statistically significant; and / or (v) in some cases, a data processor may quantify the amount of pathogen present based on a comparison of the number of reads obtained for the pathogen with the number of reads obtained by a control molecule incorporated into the sample at a known concentration prior to sequencing.

[0318] Control values ​​can be the levels of cell-free pathogen or organ-specific nucleic acids obtained from subjects (e.g., subjects with or suspected of having an infection) at different time points (e.g., time points prior to the testing time point). In such cases, comparisons of levels at different time points can indicate the presence of infection, infection in a specific organ, improvement in infection, or worsening of infection. For example, an increase in cell-free pathogen nucleic acid over time can indicate the presence of infection or worsening of infection; for instance, an increase of at least 5%, 10%, 20%, 25%, 30%, 50%, 75%, 100%, 200%, 300%, or 400% in pathogen or organ-specific cell-free nucleic acid compared to the original value can indicate the presence of infection or worsening of infection. In other instances, a decrease of at least 5%, 10%, 20%, 25%, 30%, 50%, 75%, 100%, 200%, 300%, or 400% in pathogen or organ-specific cell-free nucleic acid compared to the original value can indicate the absence of infection or improvement in infection. Typically, such measurements can be taken over a specific time period, such as daily, every other day, weekly, every other week, monthly, or every other month. For example, an increase of at least 50% in the nucleic acid of pathogens or organoid cells within a week can indicate the presence of an infection.

[0319] Control or reference values ​​can be measured as concentration or the number of sequencing reads. Control or reference values ​​can be pathogen-dependent. For example, control values ​​for *Escherichia coli* may differ from those for *Mycoplasma hominis*. Databases of levels or control values ​​can be generated based on samples obtained from one or more subjects, one or more pathogens, one or more organs, and / or one or more time points. Such databases can be targeted or proprietary. Recommended treatment options can be based on different threshold levels. For example, low levels may indicate infection, but treatment may not be necessary; moderate levels may lead to antibiotic treatment; high levels may require immediate or severe intervention.

[0320] The methods described herein can generate sequencing data with high efficiency, high accuracy, and / or high sensitivity. Typically, these methods can detect pathogens or infections that are undetectable or undetectable by other methods such as plate culture or polymerase chain reaction (PCR). These methods typically have very high sensitivity, for example, above 80%, 85%, 90%, 95%, 99%, or 99.5%. These methods typically have very low false positive rates, for example, below 5%, 4%, 3%, 2%, 1%, 0.1%, 0.05%, or 0.01%.

[0321] The methods described herein offer high specificity, high sensitivity, high positive predictive value, and / or low negative predictive value. The methods described herein offer at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher specificity (or negative percentage concordance) and / or sensitivity (or positive percentage concordance). In some cases, the nominal specificity is greater than or equal to 70%. The nominal negative predictive value (NPV) is greater than or equal to 95%. In some cases, the NPV is at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or higher.

[0322] Sensitivity, percentage positive agreement (PPA), or true positive rate (TPR) can be expressed as the equation TP / (TP+FN) or TP / (total number of infected subjects), where TP is the number of true positives and FN is the number of false negatives. When calculating the denominator of the preceding equation, this value reflects the total number of infection results based on a specific independent method for detecting infection (e.g., blood culture or PCR).

[0323] Specificity, negative percentage consistency, or true negative rate can be expressed as an equation such as TN / (TN+FP) or TN / (total number of uninfected subjects), where TN represents true negatives and FP represents false positives. When calculating the denominator of the preceding equation, this value reflects the total number of actual “non-infected” subjects identified by independent methods for detecting infection (e.g., blood culture or PCR).

[0324] In some cases, samples were identified as infected with accuracy greater than 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher. In some cases, samples were identified as infected with sensitivity greater than 95%. In some cases, samples were identified as infected with specificity greater than 95%. In some cases, samples were identified as infected with both sensitivity and specificity greater than 95%. In some cases, accuracy was calculated using a training algorithm. The diagnostic accuracy used in this paper includes specificity, sensitivity, positive predictive value, negative predictive value, and / or false detection rate. In some cases, the methods described herein have a specificity or sensitivity greater than 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or a positive or negative predictive value of at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% or higher.

[0325] When classifying samples to diagnose infection, binary classifiers typically have four possible outcomes. If both the predicted and actual values ​​are p, it's called a true positive (TP); however, if the actual value is n, it's called a false positive (FP). Conversely, a true negative occurs when both the predicted and actual values ​​are n, and a false negative occurs when the predicted value is n but the actual value is p. For tests detecting diseases or symptoms such as infection, false positives can occur when a subject tests positive but is not actually infected. On the other hand, false negatives can occur when a subject does have an infection but tests negative for that infection.

[0326] Positive predictive value (PPV), or precision or post-test probability of a disease, is the proportion of patients with a positive test result who have a correct diagnosis. PPV can be calculated by applying the following equation: PPV = TP / (TP + FP). PPV reflects the probability that a positive test reflects the underlying condition being tested. However, this value can vary depending on the prevalence of the disease. Negative predictive value (NPV) can be calculated by the following equation: TN / (TN + FN). Negative predictive value can be the proportion of patients with a negative test result who have a correct diagnosis. Both PPV and NPV measurements can be derived using appropriate disease prevalence estimates.

[0327] In some cases, the sequencing analysis results of the methods described in this paper provide a statistically correct confidence level for a given diagnosis. In some cases, such statistical confidence levels are higher than 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%.

[0328] Monitoring and treatment

[0329] The method may include monitoring whether a subject has an infection over time. For example, samples may be collected continuously at different times to determine the presence or absence of an infection. In other instances, the method may include a process for monitoring infection over time. In such cases, samples may be collected continuously at different time points during infection or illness; in some cases, the continuously collected samples are compared with each other to determine whether the infection is improving or worsening.

[0330] The methods described herein include methods for treating subjects, such as those with an infection or suspected infection. Treatment can reduce, prevent, or eliminate the infection in the subject. In some cases, treatment can reduce, prevent, or eliminate infection and / or inflammation.

[0331] Treatment may involve administering medications or other therapies to reduce or eliminate inflammation and / or infection. In some cases, medications are used to treat subjects prophylactically, for example, to prevent the development of infection or inflammation.

[0332] Any therapy (including medications) that improves or reduces symptoms of infection or inflammation may be administered to the subject. Exemplary medications include, but are not limited to, antibiotics, antiviral drugs, ampicillin, sulbactam, penicillin, vancomycin, gentamicin, aminoglycosides, clindamycin, cephalosporins, metronidazole, termetidine, ticarcillin, clavulanic acid, cefoxitin, antiretroviral drugs (e.g., highly active antiretroviral therapy (HAART), reverse transcriptase inhibitors, nucleoside / nucleotide reverse transcriptase inhibitors (NRTI), non-nucleoside RT inhibitors and / or protease inhibitors), antibody-drug conjugates, and immunoglobulins.

[0333] The methods described may include approaches to adjusting treatment regimens. For example, a subject may have a known infection and may have already been treated with medication for that infection. The methods provided herein can be used to track or monitor the efficacy of drug treatment. In some cases, the treatment regimen may be adjusted based on the results of such monitoring. For example, if the methods provided herein indicate that the infection has not improved with drug treatment, the treatment regimen may be adjusted by changing the type of medication or treatment given to the patient, discontinuing a previous medication, continuing the medication, increasing the dose of the medication, or adding a new medication or other treatment to the subject's treatment regimen. In some cases, the treatment regimen may involve specific procedures. Similarly, if the methods indicate that the infection is improving or clearing up, adjustments may involve reducing or discontinuing drug treatment.

[0334] The methods described herein may further include RNA sequencing (RNA-Seq) or a combination of methods incorporating RNA-Seq. Tissue damage or infection can lead to the release of cell-free nucleic acids from specific organs or tissues. For example, RNA can be released by apoptotic cells in tissues. RNA-Seq of cell-free RNA can indicate the health or status of different tissues in the body.

[0335] Methods including RNA sequencing can enable the detection of infections in specific organs or tissues and can be used to detect or monitor organ health. RNA-Seq can be used independently to study organ health or to provide increased confidence that an infection detected by the methods described in this paper is an infection in a specific organ. RNA-Seq tests can be performed simultaneously with, after, or before infection detection methods.

[0336] There are many possible scenarios where the pathogen detection method described herein can be combined with methods for detecting infection sites by RNA sequencing of cell-free RNA in bodily fluids. For example, the method described herein can be used to detect circulating cell-free nucleic acids from pathogens. The method may further include performing an RNA-Seq test to detect an increase in organ-specific cell-free RNA in the subject's blood. The combination of test results can indicate that a pathogen has infected an organ, and even identify which organ or tissue is infected.

[0337] After the methods described herein produce a positive test result (e.g., detection of pathogen infection), RNA-Seq testing (or a series of RNA-Seq tests) may sometimes be performed. RNA-Seq testing can be particularly useful for confirming infection or identifying the location of infection. For example, the methods described can detect the presence of a pathogen in a subject by analyzing circulating cell-free nucleic acids, but the site of infection may be unclear. In such cases, the methods may further include sequencing cell-free RNA from the subject to confirm that the infection is within an organ (e.g., by detecting increased levels of circulating cell-free RNA derived from organ tissue). The RNA sequencing test can then be repeated over time to determine whether the infection worsens or improves in a particular organ or tissue, or whether it has spread to different organs or tissues. Similarly, pathogen detection assays can also be repeated over time.

[0338] In some cases, the pathogen detection methods described herein are performed after RNA-Seq testing. For example, an increase in plasma levels of organ-associated cell-free RNA can indicate conditions such as organ infection. In this case, the method may further include detecting levels of circulating cell-free nucleic acids associated with organ infection.

[0339] The methods described herein can be repeated, for example, to monitor infection or treatment over time. The methods described herein can be repeated every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days; every 1, 2, 3, 4, 5, or 6 weeks; or every 1, 2, 3, 4, 5, 6, 7, 8, or 9 months.

[0340] In some cases, when the methods described herein produce a negative test result (e.g., no pathogen detected), the method can be repeated over time to monitor pathogen nucleic acids in the subject. In some cases, RNA-Seq assays are also repeated over time following negative pathogen test results or negative RNA-Seq results.

[0341] In some cases, when the methods described herein produce a positive test result (e.g., a pathogen is detected), a treatment regimen may be administered to the subject. Treatment regimens may include, but are not limited to, medication administration, antibiotic administration, or antiviral agent administration.

[0342] In some cases, when the methods described herein produce a positive test result, the method or test can be repeated continuously over time to monitor the infection process. For example, the treatment regimen can be adjusted based on the rise or fall of the infection. In other cases, no treatment regimen is initially initiated; for example, the infection can be monitored through a "wait and see" or "observation and waiting" approach to see if the infection clears without additional medical intervention. In some cases, when the methods described herein produce a positive test result, medication can be administered and the infection process can be monitored to detect whether the medication is working or when to discontinue treatment. In some cases, treatment can be modified as needed.

[0343] Computer control system

[0344] The present invention provides a computer control system programmed to implement the method of the present invention. Figure 7 A computer system 701 is shown that is programmed or otherwise configured to implement the methods of the present invention.

[0345] Computer system 701 includes a central processing unit (CPU, also referred to herein as a “processor” and “computer processor”) 705, which may be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 701 also includes memory or memory locations 710 (e.g., random access memory, read-only memory, flash memory), electronic storage units 715 (e.g., hard disks), communication interfaces 720 for communicating with one or more other systems (e.g., network adapters), and peripheral devices 725, such as cache memory, other memory, data storage, and / or electronic display adapters. Memory 710, storage units 715, interface 720, and peripheral devices 725 communicate with CPU 705 via a communication bus (solid line), such as a motherboard. Storage unit 715 may be a data storage unit (or data repository) for storing data. Computer system 701 may be operatively coupled to a computer network (“network”) 730 with the assistance of communication interface 720. Network 730 may be the Internet, the Internet and / or an extranet, or an intranet and / or extranet communicating with the Internet. In some cases, network 730 is a telecommunications and / or data network. Network 730 may contain one or more computer servers that enable distributed computing, such as cloud computing. In some cases, network 730 may enable a peer-to-peer network via computer system 701, which allows devices coupled to computer system 701 to act as clients or servers.

[0346] CPU 705 can execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 710. The instructions may be directed to CPU 705, which may subsequently program or otherwise configure CPU 705 to implement the methods of the present invention. Examples of operations performed by CPU 705 may include fetching, decoding, executing, and writing back.

[0347] CPU 705 may be part of a circuit such as an integrated circuit. One or more other components in system 701 may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0348] Storage unit 715 may store files, such as drivers, libraries, and saved programs. Storage unit 715 may store user data, such as user preferences and user programs. In some cases, computer system 701 may include one or more additional data storage units external to computer system 701, such as those located on a remote server communicating with computer system 701 via an intranet or the Internet.

[0349] Computer system 701 can communicate with one or more remote computer systems via network 730. For example, computer system 701 can communicate with a user's (e.g., a healthcare provider's) remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), tablet PCs, or PCBs (e.g., tablet PCs). iPad Galaxy Tab), telephone, smartphone (e.g.) iPhone, Android-enabled devices (or personal digital assistant.) Users can access computer system 701 via network 730.

[0350] The methods described herein may be implemented by machine-executable code (e.g., a computer processor) stored in an electronic storage location of computer system 701, such as memory 710 or electronic storage unit 715. This machine-executable code, or machine-readable code, may be provided in software form. During use, the code may be executed by processor 705. In some cases, the code may be retrieved from storage unit 715 and stored in memory 710 for rapid access by processor 705. In some cases, electronic storage unit 715 may be excluded, and machine-executable instructions are stored in memory 710.

[0351] The code may be pre-compiled and configured for use with a machine having a processor suitable for executing the code, or it may be compiled at runtime. The code may be provided in a programming language that can be selected to enable the code to be executed in a pre-compiled or compiled manner.

[0352] The aspects of the systems and methods provided herein, such as computer system 701, can be embodied in programming. These aspects of the technology can be considered "products" or "artifacts," typically in the form of machine (or processor) executable code and / or associated data executed or embodied in a machine-readable medium. Machine-executable code can be stored in electronic storage units, such as memory (e.g., read-only memory, random access memory, flash memory) or hard disks. "Storage" media can include any or all tangible memory of computers, processors, etc., or related modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage for software programming at any time. All or part of the software can sometimes be communicated via the Internet or various other telecommunications networks. For example, such communication allows software to be loaded from one computer or processor to another, such as from a management server or host computer to a computer platform for an application server. Therefore, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as those used in physical interfaces between local devices via wired and optical terrestrial networks and various air links. Physical elements carrying such waves, such as wired or wireless links, optical links, etc., can also be considered as media carrying the software. As used herein, unless limited to non-transitory tangible "storage" media, the term "computer or machine-readable medium" refers to any medium involved in providing instructions to a processor for execution.

[0353] Therefore, machine-readable media, such as computer-executable code, can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical discs or disks, any storage device such as any computer, such as those used to implement the database shown in the figure. Volatile storage media include dynamic memory, such as the main memory of a computer platform. Tangible transmission media include coaxial cables, copper wires, and optical fibers, including wires that contain buses within a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals or sound or light waves, such as signals generated during radio frequency (RF) and infrared (IR) data communications. Therefore, common forms of computer-readable media include, for example: floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card tapes, any other physical storage media with a perforated pattern, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chips or cartridges, carriers for transmitting data or instructions, cables or links for transmitting such carriers, or any other media from which a computer can read programming code and / or data. Many of these forms of computer-readable media may involve carrying one or more sequences of one or more instructions to a processor for execution.

[0354] Computer system 701 may include or communicate with an electronic display 735, the electronic display including a user interface (UI) 740 for providing report output, which may include a subject's diagnosis or treatment intervention for the subject. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces. Analysis may be provided as a report. This report may be provided to subjects, healthcare professionals, laboratory workers, or other individuals.

[0355] The methods and systems of the present invention can be implemented by one or more algorithms. The algorithms can be implemented in software when executed by the central processing unit 1105. These algorithms can, for example, facilitate the enrichment, sequencing, and / or detection of pathogens or other target nucleic acids.

[0356] Information about the patient or subject can be entered into the computer system, such as patient background, medical history, or medical scans. The computer system can then be used to analyze the results from the methods described herein, report the results to the patient or physician, or propose a treatment plan.

[0357] Reagents and kits

[0358] Reagents and kits for practicing one or more of the methods described herein are also provided. The reagents for the subjects and their kits can vary considerably. Reagents of interest include those specifically designed for identifying, detecting, and / or quantifying one or more pathogens or other target nucleic acids in samples obtained from the subjects. Kits may contain reagents necessary for nucleic acid extraction and / or nucleic acid detection using the methods described herein, such as PCR and sequencing. The kit may further include software for data analysis, which may include reference curves for comparison with test curves, and in particular, may include reference databases. Kits may contain reagents such as buffers and water.

[0359] Such kits may also contain information such as scientific literature references, packaging inserts, clinical trial results and / or summaries thereof, demonstrating or establishing the activity and / or benefits of the composition, and / or describing dosage, administration, side effects, drug interactions, or other information useful to healthcare providers. Such kits may also contain instructions for accessing databases. This information may be based on the results of various studies, such as studies using laboratory animals involving in vivo models and studies based on human clinical trials. The kits described herein may be available, sold, and / or promoted to healthcare providers, including physicians, nurses, pharmacists, prescribing personnel, etc. In some embodiments, the kits may also be sold directly to consumers.

[0360] This disclosure also provides kits for generating sequencing libraries. These kits may contain at least one synthetic nucleic acid as described herein and reagents for sequencing library reactions. In some cases, the kit contains one or more sequencing adaptors and one or more vector nucleic acids. The vector nucleic acids in the kit may include: i) one or more anti-end-repair vector nucleic acids; ii) one or more anti-ligation vector nucleic acids; iii) one or more anti-amplification vector nucleic acids; iv) one or more vector nucleic acids containing immobilized tags; v) one or more vector nucleic acids having a size that allows for size-based consumption; and / or vi) any combination thereof. For example, the kit may contain one or more sequencing adaptors and one or more anti-end-repair vector nucleic acids.

[0361] The ratio of the sequencing library adaptor to the amount of one or more vector nucleic acids in the kit can be specific. In some cases, this ratio is no greater than 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1, 500:1, or 1000:1. For example, the ratio may be no greater than 1:1.

[0362] Vector nucleic acid (CNA)

[0363] This disclosure provides vector nucleic acids (CNAs), particularly surreptitious CNAs, which contain features designed to exclude the CNA from one or more steps of sequencing analysis. This disclosure also provides methods for using CNAs capable of evading one or more steps of sequencing analysis. Although the CNAs provided herein may behave surreptitiously, they can generally still increase the amount of total nucleic acids in a sample, thus acting as typical “vector” nucleic acids. Vector nucleic acids typically enhance nucleic acid amounts to improve yield and / or efficiency when preparing sequencing libraries from a sample, and can ultimately improve the accuracy and / or sensitivity of sequencing analysis. The addition of vector nucleic acids (including the modified CNAs provided herein) can be particularly useful when the sample contains small amounts of target nucleic acids (e.g., less than 1 ng), as small amounts of nucleic acids can reduce the efficiency and / or yield of one or more steps in library generation (e.g., nucleic acid extraction, nucleic acid purification, nucleic acid end repair, adaptor ligation, etc.) or subsequent steps in sequencing analysis such as amplification. DNA- and / or RNA-based nucleic acids in any structural form and / or with or without one or more chemical modifications can be added as CNAs to samples of nucleic acid of interest. Typically, CNAs do not interfere with nucleic acid sequencing, for example, by suppression or by occupying prohibited portions of the sequencing throughput. In some cases, DNA CNAs are added to DNA and / or RNA samples. In other cases, RNA CNAs are added to DNA and / or RNA samples.

[0364] Table 6: Exemplary Vector Nucleic Acid Sequences

[0365]

[0366] The CNAs provided in this article can be designed or modified to avoid one or more steps in sequencing library generation, such as end repair, fragmentation, amplification, ligation, and sequencing. CNAs can be added to one or more steps in sequencing library preparation. For example, as... Figure 8 As shown, CNAs can be added directly during or after sample collection 802, during or after sample preparation (such as plasma isolation 803); before, during, or after nucleic acid isolation 804 or extraction 805, before, during, or after nucleic acid purification, before, during, or after nucleic acid end repair 806, before, during, or after ligation 807 or other procedures for attaching adaptors to nucleic acids, and / or before or during amplification 808. In some cases, CNAs can be removed from steps in sequencing analysis, such as by enzymatic digestion, affinity-based consumption, and / or size-based consumption. For example, CNAs provided herein can be physically removed from steps in sequencing analysis in a manner that excludes them from inclusion in the sequence library. In some cases, CNAs can be physically removed from the sequencing library itself.

[0367] CNA resistance to attachment

[0368] The CNAs described in this article are resistant to attachment or ligation to one or more sequencing adaptors and / or other molecules such as target nucleic acids. In some cases, CNAs can be engineered such that adaptors are preferentially ligated to target nucleic acids on the CNA. By avoiding ligation or attachment to adaptors or target nucleic acids, CNAs can also be prevented from being sequenced.

[0369] In some cases, particularly when using ligation to attach an adaptor to nucleic acids in a sample, a CNA can be designed to resist inclusion in the ligation reaction. Typically, the ligation reaction involves linking two nucleic acids via a phosphodiester bond. In some cases, the CNA can be designed with secondary structures resistant to the ligation reaction (e.g., single-stranded structures, hairpin structures). Secondary structures may contain RNA, DNA, ssDNA, dsDNA, DNA-RNA hybrids, and / or other features. In some cases, the CNA may contain blocking groups or other structures designed to prevent ligation.

[0370] The CNA described herein may contain single-stranded and / or double-stranded secondary structures designed to resist or reduce attachment or ligation. The CNA may contain one or more single-stranded regions, or may be entirely single-stranded. Single-stranded regions may appear at any location on the CNA, but in some preferred cases, the CNA contains single-stranded regions at or near one or both ends. For example, the CNA may contain single-stranded regions within 50 nucleotides from one or both ends, such as 50 nt, 45 nt, 40 nt, 35 nt, 30 nt, 25 nt, 20 nt, 15 nt, 10 nt, or 5 nt from one or both ends. In some preferred cases, the CNA may contain single-stranded regions at one or both ends (e.g., at the 5' end, at the 3' end). In some cases, the CNA may be entirely double-stranded or contain only double-stranded regions. Secondary structures (especially hairpin loops) can be used by ligases to prevent CNA binding and / or recognition. In some cases, CNA may contain Y-shaped double-stranded nucleic acids, which prevents the Y-shaped portion of the CNA from being linked to or attached to another nucleic acid.

[0371] Hairpin structures that may exist in the CNAs provided herein typically have loops and hybridization regions, such as a hairpin stem. For example, a hairpin may contain two complementary regions forming a double-stranded hybridization region and a loop connecting the two complementary regions. The complementary regions may contain at least 5, 10, 15, 20, 30, 40, or 50 nucleotides. The loop region may contain at least 3, 4, 5, 10, 15, 20, 30, 40, or 50 nucleotides. Generally, hairpin structures can be relatively easy to fabricate because they are usually just single-stranded nucleic acids without attachments. Hairpins may contain RNA or DNA.

[0372] The CNA described herein may contain circular structures that resist or reduce attachment or linkage. The circular structure may be circular DNA, circular RNA, or a circular DNA-RNA hybrid. In some cases, the circular structure is circular DNA. The circular structure may be double-stranded or single-stranded. The circular structure may have a certain length; for example, at least circular structures may have a certain length, for example, at least 5 nt, 10 nt, 20 nt, 30 nt, 32 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, 120 nt, 140 nt, 160 nt, 180 nt, 200 nt, 250 nt, 300 nt, 400 nt, 500 nt, or 1000 nt. In some cases, the circular structure contains about 30 to about 100 nucleotides. In some cases, the size of the circular structure may range from about 10 nucleotides to about 10,000 nucleotides, for example, from about 100 nucleotides to about 1,000 nucleotides. When the cyclic structure is double-stranded, the cyclic structure can have a size of at least 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp, 200 bp, 250 bp, 300 bp, 400 bp, 500 bp, or 1000 bp. In some cases, the double-stranded cyclic structure contains approximately 30 bp to 100 bp. In some cases, the size of the double-stranded cyclic structure can range from approximately 10 base pairs to approximately 10,000 base pairs, for example, from approximately 100 base pairs to approximately 1,000 base pairs. In some cases, the cyclic structure enables the CNA to resist digestion by a certain enzyme (e.g., an endonuclease). For example, a CNA may contain a double-stranded circular structure and be resistant to digestion by endonucleases, such as those that digest double-stranded linear DNA but not double-stranded circular DNA. In some cases, the CNA is predominantly or entirely circular, such as circular double-stranded DNA or circular single-stranded DNA. In some cases, the CNA contains secondary structures resistant to digestion by endonucleases (e.g., endonucleases that do not bind to and / or recognize the secondary structure of the CNA). For example, a CNA may contain double-stranded DNA that resists digestion by recognizing single-stranded DNA but not endonucleases that recognize double-stranded DNA. As another example, a CNA may contain single-stranded DNA that resists digestion by recognizing double-stranded DNA but not endonucleases that recognize single-stranded DNA.

[0373] In some cases, CNAs are double-stranded with one or more nicks. Nicks in double-stranded nucleic acid molecules can be discontinuous, where there is no phosphodiester bond between adjacent nucleotides on one strand. Nicks can be created by enzymes, such as endonucleotide reductases. In some cases, nicks can be ligated by enzymes (e.g., ligases). In other cases, nicks are protected from exonuclease digestion and / or ligation.

[0374] CNAs may contain one or more modifications (e.g., modified nucleotides) that resist ligation reactions. In some cases, the modification may be a blocking group that prevents the CNA from ligating to a nucleic acid. For example, a CNA may have blocking groups at the 3' end, 5' end, or both ends. The blocking group may contain a reverse deoxyglycan. The reverse deoxyglycan may be a reverse deoxyglycan, a reverse dideoxyglycan, or other reverse deoxyglycans. The reverse deoxyglycan may be a 3' reverse deoxyglycan or a 5' reverse dideoxyglycan. For example, the blocking group may be 3' reverse thymidine (dT), 3' reverse adenosine (dA), 3' reverse guanosine (dG), 3' reverse cytidine (dC), 3' reverse deoxyuracil (dU), 5' reverse dideoxythymidine (ddT), 5' reverse dideoxyadenosine (ddA), 5' reverse dideoxyguanosine (ddG), 5' reverse dideoxycytidine (ddC), 5' reverse dideoxyuracil (ddU), or any analogue thereof. In some cases, a CNA contains 3' reverse thymidine. In some cases, CNA contains 5'-reverse dideoxythymidine. In some cases, CNA contains 3'-reverse thymidine and / or 5'-reverse dideoxythymidine. In some cases, the blocking group contains dideoxycytidine. In some cases, the modification includes a uracil (U) base, 2'OMe-modified RNA, a C3-18 spacer region (e.g., a structure with 3-18 consecutive carbon atoms), biotin, dideoxynucleotide triphosphate, ethylene glycol, amine, and / or phosphate.

[0375] Vector nucleic acids resistant to amplification

[0376] CNAs may contain one or more nucleic acid modifications that inhibit nucleic acid amplification, thereby preventing the CNA from being amplified during sequencing reactions. In some cases, modifications prevent nucleic acid polymerases from functioning, for example, by stopping or inhibiting (e.g., slowing down) the polymerase. In some cases, modifications may contain one or more abase-free sites. An abase-free site can refer to a position in the nucleic acid that does not contain a base. For example, an abase-free site in a nucleic acid may be at the 1' end where no base is present. Abase-free sites may have purine or pyrimidine structures, base analogs, or analogs of the phosphate backbone. In some cases, abase-free sites have an N-(2-aminoethyl)-glycine backbone linked by an amide bond, tetrahydrofuran, or 1',2'-dideoxyribose (dSpacer). In some cases, modifications may contain abase-free sites and modified sugar residues, such as sugar residues with three carbon atoms, such as partial ribose structures (e.g., retaining only the 3', 4', and 5' terminal carbon atoms) to maintain connectivity along the backbone.

[0377] Abase-free sites prevent polymerase from amplifying CNA. In some cases, abase-free sites in CNA can inhibit polymerase (e.g., Taq polymerase) by an order of magnitude per abase-free site.

[0378] The CNA provided herein may contain multiple abase-free sites, such as multiple internal abase-free sites and one or more other features. The CNA may also contain features that prevent participation in one or more library preparation reactions. For example, the CNA may contain one or more internal abase-free sites, 3' reverse dT, and / or 5' reverse ddT in any combination.

[0379] In some cases, CNA may contain other modifications that inhibit nucleic acid amplification. In some cases, modifications that inhibit nucleic acid amplification include uracil (U) bases, 2'OMe-modified RNA, C3-18 spacer regions (e.g., structures with 3-18 consecutive carbon atoms, such as C3 spacer regions), ethylene glycol polymeric spacer regions (e.g., spacer region 18 (hexaethylene glycol spacer region), biotin, dideoxynucleotide triphosphates, ethylene glycol, amines, and / or phosphates.

[0380] Modification

[0381] A CNA may contain at least one, two, three, four, five, six, seven, eight, nine, ten, or more modifications (e.g., no base site). When a CNA contains multiple modifications (e.g., modifications that inhibit nucleic acid amplification), the modifications can be clustered (e.g., modifications are located consecutively adjacent to each other). In some cases, one or more modifications are located at the 5' end of the CNA. In some cases, one or more modifications are located at the 3' end of the CNA. In some cases, one or more modifications are located at both the 3' and 5' ends of the CNA. In some cases, one or more modifications are located in internal positions within the CNA. For example, a CNA may contain one or more internal dspacers (idsp).

[0382] Modifications described herein may include 2-aminopurine, 2,6-diaminopurine, 5-bromodU, deoxyuridine, reverse dT, reverse dideoxy-T, dideoxy-C, 5-methyldC, deoxyinosine, universal bases such as 5-nitroindole, 2'-O-methylRNA bases, Iso-dC, Iso-dG, ribonucleotides, morpholinonucleotides, protein nucleoside analogs, glyconucleotide analogs, locked nucleotide analogs, threonucleotide analogs, chain-terminated nucleotide analogs, thiourea nucleotide analogs, pseudouridine, dihydrouridine, q-nucleoside, γ-nucleotide, base-free sites, functional groups such as alkyne functional groups, and azides. Functional groups include azides (NHS esters), non-natural bonds such as thiophosphate bonds, spacer regions such as 2'-dideoxyribose (dSpacer), hexanediol, light-cleavable spacer regions, spacer regions of different lengths with different carbon numbers such as C3-spaced phosphoriamide, C9-spacer regions such as triethylene glycol spacer regions, and C18 and 18-atom hexaethylene glycol spacer regions. Such spacer regions may be attached to the 5' or 3' end of the CNA or the linker, or may be internally attached. Furthermore, at least one chain of the CNA may be modified by phosphorylation, for example, by containing 5' phosphate or 3' phosphate (e.g., on the complementary chain) or both.

[0383] Enzyme recognition site

[0384] The CNA may contain features that allow it to be removed from the sequencing library. Such features may include enzyme recognition sites. For example, the CNA may contain one or more enzyme recognition sites, allowing the synthetic nucleic acid to be degraded by an enzyme. In some cases, the CNA may contain one or more enzyme recognition sites that are not present in the target nucleic acid or the adaptor. Therefore, the vector nucleic acid can be removed by an enzyme targeting the recognition site without causing enzymatic degradation of the target nucleic acid or the adaptor.

[0385] In some cases, a CNA may contain a nuclease recognition site. For example, this nuclease recognition site may be an endonuclease recognition site. The endonuclease may be type I, type II (including types II and IIG), type III, or type IV endonucleases. In some cases, the endonuclease recognition site may be a restriction nuclease recognition site. For example, the recognition sites of endonucleases can be AatII, Acc65I, AccI, AcclI, AatII, Acc65I, AccI, AcclI, AfeI, AflII, AgeI, ApaI, Apali, ApoI, AscI, AsiSI, AvrII, BamHI, BclI, BglII, Bme1580I, BmtI, BsaHI, BsiEI, BsiWI, BspEI, BspHI, BsrGI, BssHII, BstBI, BstZ17I, BtgI, ClaI, DraI, EaeI, EagI, EcoRI, EcoRV, FseI, FspI, HaeII, HincII, Hind III. Recognition sites for HpaI, KasI, KpnI, MfeI, MluI, MSCI, MspA1I, NaeI, NarI, NcoI, NdeI, NgoMIV, NheI, NotI, NruI, NsiI, NspI, PacI, PciI, PmeI, PmlI, PsiI, PspOMI, PstI, PvuI, PvuII, SacI, SacII, SalI, SbfI, ScaI, SfcI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI, SwaI, XbaI, XhoI, and XmaI. Enzyme recognition sites may be sites of DNases (e.g., exonucleases) not mentioned above. Enzyme recognition sites can be sites of uracil DNA glycosylase (UDG), DNA glycosylase-lyase (endonuclease VIII), or mixtures thereof (e.g., uracil-specific excision reagent (USER) enzymes). For example, a CNA may contain one or more uracils (e.g., endouridine). Enzyme recognition sites can also be sites of RNA-directed DNases (e.g., CRISPR-associated protein nucleases, such as Cas9).In some cases, the nuclease recognition site may be a recognized site of an RNase, such as endonucleases like RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, or exonucleases like polynucleotide phosphorylases, RNase PH, RNase R, RNase D, RNase T, oligonucleotides, exonucleases I, or exonucleases II. In some specific cases, the CNA may contain a restriction enzyme recognition site, and the methods described herein may include digesting the CNA with a restriction enzyme that recognizes that site. In some cases, the CNA contains secondary or tertiary structures, ribozymes, aptamers, and DNA-based catalytic or binding polymers that can be recognized by enzymes (e.g., enzymes that bind and / or degrade the CNA). In some cases, the CNA contains one or more enzyme-recognizable, enzyme-specific binding nucleic acid sequences.

[0386] In some cases, a CNA may contain a DNA-RNA hybrid that can be degraded by DNases or RNases. In other cases, a CNA contains a DNA-RNA-DNA hybrid. Such a molecule may be double-stranded. The terminal regions of the CNA may contain deoxyribonucleotides. The internal regions may contain ribonucleotides. In some cases, the DNA-RNA hybrid can be linked to a target nucleic acid or adaptor; the DNA-RNA hybrid can then be digested by RNases before sequencing (e.g., before the amplification step). In certain specific cases, the DNA-RNA hybrid is digested (e.g., by RNases), while the target nucleic acid (e.g., DNA, such as cell-free DNA) is not digested by RNases.

[0387] If the DNA portion of the CNA is long enough to resist amplification, an RNase digestion step to remove the DNA-RNA hybrid before sequencing may not be necessary. Alternatively, if the DNA-RNA hybrid molecule is enzymatically digested and degraded before amplification, the DNA-RNA hybrid may not need to have a size or length that resists amplification.

[0388] CNA based on size consumption

[0389] CNAs can have a certain size, allowing them to be separated from sequencing libraries through size-based consumption. In some cases, the length of the CNA is greater than the length of the target nucleic acid or greater than the average length of the target nucleic acid. For example, the length of the CNA can be 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, or 50 times the length of the target nucleic acid or its average length. CNAs can have a length of at least 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 800 bp, 1 kb, 2 kb, 5 kb, or 10 kb. For example, a CNA can have a length of at least 500 bp. In some cases, the size of the CNA can range from about 150 bp to about 1000 bp. In some cases, the size of the CNA can be as high as 2 kb. In some cases, the length of the CNA is shorter than the length of the target nucleic acid or shorter than the average length of the target nucleic acid. For example, the length of the CNA can be up to 99%, 95%, 90%, 80%, 60%, 50%, 40%, 20%, or 10% of the length of the target nucleic acid or the average length of the target nucleic acid. In some cases, the size of the CNA can be up to 50% of the size of the target nucleic acid or the average size of the target nucleic acid. In some cases, the CNA has a length that is substantially the same as the length of the target nucleic acid or the average length of the target nucleic acid.

[0390] A CNA of size or length that allows for size-based consumption may also contain any of the modifications described in this disclosure, such as modifications to prevent ligation, amplification, end repair, or combinations thereof. In some cases, one or both ends of the CNA may contain one or more modifications. In some cases, the modification may be an internal modification, such as an internal absence of base sites or a combination of end modifications and internal modifications.

[0391] In certain instances, the CNA can be longer, enabling size-based consumption and modifications (e.g., end modifications) such as reverse bases that prevent linking. Other structural combinations that prevent or block linking are also possible (e.g., hairpin loops, hairpin loops combined with end modifications). In some cases, the CNA may contain one or more hairpin structures and one or more base-free sites. In certain instances, the CNA may have a size or length greater than 500 bp and have a 3' reverse dT, 5' reverse ddT, C3 spacer region, or spacer region 18 or a hairpin structure at one end. In certain instances, the CNA may have a size or length greater than 600 bp and have a 3' reverse dT or 5' reverse ddT at one end with one or more internal base-free sites.

[0392] Fixed tags

[0393] A CNA may contain one or more immobilized tags. These immobilized tags can be used to remove the CNA from a solution (e.g., a solution of a sequencing library) by affinity-based depletion. For example, the immobilized tags can be attached to a solid support, such as beads or a plate. The CNA can be removed from the solution when the solution is contacted with the solid support. A CNA containing one or more immobilized tags may be shorter than the target nucleic acid. Alternatively, the CNA molecule may be longer than the target nucleic acid, for example, to minimize CNA transfer into the sequencing reaction.

[0394] Immobilized tags may include biotin, digitoxin, nickel-aminotriacetic acid, desulfobiotin, histidine, polyhistidine, MYC, hemagglutinin (HA), FLAG, fluorescent tags, tandem affinity purification (TAP) tags, glutathione S-transferase (GST), polynucleotides, aptamers, polypeptides (e.g., antigens or antibodies), or derivatives thereof. For example, a CNA may contain biotin, such as an internally or terminally biotinylated chain. In some cases, the immobilized tag may include a magnetically susceptible material, such as a magnet or a magnetically susceptible metal. In certain instances, a biotinylated CNA may enable magnetic bead-based consumption of the CNA from a sample or sequencing library prior to the amplification step (e.g., via avidin-magnetic beads). In some cases, the CNA contains secondary or tertiary structures that can attach to a solid support or bind to the immobilized tag.

[0395] In some cases, the target nucleic acid and / or sequencing library nucleic acid contain one or more immobilized tags. In these cases, the CNA does not contain an immobilized tag or contains a different immobilized tag compared to the target nucleic acid. Therefore, different immobilized tags can be used to separate the CNA from the target nucleic acid and / or sequencing library nucleic acid through affinity-based consumption. For example, the target nucleic acid and / or sequencing library nucleic acid can be immobilized on a solid support while the CNA is washed away. In some cases, the CNA is directly or indirectly linked to the immobilized tag. In some cases, the CNA is cleaved from the immobilized tag.

[0396] CNAs may include combinations of the features and structures disclosed herein. In some cases, a CNA contains one or more modifications that inhibit nucleic acid amplification and one or more modifications that resist ligation reactions. For example, a CNA contains one or more base-free sites (e.g., internal dspacer) and reverse deoxy bases (e.g., 3' reverse thymidine). CNAs containing modifications may further contain enzyme recognition sites and / or immobilization tags. In some cases, a CNA contains a DNA-RNA hybrid with one or more immobilization tags, such as a biotinylated DNA-RNA-DNA hybrid molecule. CNAs may also have secondary and / or tertiary structures of nucleic acids with high affinity for specific enzymes or proteins, or any non-amino acid-based catalytic or affinity unit, such as ribozymes, DNA-based catalytic polymers, and molecularly imprinted polymers.

[0397] The ratio of carrier nucleic acid to nucleic acid in the sample

[0398] A certain amount of CNA can be added to a sample containing nucleic acids, for example, for preparing sequencing libraries from nucleic acids in the sample. In some cases, the ratio of the total amount of nucleic acid in the sample to the amount of CNA added to the sample is at least 1:100, 1:50, 1:10, 1:1, 10:1, 50:1, 100:1, 500:1, 1000:1, 2000:1, or 5000:1. In some cases, the ratio of the amount of target nucleic acid in the sample to the amount of CNA added to the sample is at least 1:100, 1:50, 1:10, 1:1, 10:1, 50:1, 100:1, 500:1, 1000:1, 2000:1, or 5000:1. In some cases, the ratio of total nucleic acid in the sample to the amount of CNA added to the sample is at most 10:1, 1:1, 1:10, 1:50, 1:100, 1:500, 1:1000, 1:2000, or 1:5000. In some cases, the ratio of target nucleic acid in the sample to the amount of CNA added to the sample is at most 10:1, 1:1, 1:10, 1:50, 1:100, 1:500, 1:1000, 1:2000, or 1:5000. In some cases, the ratio of total nucleic acid in the sample to the amount of CNA added to the sample is in the range of approximately 1:1 to approximately 1:100. In some cases, the ratio is a molar ratio.

[0399] Methods for using CNA when generating sequencing libraries

[0400] This document discloses methods for preparing sequencing libraries. These methods may include adding CNAs disclosed herein to improve the efficiency and / or yield of sequencing library generation. A sequencing library may refer to a population of nucleic acid molecules subjected to sequencing. The method may involve obtaining a sample containing a target nucleic acid and / or an adaptor (e.g., a sequencing adaptor) and one or more CNAs. The method may further include one or more steps for generating a sequencing library. The method may also include sequencing one or more nucleic acids in the sequencing library. Sequencing of CNAs may be omitted; for example, CNAs may be physically removed from the library, or CNAs may be engineered to not participate in one or more steps of sequencing library generation.

[0401] This method may include adding CNA to a sample containing a target nucleic acid and / or an adaptor. The amount of CNA added to the sample may be at least 0.1 ng, 0.5 ng, 1 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 150 ng, 200 ng, 300 ng, 400 ng, or 500 ng. In some cases, the amount of CNA may be from 0.1 ng to 200 ng, 1 ng to 100 ng, 5 ng to 80 ng, 10 ng to 60 ng, or 20 ng to 50 ng. The concentration of CNA in the sample may be at least 0.1 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 0.01 ng / μL, 0.05 ng / μL, 0.1 ng / μL, 0.2 ng / μL, 0.4 ng / μL, 0.8 ng / μL, 1 ng / μL, 1.2 ng / μL, 1.5 ng / μL, 2 ng / μL, 5 ng / μL, or 10 ng / μL. In some cases, the amount of CNA added to the sample may be in the range of about 1 ng / 15 μL to about 5 ng / 15 μL. In some cases, the amount of CNA added to the sample may be in the range of about 0.05 ng / μL to about 0.5 ng / μL.

[0402] The methods described herein may include adding any type of synthetic nucleic acid as described throughout this disclosure. For example, the method may include adding one or more of the following synthetic nucleic acids: synthetic nucleic acids used for generating sequencing libraries, synthetic nucleic acids used to normalize the relative abundance of target nucleic acids (e.g., synthetic nucleic acids at known concentrations), and / or synthetic nucleic acids used to determine the loss of nucleic acid diversity in a sample.

[0403] Nucleic acid extraction

[0404] This method may include extracting nucleic acids (e.g., target nucleic acids, cell-free nucleic acids) from a sample. Extraction may include separating the nucleic acids from other cellular components and contaminants that may be present in the sample, such as biological fluids or tissue samples. In some cases, extraction is performed by phenol-chloroform extraction or by precipitation with an organic solvent (e.g., ethanol or isopropanol). In some cases, extraction is performed using a nucleic acid binding column. In some cases, extraction is performed using commercially available kits, such as the Qiagen Qiamp Circulating Nucleic Acid Kit, the Qiagen Qubit dsDNA HS Assay Kit, and Agilent Technologies. TM DNA 1000 kit, TruSeq TM Sequencing library preparation or nucleic acid binding spin column (e.g., Qiagen DNAmini-prep kit). In some cases, cell-free nucleic acid extraction may involve filtration or ultrafiltration.

[0405] CNA can be added to the sample before or during extraction. For example, the vector nucleic acid can be added to the sample before mixing it with the extraction reagent (e.g., extraction buffer). Alternatively, the vector nucleic acid can be added to the extraction reagent, such as the extraction buffer that is subsequently mixed with the sample. In some cases, CNA can also be added to the mixture of the sample and the extraction reagent (e.g., extraction buffer). In these cases, both the target nucleic acid and CNA can be extracted simultaneously.

[0406] Adding CNA to a sample can improve the yield of nucleic acid extraction. The yield of target nucleic acids extracted with CNA can be higher than that extracted without CNA, for example, at least 10%, 20%, 40%, 60%, 80%, 100%, 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold. In some cases, CNA can be added to the sample containing the target nucleic acid after nucleic acid extraction. Extraction can produce at least 10 ng, 50 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng of nucleic acid.

[0407] Nucleic acid purification

[0408] This method may include purifying the target nucleic acid. Exemplary purification methods include ethanol precipitation, isopropanol precipitation, phenol-chloroform purification and column purification (e.g., affinity-based column purification), dialysis, filtration, or ultrafiltration.

[0409] CNA can be added to the sample before or during purification. For example, the vector nucleic acid can be added to the sample before mixing it with purification reagents (e.g., purification buffer). Alternatively, the vector nucleic acid can be added to purification reagents, such as purification buffer, which is then mixed with the sample. In some cases, CNA can also be added to the mixture of the sample and purification reagents (e.g., purification buffer). In these cases, both the target nucleic acid and CNA can be purified simultaneously.

[0410] Adding CNA to a sample can improve the yield of nucleic acid purification. The yield of purifying target nucleic acids with CNA can be higher than that without CNA, for example, at least 10%, 20%, 40%, 60%, 80%, 100%, 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold. In some cases, CNA can be added to a sample containing target nucleic acids after nucleic acid purification. In some cases, the purification of nucleic acids in a sample with added CNA yields at least 1 pg, 10 pg, 50 pg, 100 pg, 500 pg, 1 ng, 5 ng, 10 ng, 50 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng of total nucleic acids in the sample. In some cases, the purification of nucleic acids in samples with added CNA yields at least 1 pg, 10 pg, 50 pg, 100 pg, 500 pg, 1 ng, 5 ng, 10 ng, 50 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng of the target nucleic acid in the sample.

[0411] Fragmentation

[0412] This method may include fragmenting a target nucleic acid. Target nucleic acid fragmentation may be performed by, for example, mechanical shearing, passing the sample through a syringe, sonication, heat treatment, or a combination thereof. In some cases, target nucleic acid fragmentation is performed using an enzyme, including a nuclease or transposase. The nuclease used for fragmentation may include restriction endonucleases, homing endonucleases, nicking endonucleases, high-fidelity restriction enzymes, or any enzyme disclosed herein. This method may include fragmenting the target nucleic acid into fragments of a specified length, for example, at least 50, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500, 1000, 2000, 4000, 6000, 8000, or 10000 bp. The CNA may be added to the sample prior to target nucleic acid fragmentation. The CNA may be added to the sample after target nucleic acid fragmentation.

[0413] A-Added Tail

[0414] This method may include A-tailing of the target nucleic acid. The A-tailing reaction can be performed using one or more A-tailing enzymes. For example, an adenine (A) residue can be added by incubating a DNA fragment with dATP and a non-correcting DNA polymerase, which will add a single 3′A residue. The CNA can be added to a sample containing the target nucleic acid before A-tailing. Alternatively, the CNA can be added to a sample containing the target nucleic acid after A-tailing.

[0415] End repair

[0416] This method may include end repair of target nucleic acids. For example, end repair of target nucleic acids can be performed, thus making them applicable to other steps in sequencing library preparation. End repair reactions can be performed using one or more end-repair enzymes. Enzymes used to repair DNA may include polymerases and exonucleases. For example, polymerases can fill in missing bases in the DNA strand from the 5' to 3' direction. The resulting double-stranded DNA may have a length substantially the same as the original longest DNA strand. Exonucleases can remove the 3' overhang. The resulting double-stranded DNA may have a length substantially the same as the original shortest DNA strand.

[0417] CNA can be added to samples containing target nucleic acids before end-repair. In some cases, adding CNA improves the efficiency of the end-repair reaction, for example, by at least 10%, 20%, 40%, 60%, 80%, or 100%. In some cases, CNA can be added to samples containing target nucleic acids after end-repair. In certain situations, adding CNA can maintain the activity and / or function of an enzyme (e.g., an end-repair enzyme). For example, an enzyme may have reduced activity and / or abnormal function in a sample with a small amount of nucleic acid, and adding CNA can increase the total amount of nucleic acid in the sample, allowing the enzyme to function normally in the sample.

[0418] Connector attachment

[0419] This method may include attaching one or more adaptors to a target nucleic acid. The adaptor can be attached to the target nucleic acid via primer extension, reverse transcription, or hybridization. In some cases, the adaptor is attached to the target nucleic acid by ligation. For example, the adaptor can be attached to the target nucleic acid using a ligase. For example, the adaptor can be attached to the target nucleic acid via sticky-end ligation or blunt-end ligation. In some cases, the adaptor can be attached to the target nucleic acid using a transposase. The target nucleic acid can be attached to the adaptor at the 3' end, the 5' end, or both ends. In some cases, the target nucleic acid is attached to the same adaptor or different adaptors at both ends. In some cases, the target nucleic acid can be attached to one or more adaptors at one end.

[0420] CNAs can be added before the attachment step. Alternatively, they can be added after the attachment step. CNAs are resistant to ligation reactions. For example, CNAs can resist ligation with the target nucleic acid and / or adaptor. In these cases, when CNAs are added before the attachment step, they are not ligated with the target nucleic acid or adaptor and will not be sequenced in the sequencing step. In other cases, CNAs can be removed from the sample before the attachment step. Alternatively, CNAs can be removed after sample extraction and before the attachment step.

[0421] Before attaching the adaptor to the target nucleic acid in the sample, the sample can be treated with an enzyme. For example, an endonuclease can be used to treat the sample to create attachment sites, such as sticky ends or blunt ends. Alternatively, the sample can be treated with an enzyme after the adaptor has been attached to the target nucleic acid.

[0422] Amplification

[0423] This method may include amplifying the target nucleic acid. Amplification can refer to any method used to increase the copy number of a nucleic acid sequence. For example, amplification can be performed using a polymerase, such as in one or more polymerase chain reactions. Amplification can be performed using methods known in the art. These methods typically depend on the catalytic formation of products containing multiple copies of the nucleic acid or its complement. One such method is polymerase chain reaction (PCR), which includes AFLP (amplified fragment length polymorphism) PCR, allele-specific PCR, Alu PCR, assembly, asymmetric PCR, colony PCR, helicase-dependent PCR, hot-start PCR, inverse PCR, in situ PCR, cross-sequence-specific PCR or IS SR PCR, digital PCR, droplet digital PCR, linear exponential PCR, long PCR, nested PCR, real-time PCR, duplex PCR, multiplex PCR, quantitative PCR, or single-cell PCR. Other amplification methods may also be used, including ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), linear amplification, isothermal linear amplification, Q-β-replicaase method, 3SR, transcription-mediated amplification (TMA), strand displacement amplification (SDA), or rolling circle amplification (RCA).

[0424] CNAs can be added before amplification, or after amplification. CNAs may not be amplified. For example, a CNA might contain modifications that inhibit amplification. In these cases, when a CNA is added before amplification, it will not be amplified. Therefore, the CNA may not be in the sequencing library or will not be sequenced.

[0425] CNA removal

[0426] This method may further include removing CNAs from the sample, which typically prevents CNAs from being sequenced. In some cases, the method involves removing some or all CNAs from the sample to prepare a sequencing sample. The resulting sequencing sample may not contain CNAs and may be ready for sequencing. In some cases, the method involves preferentially removing at least one CNA from other nucleic acids in the sample, such as target nucleic acids, adaptors, or multimers of adaptors.

[0427] Enzymes can be used to remove CNA. For example, CNA can be degraded by enzymes, such as through enzymatic digestion. In some cases, the method includes the use of nucleases to remove CNA. For example, the method may include the use of endonucleases to remove CNA, such as type I, type II (including type IIS, type IIG), type III, or type IV endonucleases. This method may include the use of restriction endonucleases such as AatII, Acc65I, AccI, AcclI, AatII, Acc65I, AccI, AcclI, AfeI, AflII, AgeI, ApaI, ApaliI, ApoI, AscI, AsiSI, AvrII, BamHI, BclI, BglII, Bme1580I, BmtI, BsaHI, BsiEI, BsiWI, BspEI, BspHI, BsrGI, BssHII, BstBI, BstZ17I, BtgI, ClaI, DraI, EaeI, EagI, EcoRI, EcoRV, FseI, FspI, HaeII, HincII, HindI II. CNA removal using HpaI, KasI, KpnI, MfeI, MluI, MSCI, MspA1I, NaeI, NarI, NcoI, NdeI, NgoMIV, NheI, NotI, NruI, NsiI, NspI, PacI, PciI, PmeI, PmlI, PsiI, PspOMI, PstI, PvuI, PvuII, SacI, SacII, SalI, SbfI, ScaI, SfcI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI, SwaI, XbaI, XhoI, XmaI, or any combination thereof. This method may include CNA removal using a DNase not mentioned above (e.g., an exonuclease). This method may include removing CNA using uracil DNA glycosylase (UDG), DNA glycosylase-lyase (endonuclease VIII), or a mixture thereof (e.g., uracil-specific excision reagent (USER) enzyme). This method may also include removing CNA using RNA-directed DNases (e.g., CRISPR-associated protein nucleases, such as Cas9).The method may include using an RNase to remove vector-synthesized nucleic acids, such as endonucleases like RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, or exonucleases like polynucleotide phosphorylases, RNase PH, RNase R, RNase D, RNase T, oligonucleotides, exonucleases I or II, or any combination thereof. In some cases, the method includes removing CNA using any nucleic acid degradation reagent known in the art. In some cases, the method may include subjecting CNA to physical treatment (e.g., heating, cooling, or shearing) to remove CNA. In some cases, the method for removing CNA does not remove target nucleic acids, adaptors, or any other molecules from the sequencing library in the sample. In some cases, CNA removal is not performed by enzymatic degradation (e.g., endonuclease digestion).

[0428] To remove CNA, the method may include incubating the CNA with the enzyme at the temperature at which the enzyme functions. For example, the method may include incubating the CNA with the enzyme at temperatures ranging from 10°C to 80°C, such as 20°C to 60°C, 20°C to 40°C, 30°C to 40°C, or 20°C to 25°C. The method may also include incubating the CNA with the enzyme at temperatures of at least 10°C, 20°C, 25°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 50°C, 60°C, or 70°C. In some cases, the method may include incubating the CNA with the enzyme at a temperature of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C.

[0429] To remove CNA, the method may include incubating the CNA with an enzyme for a period of time to allow the enzyme to function. In some cases, the method may include incubating the CNA with the enzyme for at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours.

[0430] This method may include removing carrier-synthesized nucleic acids (CNAs) by affinity-based consumption. Affinity-based consumption can be performed on the carrier-synthesized nucleic acid, which may contain one or more immobilized tags. In these cases, the method may include removing the CNA by attaching the immobilized tags to a solid support. Such a solid support may be paper, glass (e.g., controlled-pore glass (CPG)), plastic (e.g., polymethyl methacrylate, polyethylene, polypropylene, polyacrylate, polymethyl methacrylate, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polycarbonate macroporous polystyrene (MPPS), or nylon), polyacrylamide, cellulose acetate, cellulose nitrate, silicon, or other metals or optical fibers.

[0431] Solid supports based on affinity consumption can be molded into any shape and form. In some cases, solid supports can be manufactured in the form of planar devices having discrete isolated areas in the form of wells, channels, bases, hydrophobic or hydrophilic patches, die-cut adhesive reservoirs, or other physical barriers to fluid flow. Examples of such solid supports include glass slides, microplates, sheets, membranes, test strips, etc.

[0432] In other cases, the solid support may be in the form of beads or pellets containing a coated cationic surface. Beads provide a means of increasing probe density on the coated solid support. Beads may offer a variety of surface chemistry or functions suitable for cationizing the beads by, for example, amination (e.g., amine, carboxyl, or hydroxyl groups). Suitable bead compositions include, for example, plastics such as polystyrene, methylstyrene, acrylic polymers, ceramics, glass, polymeric materials such as cross-linked dextran, cellulose, nylon and latex, paramagnetic materials, titanium dioxide, and latex. Beads may include any type of solid or hollow spheres, balls, bearings, cylinders, or other solid configurations. Beads may be porous or non-porous in nature. The use of porous beads increases the surface area of ​​beads that can be used for nucleic acid detection. Bead sizes may range from 100 nm to 5 mm, for example, 0.2 μm to 200 μm or 0.5 μm to 5 μm. In some cases, the solid support may be magnetic or susceptible to magnetic influence. The solid support may be coated. The coating may be combined with an immobilized tag. For example, a solid support can be coated with a binding partner of an immobilized tag, such as streptavidin, an antigen, an antibody (e.g., an anti-multihistidine antibody), glutathione S-transferase, or an analogue thereof.

[0433] This method may include removing CNAs by size-based consumption. For example, size-based consumption may be performed using porous beads (e.g., solid-phase reversible immobilization (SPRI) magnetic beads, electrophoretic gel purification (e.g., agarose gel purification), and / or gel filtration). In some cases, this method may include removing synthetic nucleic acids with lengths of at least 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 5 kb, or 10 kb. For example, this method may include removing synthetic nucleic acids with a length of at least 500 bp.

[0434] sequencing

[0435] This method may include sequencing target nucleic acids and / or adaptors in the sequencing library. Sequencing can be performed using basic sequencing methods, including Maxam-Gilbert sequencing, chain termination sequencing, shotgun sequencing, or bridge PCR. Sequencing can also be performed using massively parallel sequencing methods (e.g., next-generation sequencing), which include high-throughput sequencing, pyrosequencing, sequencing synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, synthetic single-molecule sequencing (SMSS) (Helicos), massively parallel sequencing, cloned single-molecule array (Solexa), shotgun sequencing, Maxam-Gilbert or Sanger sequencing, primer walking, and sequencing using Illumina, PacBio, SOLiD, Ion Torrent, 454, or nanopore platforms. In the case of sequencing using next-generation sequencing methods, the sequencing library generated in this paper is a next-generation sequencing library.

[0436] As used throughout this specification, when referring to a number or range of numbers, the term "about" means that the number or range of numbers mentioned is an approximation within experimental variability (or statistical experimental error), and that the number or range of numbers may vary, for example, between 1% and 15% of the stated number or range. In an example, the term "about" means ±10% of the stated number or value.

[0437] As used herein, unless otherwise stated, the term "or" is used to refer to a non-exclusive "or", such as "A or B" including "A but not B", "B but not A" and "A and B".

[0438] Example

[0439] Example 1: Diagnosis via cell-free DNA sequencing analysis

[0440] Cell-free plasma samples were prepared. DNA libraries for next-generation sequencing were prepared as previously described (De Vlaminck I, Khush KK, Strehl C, et al. Temporal response of the human virome to immunosuppression and antiviral therapy. Cell 2013; 155(5):1178-87.; De Vlaminck I, Martin L, Kertesz M, et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proceedings of the National Academy of Sciences of the United States of America 2015; 112(43):13336-41.; each of which is incorporated herein by reference in its entirety). Sequencing and analysis were performed on an Illumina NextSeq instrument. Briefly, after removing low-quality reads, the reads were mapped to a human reference genome (e.g., hg19). The remaining reads were mapped to a curated reference database of viruses, bacteria, fungi, and other eukaryotic pathogens. The abundance of an individual pathogen is expressed as genome copies per volume, an absolute measure of the amount of nucleic acid from a specific pathogen per 1 ml of plasma. Further analysis can be performed to identify known resistance-conferring sequences.

[0441] Direct next-generation sequencing (NGS) of patient plasma

[0442] Prior to DNA extraction, synthetic DNA molecules of known concentration were incorporated into the plasma sample. DNA was extracted using a modified magnetic bead-based method (Omega Biotek, Norcross, GA). NGS libraries were constructed using a modified library preparation kit (NuGEN, SanCarlos, CA). Negative (containing buffer but no plasma) and positive (containing plasma from a healthy donor plus a known concentration of spliced, lab-derived pathogen DNA) control samples were processed along with the samples. All three DNA library types were multiplexed and sequenced using a 75-cycle single-end double-exponential sequencing kit on Illumina NextSeq.

[0443] Bioinformatics Analysis

[0444] Pathogen reads were quantified from the NGS read set. In short, after discarding low-quality reads, human reads were removed by alignment with human reference sequences (e.g., hg19). Synthetic incorporated reads were identified by alignment with a database of complete incorporated sequences. The remaining reads were aligned with a targeted database of over 8,000 reference sequences for viruses, prokaryotes, and eukaryotes (including fungi, protozoa, and parasites). Based on the alignments, duplicate reads presumed to originate from PCR replication or sequencing instrument errors were identified and removed. The relative abundance of an organism is expressed as the estimated duplicate reads (EDR), or per million reads (RPM, normalized relative to the total reads in the sample), or reads per volume of sample (MPM, molecules per microliter). MPM is a normalized quantity that calculates the estimated number of nucleic acid fragments represented by each organism in 1 microliter of plasma. This calculation is derived from the number of sequences present in each organism, normalized relative to the known amount of synthetic DNA incorporated into the plasma at the start of extraction.

[0445] The processing of the two patient samples is described as follows: Spank-75B (SEQ ID NO:120), Spark-32 / 52 / 75 / 100 / 125 / 150 / 175 / 350 (SEQ ID NO:111-118) and ID-Spike (each incorporated at 3 × 10⁻⁶ ppm per μL of plasma) were added. 5 A mixture of (number of molecules) of the Spank / Spark mixture was incorporated into the plasma. Each sample received the same Spank / Spark mixture but a different ID-Spike. The incorporated plasma was centrifuged at 16,000g for 10 minutes, and the supernatant consisting of cell-free plasma was transferred to a new tube. After cell-free DNA extraction and library preparation to add the double-exponential Illumina adaptor, the samples, along with parallel negative and positive control samples, were pooled and then sequenced on Illumina NextSeq. Typically, approximately 400 million reads are distributed across the samples in a batch, with the number of reads in any single sample proportional to the fraction of the total library pool contained in the sample, which in turn is proportional to the amount of DNA in the cell-free plasma.

[0446] Computational analysis: Reads in individual samples were identified based on corresponding adaptor barcode sequences (“multipath decomposition”). After removing adaptor dimer sequences and quality-based read pruning, the probable origin of the read sequences was determined by alignment with human genome, incorporation, and pathogen genome reference sequences. Incorporation alignments were used to count the number of ID-Spike and SPANK-75B reads; SPANK-75B reads were deduplicated using random sequence tags embedded within each incorporation molecule. Pathogen alignments were performed based on genomic positional repetitions, and machine learning methods were used to determine the most probable taxonomic origin of each read to obtain estimated repeat reads attributed to a specific pathogen. Normalized pathogen abundance was expressed as concentration in pathogen molecules per microliter (MPM-Spank) and calculated as follows: MPM-Spank = (estimated repeat reads / number of SPANK-75B reads) × c, where c is the concentration of SPANK-75B reads incorporated into the sample, i.e., 3 x 10^6 reads per microliter. 5 One read.

[0447] For each sample in a sequencing batch, ID-Spike can be a unique incorporation type. SPANK molecules can be incorporated at a constant concentration in all libraries. Therefore, the number of repeating SPANK molecules detected in a particular library can represent the minimum detectable concentration in that library. More generally, it can be proportional to the efficiency with which the library transforms nucleic acid (e.g., DNA) molecules in the original sample to read nucleic acid sequencing data. The purpose of SPANK molecules can be to help determine the relative abundance of target (e.g., pathogen or disease-related) molecules within the mixture shown in the sample.

[0448]

[0449]

[0450] Example 2: Synthesis and processing of ID Spike

[0451] 110 exemplary ID Spikes (ID Spikes 28-137, sequences listed in Table 1) were constructed by annealing complementary 100mer oligonucleotide pairs synthesized by Integrated DNA Technologies. These sequences were then divided into four groups, added to four aliquots of human plasma, and extracted and processed together with cell-free DNA from the plasma into sequencing libraries, which were then sequenced on an Illumina NextSeq 500 instrument. The number of reads mapped to each of the 110 ID Spikes was determined and then normalized to per million total reads. Figure 9 The graph shows a minimum of approximately 12,000 and a maximum of approximately 88,000 per million total reads.

[0452] The signal from ID Spike can be adjusted. This can be achieved, for example, based on... Figure 9 The median signal shown is used to adjust the input amount for each IDSpike to normalize the IDSpike signal. For example... Figure 10 As shown, retesting the normalized ID Spike signal results in a more uniform read count. In this case, the number of reads mapped to each ID Spike ranges from 10,000 to 25,000 per million total reads, and most ID Spikes have a number of reads ranging from 15,000 to 20,000 per million total reads. The benefit of balancing or normalizing the signals of different ID Spikes is that it can improve the accuracy of cross-contamination detection. For example, if one ID Spike typically provides 70,000 reads per million while another ID Spike provides 19,000, the latter may be a less sensitive cross-contamination tracker; normalizing the signal between the two ID Spikes can provide greater uniformity.

[0453] Example 3: Measuring cross-contamination of samples using ID Spike

[0454] Four samples were prepared. ID Spike 124-127 was added to sample 1. ID Spike 128-131 was added to sample 2. ID Spike 132-134 was added to sample 3. ID Spike 135-137 was added to sample 4. The four samples were processed and sequenced. As shown in the subsets of ID Spike in Table 7, cross-contamination (e.g., attributed to ID Spike reads in samples where no ID Spike was intentionally added) was less than 1:10,000.

[0455] Table 7:

[0456]

[0457] Example 4: Measuring cross-contamination of samples using ID Spike

[0458] Four samples were prepared. ID Spike 124 was added to sample A. ID Spike 123 was added to sample B. ID Spike 122 was added to sample C. ID Spike 119 was added to sample D. The four samples were processed and sequenced. As shown in Table 8, the large number of reads of ID Spike 123 and ID Spike 124 from samples A and B indicates cross-contamination between the two ID Spike stock solu...

Claims

1. A method for determining the abundance of nucleic acids in an initial sample containing target nucleic acids, the method comprising: (a) At least 10 of the starting amount 4 A synthetic nucleic acid is added to the sample, wherein at least 10 4 Each of the synthetic nucleic acids contains a unique variable region, wherein at least 10 4 The synthetic nucleic acid cannot hybridize with the target nucleic acid; (b) At least 10 of the target nucleic acids and a portion of the sample. 4 Sequencing analysis is performed on a synthetic nucleic acid to obtain target nucleic acid sequence readouts and synthetic nucleic acid sequence readouts, wherein the synthetic nucleic acid sequence readouts include unique variable region sequences; (c) Detect the at least 10 by the following steps 4 Loss of diversity in synthetic nucleic acids: (i) Quantify the number of different variable region sequences within the readout of the synthesized nucleic acid sequence to obtain unique sequencing values; as well as (ii) the at least 10 4 The initial amount of each synthetic nucleic acid is compared with the unique sequencing value to obtain the at least 10 4 Loss of diversity in synthetic nucleic acids; as well as (d) Using the aforementioned at least 10 4 The abundance of the target nucleic acid in the initial sample is calculated by the diversity loss of the synthetic nucleic acid.

2. The method of claim 1, wherein the at least 10 4 A synthetic nucleic acid contains DNA.

3. The method of claim 1, wherein the at least 10 4 Each synthetic nucleic acid includes a double-stranded nucleic acid with a length of less than 500 base pairs, or wherein at least 10 14 A synthetic nucleic acid includes a single-stranded nucleic acid with a length of less than 500 nucleotides.

4. The method of claim 1, wherein the at least 10 4 Each synthetic nucleic acid includes a double-stranded nucleic acid with a length of less than 200 base pairs, or wherein at least 10 4 A synthetic nucleic acid includes a single-stranded nucleic acid with a length of less than 200 nucleotides.

5. The method according to claim 1, wherein the sample is an isolated nucleic acid sample.

6. The method of claim 1, further comprising generating a sequencing library from the sample, wherein the at least 10 4 A synthetic nucleic acid was added to the sample.

7. The method of claim 1, wherein the at least 10 4 The loss of diversity of a synthetic nucleic acid indicates the loss of one or more nucleic acids during sample processing of the sample.

8. The method of claim 1, wherein the at least 10 4 Each synthetic nucleic acid contains a tag sequence.

9. The method of claim 8, wherein quantifying the number of different variable region sequences within the synthetic nucleic acid sequence read includes detecting the sequence read from the tag sequence.

10. The method of claim 1, wherein the sample is derived from a human subject.

11. The method of claim 1, wherein quantifying the number of different variable region sequences within the synthetic nucleic acid sequence read comprises determining a read count for each of the different variable region sequences within the synthetic nucleic acid sequence read.

12. The method of claim 1, wherein the at least 10 4 Each synthetic nucleic acid contains at least 10 8 A synthetic nucleic acid.

13. The method of claim 1, further comprising adding additional synthetic nucleic acids having at least three different lengths.

14. The method of claim 1, further comprising adding a first set of additional synthetic nucleic acids having a first length, a second set of additional synthetic nucleic acids having a second length, and a third set of additional synthetic nucleic acids having a third length, wherein each of the first, second, and third sets of additional synthetic nucleic acids comprises synthetic nucleic acids having at least three different GC contents.

15. The method of claim 14, further comprising using the additional synthetic nucleic acid to calculate the abundance of the target nucleic acid in the sample.

16. The method of claim 13, further comprising using the additional synthetic nucleic acid to calculate the abundance of the target nucleic acid in the sample based on the length of the additional synthetic nucleic acid, the GC content, or both the length and the GC content.

17. The method of claim 1, wherein the at least 10 4 A synthetic nucleic acid was added to the sample.

18. The method of claim 17, further comprising, in the second sample processing step, adding an additional pool of at least 10 4 A synthetic nucleic acid is added to the sample, wherein the second sample processing step differs from the first sample processing step, wherein the additional pool contains at least 10 4 Each of the synthetic nucleic acids contains a unique variable region.

19. The method of claim 18, further comprising calculating at least 10 additional pools. 4 The loss of diversity in synthetic nucleic acids.

20. The method of claim 19, further comprising by means of the at least 10 4 The loss of diversity in the synthetic nucleic acids is related to the additional pool of at least 10 4 The diversity loss of each synthetic nucleic acid is compared to identify sample processing steps with relatively high diversity loss.

21. The method of claim 1, further comprising adding a sample-identifying nucleic acid to the sample.

22. The method of claim 1, wherein the calculated abundance is a relative abundance.

23. The method of claim 1, wherein the calculated abundance is an absolute abundance.

24. The method of claim 14, wherein the at least three different GC contents include a first GC content between 10% and 40%, a second GC content between 40% and 60%, and a third GC content between 60% and 90%.

25. The method of claim 14, wherein the content of each of the at least three different GCs is between 10% and 50%.

26. The method of claim 1, wherein the at least 10 4 Each synthetic nucleic acid includes a double-stranded nucleic acid with a length of less than 100 base pairs, or wherein at least 10 4 A synthetic nucleic acid includes a single-stranded nucleic acid with a length of less than 100 nucleotides.

27. The method of claim 1, wherein the at least 10 4 Each synthetic nucleic acid contains double-stranded DNA.

28. The method of claim 1, further comprising using the at least 10 4 A synthetic nucleic acid is used to monitor the denaturation of the target nucleic acid.

29. The method of claim 1, further comprising normalizing the relative abundance or initial abundance of the target nucleic acid by using a weighting factor.

30. The method of claim 1, wherein the at least 10 14 Synthetic nucleic acids cannot be degraded by nucleases.

31. The method of claim 1, wherein the results of the method are reportable.

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