Method and system for detecting contamination between samples

Abstract

Provided herein are various methods and related systems for detecting the presence / absence of contamination of a first sample by a second sample. For example, in some embodiments, the method includes (a) sequencing a collection of polynucleotides to generate more than one sequencing read, (b) aligning more than one sequencing read with a reference sequence, (c) grouping more than one sequencing read into more than one family, (d) generating family identifiers for more than one family, (e) screening a collection of common family identifiers, (f) determining a quantitative measure of the collection of common family identifiers, and (g) classifying the first sample as contaminated by the second sample or not contaminated by the second sample based on the quantitative measure of the common family identifier.

Description

[0001] Cross-references

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 724,622, filed on August 30, 2018, which is incorporated herein by reference in its entirety.

[0003] background

[0004] Cancer is usually caused by the accumulation of mutations within an individual's normal cells, at least some of which result in improper regulation of cell division. Such mutations usually include single nucleotide variations (SNVs), gene fusions, insertions and deletions (indels), transversions, translocations, and inversions.

[0005] Cancer is usually detected by tissue biopsy of tumor, and then analyzing cell pathology, biomarkers or DNA extracted from cells. But it has been proposed recently that cancer can also be detected by cell-free nucleic acids (e.g., circulating nucleic acids, circulating tumor nucleic acids, exosomes, nucleic acids from apoptotic cells and / or necrotic cells) in body fluids such as blood or urine (see, e.g., Siravegna et al., Nature Reviews, 14: 531-548 (2017)). Such tests have the advantage that they are non-invasive and can be performed without biopsy to identify suspicious cancer cells and sample nucleic acids from all parts of cancer. However, due to the low and variable fact that the amount of nucleic acid released into body fluids is low, such tests are complicated, and so is the recovery of nucleic acids in an analyzable form from such body fluids. These tests are designed so that they can detect very low frequency sequences, which are represented by 1 in 1000 molecules at a given locus. Therefore, such tests may be prone to false positive results based on low-level molecular contamination from other samples.

[0006] Samples may be contaminated by various sources, such as, but not limited to: physical carryover of liquids between samples (e.g., pipetting, automated liquid handling via sample prep or sequencer, manipulation of amplified material); demultiplexing artifacts (e.g., base call errors that confound sample indexes with finite pairwise Hamming distances; insertions / deletions that confound sample indexes with finite pairwise edit distances), and reagent impurities (e.g., sample index oligonucleotides with some loss of oligonucleotides synthesized in the same batch; sample index oligonucleotides contaminated with oligonucleotides containing another sample index (through carryover of synthesis errors)).

[0007] Overview

[0008] The application discloses a method and system for detecting the pollution between two samples.Previous sample contamination detection methods are based on the detection of certain molecules, which can only exist in high abundance or not exist at all in uncontaminated samples, but if low abundance is observed, pollution is indicated.Two such types of molecules are molecules or Y chromosome molecules carrying common germline single nucleotide polymorphisms (SNPs).These methods are limited by the following facts, that is, the above molecules are usually only a small part of all contaminating molecules, and their amount may not be enough for detection in the presence of sequencing errors and sampling errors.In addition, in the case of high contamination rates, germline SNVs based on contamination may not be distinguished from the natural germline SNVs of contaminated samples.Because Y chromosome molecules are naturally present only in male patients, using Y chromosome molecules as a detection mechanism is further limited to female patient samples being contaminated by male patient samples.In addition to physical contamination, when sample index is easily converted into another index that is then misassigned in the algorithm, digital cross contamination may occur.This problem can be alleviated by adding dual indexing (dual indexing), but the method has its own shortcomings.

[0009] The present disclosure provides methods, compositions, and systems for detecting the presence or absence of contamination of a first sample by a second sample.

[0010] In one aspect, the present disclosure provides a system for detecting the presence or absence of contamination of a first sample by a second sample, the system comprising: a communication interface that receives more than one sequencing read of a set of tagged polynucleotides from a sample generated by a nucleic acid sequencer through a communication network, wherein the sequencing read comprises a tag sequence and a sequence derived from the polynucleotide; and a computer that communicates with the communication interface, wherein the computer comprises one or more computer processors and a computer-readable medium containing machine executable code, which when executed by the one or more computer processors implements a method, the method comprising: (a) receiving more than one sequencing read of a set of tagged polynucleotides from a sample generated by a nucleic acid sequencer through a communication network; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; and (c) for each sample, grouping the more than one sequencing read into In more than one family, the grouping features include at least one of the following (i), (ii), (iii) and (iv): (i) a tag, (ii) a start region, (iii) an end region and (iv) a length of a polynucleotide, wherein each family in the sample includes sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in a set of polynucleotides in the sample; (d) generating family identifiers for more than one family; (e) screening out a set of common family identifiers, wherein a given common family identifier is a family identifier of a first sample that is identical or substantially identical to a family identifier of a second sample; (f) determining a quantitative measure of the set of common family identifiers; and (g) classifying the first sample as contaminated by the second sample if the quantitative measure of the set of common family identifiers is above a predetermined threshold, or classifying the first sample as not contaminated by the second sample if the quantitative measure of the set of common family identifiers is at or below a predetermined threshold.

[0011] In another aspect, the present disclosure provides a system comprising a controller, the controller comprising or having access to a computer-readable medium containing non-transitory computer-executable instructions, the non-transitory computer-executable instructions, when executed by at least one electronic processor, performing a method, the method comprising: (a) sequencing a collection of polynucleotides from a first sample and a second sample to generate more than one sequencing read; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising at least one of the following (i), (ii), and (iii): (i) a start region, (ii) a end region and (iii) the length of the polynucleotide, wherein each family in the sample includes sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the sample; (d) generating family identifiers for more than one family; (e) screening a set of common family identifiers, wherein a given common family identifier is a family identifier of a first sample that is identical or substantially identical to a family identifier of a second sample; (f) determining a quantitative measure of the set of common family identifiers; and (g) if the quantitative measure of the set of common family identifiers is above a predetermined threshold, classifying the first sample as contaminated by the second sample, or if the quantitative measure of the set of common family identifiers is at or below a predetermined threshold, classifying the first sample as not contaminated by the second sample.

[0012] In another aspect, the present disclosure provides a system comprising a controller comprising or having access to a computer-readable medium containing non-transitory computer-executable instructions, wherein the non-transitory computer-executable instructions, when executed by at least one electronic processor, perform a method comprising: (a) sequencing a collection of polynucleotides from a sample to generate more than one sequencing read; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) grouping the more than one sequencing reads of two samples together into more than one family based on a grouping feature, wherein the grouping feature comprises at least one of the following (i), (ii) and (iii): (i) starting (i) a starting region, (ii) an ending region, and (iii) a length of a polynucleotide, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the sample; (d) screening more than one family to identify a set of shared families, wherein a given shared family comprises at least one sequencing read from the first sample and at least one sequencing read from the second sample; (e) determining a quantitative measure of the set derived from the shared family; and (f) classifying the first sample as contaminated by the second sample if the quantitative measure of the set of the shared family is above a predetermined threshold, or classifying the first sample as not contaminated by the second sample if the quantitative measure of the set of the shared family is at or below a predetermined threshold.

[0013] In another aspect, the present disclosure provides a system comprising a controller comprising or having access to a computer-readable medium containing non-transitory computer-executable instructions, which when executed by at least one electronic processor perform a method comprising: (a) sequencing a collection of tagged polynucleotides from a sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read to a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, wherein the more than one sequencing read comprises a tag and a polynucleotide; (d) aligning the more than one sequencing read to a reference sequence, thereby determining a start region and an end region of the alignment; and (e) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature. The grouping features include tags, wherein each family in the sample includes sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in the set of tagged polynucleotides in the sample; (d) selecting a set of common family identifiers, wherein a given common family identifier is a family identifier of a first sample that is the same or substantially the same as a family identifier of a second sample; (e) determining a quantitative measure of the set of common family identifiers; and (f) classifying the first sample as contaminated by the second sample if the quantitative measure of the set of common family identifiers is above a predetermined threshold, or classifying the first sample as not contaminated by the second sample if the quantitative measure of the common family identifiers is at or below a predetermined threshold.

[0014] In another aspect, the present disclosure provides a system comprising a controller comprising or having access to a computer-readable medium containing non-transitory computer-executable instructions, wherein the non-transitory computer-executable instructions, when executed by at least one electronic processor, perform a method comprising: (a) sequencing a collection of polynucleotides from a sample to generate more than one sequencing read; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, wherein the grouping feature comprises at least one of the following (i), (ii), and (iii): (i) a start region, (ii) an end region and (iii) a length of the polynucleotide, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the sample; (d) screening more than one family to identify a set of shared families, wherein a given shared family is a family of the first sample having a grouping characteristic that is the same or substantially the same as a grouping characteristic of a family of the second sample; (e) determining a quantitative measure of the set of shared families of the first sample; and (f) classifying the first sample as contaminated by the second sample if the quantitative measure of the set of shared families is above a predetermined threshold, or classifying the first sample as not contaminated by the second sample if the quantitative measure of the set of shared families is at or below a predetermined threshold.

[0015] In some embodiments, the sequencing read comprises (i) a tag sequence, and (ii) a sequence derived from a polynucleotide. In some embodiments, the system further comprises, for each sample, grouping more than one sequencing read into more than one family based on information from at least one of the following (i), (ii), (iii), and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) a length of the polynucleotide, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the sample.

[0016] In another aspect, the present disclosure provides a system comprising a controller comprising or having access to a computer-readable medium containing non-transitory computer-executable instructions, which when executed by at least one electronic processor perform a method comprising: (a) sequencing a collection of tagged polynucleotides from a sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read to a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, wherein the grouping feature The method comprises: (a) screening more than one family to identify a set of shared families, wherein the shared family is a family of the first sample having a grouping characteristic that is the same or substantially the same as a grouping characteristic of a family of the second sample; (b) determining a quantitative measure of the set of shared families of the first sample; and (c) classifying the first sample as being contaminated by the second sample if the quantitative measure of the set of shared families is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of shared families is at or below a predetermined threshold.

[0017] In another aspect, the present disclosure provides a system comprising a controller comprising or having access to a computer-readable medium containing non-transitory computer-executable instructions, wherein the non-transitory computer-executable instructions, when executed by at least one electronic processor, perform a method comprising: (a) sequencing a collection of tagged polynucleotides from a sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read to a reference sequence, thereby determining a start region and an end region of the alignment; (c) grouping the more than one sequencing reads of two samples together into more than one family based on a grouping feature , wherein the grouping features include tags, wherein each family in the sample includes sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in the set of tagged polynucleotides in the sample; (d) screening more than one family to identify a set of shared families, wherein a given shared family includes at least one sequencing read from a first sample and at least one sequencing read from a second sample; (e) determining a quantitative measure of the set derived from the shared family; (f) if the quantitative measure of the set of shared families is above a predetermined threshold, classifying the first sample as contaminated by the second sample, or if the quantitative measure of the shared family is at or below a predetermined threshold, classifying the first sample as not contaminated by the second sample.

[0018] In some embodiments, the system further comprises detecting somatic genetic variation in a polynucleotide of the first sample by excluding sequencing reads of common families of the first sample, wherein the first sample is classified as contaminated by the second sample.

[0019] In some embodiments, the system further comprises generating a report, which optionally includes information about the contamination status of the sample and / or information derived from the contamination status of the sample.

[0020] In some embodiments, the system further comprises transmitting the report to a third party, such as the subject from whom the sample originated or a health care practitioner.

[0021] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of polynucleotides from the sample to generate more than one sequencing read; (b) aligning the more than one sequencing reads with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing reads into more than one family based on a grouping feature, the grouping feature comprising at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region, and (iii) a length of the polynucleotide, wherein each family in the sample comprises a collection of polynucleotides from the sample; (d) generating family identifiers for more than one family; (e) screening a set of shared family identifiers, wherein a given shared family identifier is a family identifier for the first sample that is identical or substantially identical to a family identifier for the second sample; (f) determining a quantitative measure for the set of shared family identifiers; and (g) classifying the first sample as being contaminated by the second sample if the quantitative measure for the set of shared family identifiers is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure for the set of shared family identifiers is at or below a predetermined threshold.

[0022] On the other hand, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) accessing sequence information including more than one sequencing reads from the first sample and the second sample through a computer system; (b) aligning the more than one sequencing reads with a reference sequence through a computer system, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing reads into more than one family based on a grouping feature through a computer system, the grouping feature comprising at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region and (iii) a length of a polynucleotide, wherein each family in the sample comprises polynucleotides from the sample (d) generating family identifiers for more than one family by a computer system; (e) screening a set of shared family identifiers by a computer system, wherein a given shared family identifier is a family identifier of a first sample that is identical or substantially identical to a family identifier of a second sample; (f) determining a quantitative measure of the set of shared family identifiers by a computer system; and (g) classifying the first sample as being contaminated by the second sample by the computer system if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample by the computer system if the quantitative measure of the set of shared family identifiers is at or below a predetermined threshold.

[0023] On the other hand, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) obtaining sequence information comprising more than one sequencing read from the first sample and the second sample; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region and (iii) a length of a polynucleotide, wherein each family in the sample comprises from (d) generating family identifiers for more than one family; (e) screening a set of shared family identifiers, wherein a given shared family identifier is a family identifier for a first sample that is identical or substantially identical to a family identifier for a second sample; (f) determining a quantitative measure of the set of shared family identifiers; and (g) classifying the first sample as being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is at or below a predetermined threshold.

[0024] In some embodiments, the method further comprises, prior to a), tagging the set of polynucleotides to produce tagged polynucleotides, wherein each tagged polynucleotide comprises a tag and a polynucleotide. In some embodiments, the method further comprises, for each sample, grouping more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising at least one of (i), (ii), (iii), and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) a length of the polynucleotide, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the sample.

[0025] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a tagged polynucleotide or a collection of polynucleotides from a sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising a tag, wherein each family in the sample comprises a set of tagged polynucleotides from the sample; (d) generating family identifiers for more than one family; (e) screening a set of shared family identifiers, wherein a given shared family identifier is a family identifier for a first sample that is identical or substantially identical to a family identifier for a second sample; (f) determining a quantitative measure of the set of shared family identifiers; and (g) classifying the first sample as being contaminated by the second sample if the quantitative measure of the shared family identifier is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is at or below a predetermined threshold.

[0026] In some embodiments, the quantitative measure of the set of shared family identifiers is the number of shared family identifiers in the first sample. In some embodiments, the quantitative measure of the set of shared family identifiers includes the ratio of the number of shared family identifiers in the first sample to the total number of family identifiers in the first sample. In some embodiments, the quantitative measure of the set of shared family identifiers does not include the following shared identifiers in the first sample: those shared family identifiers whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample. In some embodiments, the quantitative measure of the set of shared family identifiers in the first sample does not include shared family identifiers at over-represented genome start positions and genome end position pairs. In some embodiments, the total number of family identifiers in the first sample does not include family identifiers at over-represented genome start positions and genome end position pairs.

[0027] In some embodiments, the over-represented genomic start position and genomic end position are determined by: (a) providing more than one sample, wherein more than one sample includes a distribution of genomic start positions and genomic end positions that are identical or substantially identical to the first sample and / or the second sample; (b) determining the family identifiers in more than one sample; (c) quantifying the number of family identifiers that have a pair of genomic start positions and genomic end positions in more than one sample; and (d) if the number of family identifiers exceeds a set threshold, the pair of genomic start positions and genomic end positions are classified as over-represented. In some embodiments, more than one sample does not include the first sample or the second sample. In some embodiments, more than one sample does not include the first sample and the second sample. In some embodiments, more than one sample includes a sample processed in the same flow cell as the first sample. In some embodiments, more than one sample includes a training sample. In some embodiments, the threshold is set at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 families.

[0028] On the other hand, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of polynucleotides from the sample to generate more than one sequencing read; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region, and (iii) a length of the polynucleotide, wherein each of the samples in the sample (d) screening more than one family to identify a set of shared families, wherein a given shared family is a family of the first sample having a grouping characteristic that is the same or substantially the same as a grouping characteristic of a family of the second sample; (e) determining a quantitative measure of the set of shared families of the first sample; and (f) classifying the first sample as being contaminated by the second sample if the quantitative measure of the set of shared families is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of shared families is at or below a predetermined threshold.

[0029] On the other hand, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of polynucleotides from the samples to generate more than one sequencing read; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) grouping the more than one sequencing reads of the two samples together into more than one family based on a grouping feature, wherein the grouping feature comprises at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region and (iii) a length of the polynucleotide. (d) screening more than one family to identify a set of shared families; wherein the shared family includes at least one sequencing read from the first sample and at least one sequencing read from the second sample; (e) determining a quantitative measure of the set derived from the shared family; and (f) classifying the first sample as contaminated by the second sample if the quantitative measure of the set of shared families is above a predetermined threshold, or classifying the first sample as not contaminated by the second sample if the quantitative measure of the set of shared families is at or below a predetermined threshold.

[0030] In some embodiments, the method further comprises, prior to sequencing, tagging the collection of polynucleotides to produce tagged polynucleotides, wherein each tagged polynucleotide comprises a tag and a polynucleotide.

[0031] In some embodiments, the method includes, for each sample, grouping more than one sequencing read into more than one family based on grouping features, wherein the grouping features include at least one of the following (i), (ii), (iii), and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) a length of the polynucleotide, wherein each family in the sample includes sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in a set of polynucleotides in the sample.

[0032] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of tagged polynucleotides from the sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising the tag, wherein the family in the sample comprises the tagged polynucleotides from the sample (d) screening more than one family to identify a set of shared families, wherein a given shared family is a family of the first sample having a grouping characteristic that is the same or substantially the same as a grouping characteristic of a family of the second sample; (e) determining a quantitative measure of the set of shared families of the first sample; and (f) classifying the first sample as being contaminated by the second sample if the quantitative measure of the set of shared families is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of shared families is at or below a predetermined threshold.

[0033] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of tagged polynucleotides from the samples to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) grouping the more than one sequencing reads of the two samples together into more than one family based on a grouping feature, wherein the grouping feature comprises a tag, wherein each family in the sample comprises a tagged polynucleotide from the sample; (d) screening more than one family to identify a set of shared families, wherein a given shared family includes at least one sequencing read from a first sample and at least one sequencing read from a second sample; (e) determining a quantitative measure of the set derived from the shared family; and (f) classifying the first sample as contaminated by the second sample if the quantitative measure of the set of the shared family is above a predetermined threshold, or classifying the first sample as not contaminated by the second sample if the quantitative measure of the set of the shared family is at or below a predetermined threshold.

[0034] In some embodiments, the quantitative measure includes the number of shared families in the first sample. In some embodiments, the quantitative measure includes the ratio of the number of sequencing reads of the first sample in the shared family to the number of sequencing reads of the second sample. In some embodiments, the quantitative measure includes the ratio of the number of shared families in the first sample to the total number of families in the first sample. In some embodiments, the quantitative measure of the set of shared families does not include the following shared families in the first sample: those shared families whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample. In some embodiments, the quantitative measure of the set of shared families in the first sample does not include the shared families at the over-represented genome start position and genome end position pairs. In some embodiments, the total number of families in the first sample does not include the families at the over-represented genome start position and genome end position pairs. In some embodiments, the over-represented genomic start position and genomic end position are determined by: (a) providing more than one sample, wherein the more than one sample includes a distribution of genomic start positions and genomic end positions that are identical or substantially identical to the first sample and / or the second sample; (b) determining the families in more than one sample; (c) quantifying the number of families with a pair of genomic start positions and genomic end positions in more than one sample; and (d) if the number of families exceeds a set threshold, the pair of genomic start positions and genomic end positions are classified as over-represented. In some embodiments, more than one sample does not include the first sample or the second sample. In some embodiments, more than one sample does not include the first sample and the second sample. In some embodiments, more than one sample includes a sample processed in the same flow cell as the first sample. In some embodiments, more than one sample includes a training sample. In some embodiments, the threshold is set at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 families. In some embodiments, the threshold is set at about 5 families. In some embodiments, the threshold is set at about 10 families. In some embodiments, the threshold is set at about 15 families. In some embodiments, the threshold is set at about 20 families. In some embodiments, the threshold is set at about 30 families. In some embodiments, the threshold is set at about 40 families. In some embodiments, the threshold is set at about 50 families. In some embodiments, the threshold can be at least 10 of the total families observed in more than one sample. -3 , at least 10 -4 , at least 10 -5 , at least 10 -6 , at least 10-7 , at least 10 -8 or at least 10 -9 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -4 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -5 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -6 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -7 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -8 .

[0035] In some embodiments, the starting region includes the genomic starting position of the sequencing read, at which the 5' end of the sequencing read is determined to start the alignment with the reference sequence, and the ending region includes the genomic ending position of the sequencing read, at which the 3' end of the sequencing read is determined to terminate the alignment with the reference sequence. In some embodiments, the starting region includes the first 1, first 2, first 5, first 10, first 15, first 20, first 25, first 30, or at least the first 30 base positions of the 5' end of the sequencing read aligned with the reference sequence. In some embodiments, the ending region includes the last 1, last 2, last 5, last 10, last 15, last 20, last 25, last 30, or at least the last 30 base positions of the 3' end of the sequencing read aligned with the reference sequence.

[0036] In some embodiments, the label includes one or more molecular barcodes attached to the ends of the polynucleotides. In some embodiments, the length of the one or more molecular barcodes is at least 2, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, or at least 20 nucleotides. In some embodiments, the one or more molecular barcodes attached to the polynucleotides of the first sample are different from the one or more molecular barcodes attached to the polynucleotides of the second sample. In some embodiments, the polynucleotides of the sample are labeled with at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 50,000, or at least 100,000 different molecular barcodes.

[0037] In some embodiments, the first sample and the second sample are sequenced in the same flow cell. In some embodiments, the second sample is sequenced in a flow cell different from the first sample. In some embodiments, the second sample is processed at different times from the first sample on the same day as the first sample. In some embodiments, the second sample is processed at least 1 minute, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours or at least 4 hours after processing the first sample. In some embodiments, the first sample and the second sample are processed on different dates. In some embodiments, the first sample and the second sample are in the same sample batch. In some embodiments, the second sample is processed with the same batch of reagents as the first sample. In some embodiments, the first sample and the second sample are processed in different geographical locations.

[0038] In some embodiments, the set of tagged polynucleotides of a sample is uniquely tagged. In some embodiments, the set of tagged polynucleotides of a sample is non-uniquely tagged. In some embodiments, the first sample is obtained from a body fluid of one subject and the second sample is obtained from a body fluid of another subject.

[0039] In some embodiments, the polynucleotide is a cell-free polynucleotide. In some embodiments, the cell-free polynucleotide is cell-free DNA. In some embodiments, at least one of the subjects suffers from a disease. In some embodiments, the disease is cancer.

[0040] In some embodiments, the set of the polynucleotide of the sample is amplified before sequencing, thereby producing amplified progeny polynucleotides. In some embodiments, the method also includes selectively enriching at least a portion of the amplified progeny polynucleotides from the region of the genome or transcriptome of the subject before sequencing. In some embodiments, the method also includes attaching one or more sample indexes to one end or both ends of the amplified progeny polynucleotides before sequencing, wherein the sample index distinguishes the first sample and the second sample. In some embodiments, the predetermined threshold is at least 0.001%, at least 0.005%, at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5% or at least 10% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.01% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.05% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.1% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.5% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 1% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 2% of the total number of families in the first sample.

[0041] In some embodiments, the method further comprises detecting somatic genetic variation of polynucleotides of the first sample by excluding sequencing reads of the common family identifier of the first sample, wherein the first sample is classified as contaminated by the second sample. In some embodiments, the method further comprises detecting somatic genetic variation of polynucleotides of the first sample by excluding sequencing reads of the common family of the first sample, wherein the first sample is classified as contaminated by the second sample.

[0042] In some embodiments, the method further comprises generating a report, the report optionally comprising information about the contamination state of the sample and / or information derived from the contamination state of the sample. In some embodiments, the method comprises transmitting the report to a third party, such as a subject or a health care practitioner from whom the sample is derived.

[0043] The embodiments as described herein may be used or applied to both the methods and systems described herein.

[0044] In some embodiments, the result of the system and / or method disclosed herein is used as input to generate a report. The report can be in paper format or electronic format. For example, information about the contamination state of the first sample and / or information derived from the contamination state of the first sample as determined by the method or system disclosed herein can be presented in such a report. The method or system disclosed herein can also include the step of transmitting the report to a third party, such as a subject or a health care practitioner from whom the sample is derived.

[0045] The various steps of the methods disclosed herein, or the steps performed by the systems disclosed herein, can be performed at the same time or at different times, and / or at the same geographical location or at different geographical locations (e.g., countries). The various steps of the methods disclosed herein can be performed by the same person or by different people.

[0046] In certain aspects, the present disclosure provides a non-transitory computer-readable medium containing non-transitory computer-executable instructions that, when executed by at least one electronic processor, may perform one or more steps or methods described herein.

[0047] In another aspect, the present disclosure provides a non-transitory computer-readable medium comprising non-transitory computer-executable instructions, which when executed by at least one electronic processor can perform at least the following: (a) obtaining more than one sequencing read of a set of tagged polynucleotides from a sample generated by a nucleic acid sequencer; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, wherein the grouping feature includes at least one of the following (i), (ii), (iii) and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) a sequence of sequences of interest. (iv) the length of the polynucleotides, wherein each family in the sample comprises sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the sample; (d) generating family identifiers for more than one family; (e) screening a set of common family identifiers, wherein a given common family identifier is a family identifier for a first sample that is identical or substantially identical to a family identifier for a second sample; (f) determining a quantitative measure for the set of common family identifiers; and (g) classifying the first sample as contaminated by the second sample if the quantitative measure for the set of common family identifiers is above a predetermined threshold, or classifying the first sample as not contaminated by the second sample if the quantitative measure for the set of common family identifiers is at or below a predetermined threshold.

[0048] In certain aspects, the methods, systems, and / or computer-readable media described herein can be used as a quality control metric for assay performance and / or for assessing the quality of obtained sequencing data to ensure reliable detection of somatic variation in a sample.

[0049] From the following detailed description, other aspects and advantages of the present disclosure will become readily apparent to those skilled in the art, in which only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure can have other and different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and description are considered to be illustrative in nature, rather than restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The accompanying drawings illustrate certain embodiments and are used together with the written description to explain certain principles of the methods, computer-readable media, and systems disclosed herein, and are incorporated into and constitute a part of this specification. The description provided herein is better understood when read in conjunction with the accompanying drawings, which are included by way of example and not by way of limitation. It will be understood that, unless the context otherwise indicates, similar reference numerals identify similar components throughout the accompanying drawings. It will also be understood that some or all of the drawings may be schematic representations for illustrative purposes and do not necessarily depict the actual relative sizes or positions of the elements shown.

[0052] Figure 1 is a flow chart representation of a method for detecting the presence or absence of contamination between two samples according to an embodiment of the present disclosure.

[0053] Figure 2 is a flow chart representation of a method for detecting the presence or absence of contamination between two samples according to an embodiment of the present disclosure.

[0054] Figure 3 is a schematic diagram illustrating grouping sequencing reads into families and thereby detecting the presence or absence of contamination between two samples according to an embodiment of the present disclosure.

[0055] Figure 4 is a schematic diagram of an exemplary system suitable for use with some embodiments of the present disclosure.

[0056] Definition of terms

[0057] Although various embodiments of the present disclosure have been shown and described herein, it will be appreciated by those skilled in the art that such embodiments are provided by way of example only. It will be appreciated by those skilled in the art that many variations, changes and substitutions may be made without departing from the present disclosure. It should be appreciated that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0058] In order to more easily understand the present disclosure, some terms are first defined below. The following terms and other definitions of other terms can be set forth through the specification. If the definition of a term set forth below is inconsistent with the definition in an application or patent incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0059] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of a type described herein and / or which will become apparent to one of ordinary skill in the art upon reading this disclosure, and so forth.

[0060] It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting. In addition, unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. When describing and claiming methods, computer-readable media, and systems, the following terms and their grammatical variations will be used according to the definitions set forth below.

[0061] About: As used herein, "about" or "approximately" refers to a value or element similar to a stated reference value or element when applied to one or more values ​​or elements of interest. In certain embodiments, the term "about" or "approximately" refers to a range of values ​​or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a stated reference value or element in either direction (greater than or less than), unless otherwise stated or otherwise apparent from context (except when such a number would exceed 100% of a possible value or element).

[0062] Adaptor: As used herein, "adaptor" refers to a short nucleic acid (e.g., less than about 500 nucleotides, less than about 100 nucleotides, or less than about 50 nucleotides in length), which is typically at least partially double-stranded and is used to connect either end or both ends of a given sample nucleic acid molecule. An adaptor may include a nucleic acid primer binding site and / or a sequencing primer binding site that allows amplification of nucleic acid molecules flanked by adaptors at both ends, and / or the sequencing primer binding site includes primer binding sites for sequencing applications such as various next generation sequencing (NGS) applications. An adaptor may also include a binding site for a capture probe such as an oligonucleotide attached to a flow cell support. An adaptor may also include a nucleic acid tag as described herein. Nucleic acid tags are typically positioned relative to the binding sites of amplification primers and sequencing primers so that the nucleic acid tag is included in the amplicon and sequencing reads of a given nucleic acid molecule. The same or different adaptors may be connected to the corresponding ends of a nucleic acid molecule. In some embodiments, adaptors of the same sequence that are different except for the nucleic acid tag are connected to the corresponding ends of a nucleic acid molecule. In some embodiments, the adapter is a Y-shaped adapter, one end of which is blunt-ended or tailed as described herein, for connecting nucleic acid molecules, which are also blunt-ended or tailed with one or more complementary nucleotides. In still other exemplary embodiments, the adapter is a bell-shaped adapter, which comprises a blunt-ended or tailed end for connection to the nucleic acid molecule to be analyzed. Other examples of adapters include T-tailed and C-tailed adapters.

[0063] Amplification: As used herein, "amplify" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), wherein the amplification product or amplicon is typically detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes.

[0064] Barcode: As used herein, "barcode" or "molecular barcode" in the context of nucleic acids refers to a nucleic acid molecule that contains a sequence that can be used as a molecular identifier. For example, during next-generation sequencing (NGS) library preparation, a separate "barcode" sequence is typically added to each DNA fragment so that each read can be identified and sorted prior to final data analysis.

[0065] Cancer type: As used herein, "cancer type" refers to a type or subtype of cancer, for example, as defined by histopathology. Cancer type can be defined by any conventional criteria, such as based on occurrence in a given tissue (e.g., blood cancer, central nervous system (CNS) cancer, brain cancer, lung cancer (small cell and non-small cell), skin cancer, nose cancer, laryngeal cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, intestinal cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, breast cancer, prostate cancer, ovarian cancer, lung cancer, small intestine cancer, soft tissue cancer, neuroendocrine cancer, gastroesophageal cancer, head and neck cancer, gynecological cancer, colorectal cancer, urothelial cancer, solid state cancers, heterogeneous cancers, homogeneous cancers Cancer)), unknown primary origin, etc., and / or having the same cell lineage (e.g., carcinoma, sarcoma, lymphoma, cholangiocarcinoma, leukemia, mesothelioma, melanoma or glioblastoma) and / or showing cancer markers such as Her2, CA15-3, CA19-9, CA-125, CEA, AFP, PSA, HCG, hormone receptors and NMP-22. Cancer can also be classified by stage (e.g., 1st, 2nd, 3rd or 4th phase) and whether it is a primary or secondary source.

[0066] Cell-free nucleic acid: As used herein, "cell-free nucleic acid" refers to nucleic acids that are not contained in cells or otherwise bound to cells, or in some embodiments, nucleic acids that remain in a sample after the removal of intact cells. Cell-free nucleic acids may include, for example, all unencapsulated nucleic acids derived from body fluids from a subject (e.g., blood, plasma, serum, urine, cerebrospinal fluid (CSF), etc.). Cell-free nucleic acids include DNA (cfDNA), RNA (cfRNA), and their hybrids, including genomic DNA, mitochondrial DNA, circulating DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi interacting RNA (piRNA), long non-coding RNA (long ncRNA), and / or fragments of any of these. Cell-free nucleic acids may be double-stranded, single-stranded, or hybrids thereof. Cell-free nucleic acids may be released into body fluids by secretion or cell death processes, such as cell necrosis, apoptosis, etc. Some cell-free nucleic acids are released from cancer cells into body fluids, for example, circulating tumor DNA (ctDNA). Others are released from healthy cells. ctDNA can be a fragmented DNA of tumor origin that is not encapsulated. Another example of cell-free nucleic acids is fetal DNA that circulates freely in the maternal bloodstream, also referred to as cell-free fetal DNA (cffDNA). Cell-free nucleic acids can have one or more epigenetic modifications, for example, cell-free nucleic acids can be acetylated, 5-methylated, ubiquitinated, phosphorylated, sumoylated, ribosylated and / or citrullinated.

[0067] Cellular nucleic acid: As used herein, "cellular nucleic acid" means nucleic acid that is located within one or more cells from which the nucleic acid is produced, at least at the point at which a sample is obtained or collected from a subject, even if such nucleic acid is subsequently removed (e.g., via cell lysis) as part of a given analytical procedure.

[0068] Contamination of a sample: As used herein, the term "contamination" or "contamination of a sample" refers to any chemical or digital contamination of one sample by another sample. Contamination may be due to a variety of sources, such as, but not limited to: physical carryover of liquids between samples (e.g., pipetting, automated liquid handling via sample preparation or sequencer systems, manipulation of amplified material); demultiplexing artifacts (e.g., base call errors that confuse sample indexes with finite pairwise Hamming distances; insertions / deletions that confuse sample indexes with finite pairwise edit distances) and reagent impurities (e.g., contamination of a sample index oligonucleotide with an oligonucleotide containing another sample index (through carryover of synthesis errors)).

[0069] Deoxyribonucleic acid or ribonucleic acid: As used herein, "deoxyribonucleic acid" or "DNA" refers to a natural or modified nucleotide having a hydrogen group at the 2'-position of the sugar portion. DNA generally includes a nucleotide chain comprising the following four types of nucleotide bases: adenine (A), thymine (T), cytosine (C) and guanine (G). As used herein, "ribonucleic acid" or "RNA" refers to a natural or modified nucleotide having a hydroxyl group at the 2'-position of the sugar portion. RNA generally includes a nucleotide chain comprising the following four types of nucleotide bases: A, uracil (U), G and C. As used herein, the term "nucleotide" refers to a natural nucleotide or a modified nucleotide. Certain nucleotide pairs specifically bind to each other in a complementary manner (referred to as complementary base pairing). In DNA, adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U) and cytosine (C) pairs with guanine (G). When the first nucleic acid chain is combined with a second nucleic acid chain consisting of nucleotides complementary to those in the first chain, the two chains combine to form a double strand. As used herein, "nucleic acid sequencing data", "nucleic acid sequencing information", "sequence information", "nucleic acid sequence", "nucleotide sequence", "genomic sequence", "gene sequence", or "fragment sequence", or "nucleic acid sequencing read" means any information or data indicating the order and identity of nucleotide bases (e.g., adenine, guanine, cytosine, and thymine or uracil) in a molecule (e.g., a whole genome, a whole transcriptome, an exome, an oligonucleotide, a polynucleotide, or a fragment) of a nucleic acid such as DNA or RNA. It should be understood that the present teachings contemplate the use of all available various techniques, platforms or technologies, including but not limited to the following sequence information obtained: capillary electrophoresis, microarrays, ligation-based systems, polymerase-based systems, hybridization-based systems, direct or indirect nucleotide identification systems, pyrophosphate sequencing, ion-based or pH-based detection systems, and electronic signature-based systems.

[0070] Family: As used herein, the term "family" refers to one or more sequencing reads derived from a single polynucleotide molecule. Bioinformatically, one or more sequencing reads derived from a single polynucleotide molecule will have the same or substantially the same grouping characteristics, wherein the grouping characteristics include at least one of the following: (i) a label (i.e., a molecular barcode), (ii) the start region of the alignment, (iii) the end region of the alignment, and (iv) the length of the polynucleotide. Those sequencing reads with the same or substantially the same grouping characteristics can be grouped together into a family. In some embodiments, although there is a low probability, at least two molecules may have the same grouping characteristics, and therefore sequencing reads derived from at least two molecules can be grouped into a single family.

[0071] In some embodiments, the sequencing reads derived from a single polynucleotide molecule are only detected in a single sample. In some embodiments, in the presence of contamination of at least two samples, the sequencing reads derived from a single polynucleotide molecule (of a single sample) can be detected in at least two samples. In these embodiments, in the case of grouping of sequencing reads independently for each sample, the sequencing reads derived from a single polynucleotide molecule detected in each sample will be grouped into a separate family in the sample. In other embodiments, in the case of grouping of sequencing reads for all at least two samples together, the sequencing reads derived from a single polynucleotide molecule detected in at least two samples will be grouped into a single family.

[0072] The grouping feature of a family represents the grouping feature of the sequencing reads in the family. In some embodiments, if the family includes sequencing reads with the same grouping feature, the grouping feature of any sequencing read is the grouping feature of the family. In other embodiments, if the family includes sequencing reads with the same and substantially the same grouping feature, the grouping feature of the family can be one of the following but not limited to the following or a combination of the following but not limited to the following: (i) the most frequently represented grouping feature of the sequencing reads; (ii) the average value of the grouping feature of the sequencing reads; (iii) the most frequently represented nucleotide base in the molecular barcode; (iv) the maximum likelihood value of the molecular barcode and / or the start region and / or end region of the sequencing read.

[0073] In some embodiments, a family includes at least two sequencing reads derived from a single polynucleotide molecule. In some embodiments, a family may include a sequence read derived from a single strand of a double-stranded polynucleotide molecule. In some embodiments, a family includes a sequence read derived from two strands (sense strand and antisense strand) of a double-stranded polynucleotide molecule. In an example, molecular barcodes, genome start positions, and genome termination positions are considered to be grouping features of a family. In this example, if a family has 10 sequence reads and all sequence reads have the same molecular barcode and genome start position, but the genome termination position is not identical, then the molecular barcode and genome start position become the grouping features of the family, and for the genome termination position, the genome termination position represented by most of the sequencing reads in the family will be considered to be the genome termination position of the family (it is a part of the grouping features of the family).

[0074] Family identifier: As used herein, the term "family identifier" refers to an identifier that uniquely identifies each family, and it includes grouping characteristics and / or information derived from grouping characteristics of the family. In some embodiments, the family identifier may include an integer, a letter, or a combination of both. In some embodiments, the family identifier is assigned to the sequencing reads in the family.

[0075] Germline mutation: As used herein, the terms "germline mutation" or "germline variation" are used interchangeably and refer to an inherited mutation (i.e., a mutation that does not arise after conception). A germline mutation may be the only mutation that can be passed on to offspring and may be present in every somatic and germline cell in the offspring.

[0076] Indel: As used herein, "Indel" refers to a mutation involving the insertion or deletion of nucleotides in the genome of a subject.

[0077] Mutant Allele Fraction: As used herein, "mutant allele fraction", "mutation dose" or "MAF" refers to the fraction of nucleic acid molecules carrying allele changes or mutations at a given genomic position / locus in a given sample. MAF is usually expressed as a fraction or percentage. For example, the MAF of somatic variants may be less than 0.15.

[0078] Mutation: As used herein, "mutation" refers to a variation from a known reference sequence, and includes mutations such as, for example, single nucleotide variations (SNVs) and insertions or deletions (gains and losses). Mutations can be germline mutations or somatic mutations. In some embodiments, the reference sequence for comparison purposes is the wild-type genomic sequence of the species of the subject providing the test sample, typically the human genome.

[0079] Neoplasm: As used herein, the terms "neoplasm" and "tumor" are used interchangeably. They refer to an abnormal growth of cells in a subject. A neoplasm or tumor can be benign, potentially malignant, or malignant. A malignant tumor refers to a cancer or cancerous tumor.

[0080] Next generation sequencing: As used herein, "next generation sequencing" or "NGS" refers to sequencing technologies with increased throughput compared to traditional Sanger and capillary electrophoresis-based methods, e.g., sequencing technologies with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next generation sequencing technologies include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization.

[0081] Nucleic acid tag: As used herein, "nucleic acid tag" refers to a short nucleic acid (e.g., less than about 500 nucleotides, about 100 nucleotides, about 50 nucleotides, or about 10 nucleotides in length) that is used to distinguish nucleic acids from different samples (e.g., representing a sample index), or different nucleic acid molecules of different types or undergoing different treatments in the same sample (e.g., representing a molecular barcode). Nucleic acid tags contain predetermined, fixed, non-random, random, or semi-random oligonucleotide sequences. Such nucleic acid tags can be used to label different nucleic acid molecules or different nucleic acid samples or subsamples. Nucleic acid tags can be single-stranded, double-stranded, or at least partially double-stranded. Nucleic acid tags optionally have the same length or different lengths. Nucleic acid tags can also include double-stranded molecules with one or more blunt ends, including 5' or 3' single-stranded regions (e.g., overhangs), and / or include one or more other single-stranded regions at other positions within a given molecule. Nucleic acid tags can be attached to one or both ends of other nucleic acids (e.g., sample nucleic acids to be amplified and / or sequenced). Nucleic acid tags can be decoded to reveal information such as the sample source, form or processing of given nucleic acid. For example, nucleic acid tags can also be used to realize the collection and / or parallel processing of multiple samples of nucleic acids containing different molecular bar codes and / or sample indexes, wherein nucleic acid is subsequently deconvolved by detecting (for example, reading) nucleic acid tags. Nucleic acid tags can also be referred to as identifiers (for example, molecular identifiers, sample identifiers). Additionally or alternatively, nucleic acid tags can be used as molecular bar codes (for example, to distinguish the amplicon of different molecules or different parent molecules in the same sample or subsample). This includes, for example, uniquely labeling different nucleic acid molecules in a given sample, or non-uniquely labeling such molecules. In the case of non-unique labeling applications, a limited number of labels (for example, molecular bar codes) can be used to label nucleic acid molecules so that different molecules can be distinguished based on their endogenous sequence information (for example, it is mapped to the starting position and / or termination position of the selected reference genome, one end of the sequence or the subsequence of the two ends, and / or the length of the sequence) and a combination of at least one molecular bar code. Typically, a sufficient number of different molecular barcodes are used so that the probability that any two molecules may have the same endogenous sequence information (e.g., start position and / or end position, subsequence at one or both ends of the sequence, and / or length) and also have the same molecular barcode is low (e.g., less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% probability).

[0082] Over-represented genomic start position and genomic end position pair: As used herein, the term "over-represented genomic start position and genomic end position pair" or "over-represented pair" refers to a genomic start position and genomic end position pair in which the number or frequency of families with a common genomic start position and genomic end position pair in more than one sample exceeds a set threshold. In some embodiments, more than one sample includes a sample run in a flow cell, and the first sample and the second sample are run in the flow cell. For example, more than one sample can be a training sample or a sample processed in a specific flow cell of a nucleic acid sequencer associated with the first sample and / or the second sample being analyzed. In some embodiments, more than one sample does not include the first sample and / or the second sample. In some embodiments, the set threshold can be any value between 2 and 100. In some embodiments, the set threshold can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, at least 21, at least 25, at least 30, at least 35, at least 40, or at least 50. In some embodiments, the set threshold may be 5. In some embodiments, the set threshold may be 10. In some embodiments, the set threshold may be 15. In some embodiments, the set threshold may be 20. In some embodiments, the set threshold may be at least 10 of the total families observed in more than one sample. -3 , at least 10 -4 , at least 10 -5 , at least 10 -6 , at least 10 -7 , at least 10 -8 or at least 10 -9 In some embodiments, the threshold may be 10% of the total family observed in more than one sample. -4 In some embodiments, the threshold may be 10% of the total family observed in more than one sample. -5 In some embodiments, the threshold may be 10% of the total family observed in more than one sample. -6 In some embodiments, the threshold may be 10% of the total family observed in more than one sample. -7 In some embodiments, the threshold may be 10% of the total family observed in more than one sample. -8 .

[0083] Polynucleotide: As used herein, "polynucleotide", "nucleic acid", "nucleic acid molecule" or "oligonucleotide" refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or their analogs) connected by internucleoside linkages. Typically, a polynucleotide contains at least three nucleosides. The size of an oligonucleotide typically ranges from a few monomer units (e.g., 3-4) to hundreds of monomer units. Whenever a polynucleotide is represented by a string of letters such as "ATGCCTG", it will be understood that the nucleotides are in the order of 5'→3' from left to right, and in the case of DNA, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents deoxythymidine, unless otherwise specified. As standard in the art, the letters A, C, G and T can be used to refer to the bases themselves, nucleosides, or nucleotides containing these bases.

[0084] Reference sequence: As used herein, "reference sequence" refers to a known sequence for the purpose of comparing with an experimentally determined sequence. For example, a known sequence can be a whole genome, a chromosome, or any segment thereof. A reference sequence typically includes at least about 20, at least about 50, at least about 100, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 1000, or more than 1000 nucleotides. A reference sequence can be aligned with a single continuous sequence of a genome or chromosome, or can include non-continuous segments aligned with different regions of a genome or chromosome. Examples of reference sequences include, for example, a human genome, such as, hG19 and hG38.

[0085] Sample: As used herein, "sample" means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0086] Sequencing: As used herein, "sequencing" refers to any of a number of techniques used to determine the sequence (eg, the identity and order of monomeric units) of a biological molecule, eg, a nucleic acid such as DNA or RNA. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy terminator sequencing, whole genome sequencing, hybridization sequencing, pyrosequencing, capillary electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing, emulsion PCR, low denaturing temperature co-amplification PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired-end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD TM Sequencing, MS-PET sequencing, and combinations thereof. In some embodiments, sequencing can be performed by a genetic analyzer, such as, for example, commercially available genetic analyzers from Illumina, Inc., Pacific Biosciences, Inc., or Applied Biosystems / Thermo Fisher Scientific, among many other companies.

[0087] Sequence information: As used herein, "sequence information" in the context of a nucleic acid polymer means the order and identity of the monomeric units (eg, nucleotides, etc.) in the polymer.

[0088] Shared family: If grouping of sequencing reads into families is performed independently for a first sample and a second sample, the term "shared family" refers to a family in the first sample whose grouping characteristics are the same or substantially the same as the grouping characteristics of the family in the second sample. Alternatively, if grouping of sequencing reads into families is performed together for both the first sample and the second sample, the term "shared family" refers to a family that includes at least one sequencing read from the first sample and at least one sequencing read from the second sample.

[0089] In some embodiments, in the presence of contamination of at least two samples, sequencing reads derived from a single polynucleotide molecule (of a single sample) can then be detected in at least two samples. In these embodiments, in the case of grouping sequencing reads independently for each sample, then the sequencing reads derived from a single polynucleotide molecule detected in each sample will be grouped into separate families in the sample. In these embodiments, a shared family refers to a family in the first sample whose grouping characteristics are the same or substantially the same as the grouping characteristics of the family in the second sample.

[0090] Alternatively, in other embodiments, when all at least two samples are grouped together for sequencing reads, then sequencing reads derived from a single polynucleotide molecule detected in at least two samples are grouped into a single family. In these embodiments, a common family refers to a family having at least one sequencing read from at least two samples.

[0091] In some embodiments, the first sample and the second sample can be in the same flow cell or in different flow cells.

[0092] Shared family identifier: As used herein, the term "shared family identifier" refers to a family identifier for a family in a first sample that is the same or substantially the same as a family identifier for a family in a second sample, i.e., a grouping feature for a family in a first sample that is the same or substantially the same as a grouping feature for a family in a second sample. In some embodiments, the first sample and the second sample can be in the same flow cell or in different flow cells.

[0093] Single nucleotide polymorphism: As used herein, the terms "single nucleotide polymorphism" or "SNP" are used interchangeably. They refer to variations of a single nucleotide occurring at a specific position in a genome, each of which is present in a population to an appreciable degree (e.g., greater than about 1%).

[0094] Single nucleotide variation: As used herein, "single nucleotide variation" or "SNV" means a mutation or variation of a single nucleotide occurring at a specific position in the genome.

[0095] Somatic mutation: As used herein, the terms "somatic mutation" or "somatic variation" are used interchangeably. They refer to mutations in the genome that occur after conception. Somatic mutations can occur in any cell of the body except germ cells and are therefore not passed on to offspring.

[0096] Subject: As used herein, "subject" refers to an animal, such as a mammalian species (e.g., a human), or an avian (e.g., bird) species, or other organisms, such as a plant. More specifically, a subject can be a vertebrate, for example, a mammal, such as a mouse, a primate, an ape, or a human. Animals include farm animals (e.g., production cattle, dairy cows, poultry, horses, pigs, etc.), sports animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual suffering from or suspected of suffering from a disease or having a tendency to suffer from the disease, or an individual in need of treatment or suspected of needing treatment. The terms "individual" or "patient" are intended to be interchangeable with "subject."

[0097] For example, the subject can be an individual who has been diagnosed with cancer, is about to receive cancer treatment and / or has received at least one cancer treatment. The subject can be in cancer remission. As another example, the subject can be an individual diagnosed with an autoimmune disease. As another example, the subject can be a female individual who is pregnant or planning pregnancy, and it may have been diagnosed with or suspected of having a disease, such as cancer, autoimmune disease.

[0098] Substantially identical: As used herein, the term "substantially identical" refers to two different entities that are 99.9% identical, at least 95% identical, at least 90% identical, at least 85% identical, at least 80% identical, at least 75% identical, at least 70% identical, at least 60% identical, or at least 50% identical. For example, when a family in a first sample is substantially identical to a family in a second sample, then the grouping characteristics of the family in the first sample are 99.9% identical, at least 95% identical, at least 90% identical, at least 85% identical, at least 80% identical, at least 75% identical, at least 70% identical, at least 60% identical, or at least 50% identical to the grouping characteristics of the family in the second sample. In the case where the entity is a molecular barcode, then the term "substantially identical" refers to two different molecular barcodes having a Hamming distance or edit distance of less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, or less than 8. In the case where the entity is a start region or an end region, the term "substantially identical" refers to two different regions within 1 bp, within 2 bp, within 3 bp, within 4 bp, within 5 bp, within 6 bp, within 7 bp, within 8 bp, within 9 bp, within 10 bp, within 11 bp, within 15 bp, within 20 bp, or within 25 bp. In the case where the entity is the length of a polynucleotide, the term "substantially identical" refers to two different lengths within 1 bp, within 2 bp, within 3 bp, within 4 bp, within 5 bp, within 6 bp, within 7 bp, within 8 bp, within 9 bp, within 10 bp, within 11 bp, within 15 bp, within 20 bp, within 25 bp, within 30 bp, within 40 bp, or within 50 bp.

[0099] Threshold: As used herein, "threshold" refers to a predetermined value that is used to characterize experimentally determined values ​​of the same parameter for different samples, depending on their relationship to the threshold. For example, a threshold for a p-value can refer to any predetermined value between 0 and 1, and is used to identify the source of nucleic acid variation.

[0100] Training samples: As used herein, "training samples" refers to a set of samples having similar properties, parameters and / or composition as the first sample and / or second sample being analyzed for the presence or absence of contamination.

[0101] Variation: As used herein, "variation" can refer to an allele. Variations are typically present at a frequency of 50% (0.5) or 100% (1), depending on whether the allele is heterozygous or homozygous. For example, germline variation is inherited and typically has a frequency of 0.5 or 1. However, somatic variation is acquired variation and typically has a frequency of less than about 0.5. The major allele and minor allele of a genetic locus refer to a nucleic acid containing the locus, wherein the locus is occupied by nucleotides of a reference sequence and variant nucleotides that are different from the reference sequence, respectively. Measurements at a locus can take the form of an allele fraction (AF), which measures the frequency with which an allele is observed in a sample.

[0102] Details

[0103] I. Overview

[0104] When processing samples for analysis, false positive results may be introduced by chemical or digital cross-contamination of samples processed in the same batch or in close temporal and spatial proximity by spreading molecules present in a sample into another sample. In the case of assaying cell-free nucleic acids from samples containing contaminants or a second genome (i.e., a genome other than the subject's genome and a genome generated from, for example, a transplant, a transfusion, or a fetus), the sample may require additional manual inspection or even an additional sequencing run.

[0105] The present disclosure provides methods and systems for detecting the presence or absence of contamination of a first sample by a second sample.

[0106] In one aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) accessing sequence information comprising more than one sequencing reads from the first sample and the second sample through a computer system; (b) aligning the more than one sequencing reads with a reference sequence through a computer system, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing reads into more than one family based on a grouping feature through a computer system, the grouping feature comprising at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region and (iii) a length of the sequence read, wherein each family in the sample comprises reads from the first sample and the second sample; (d) generating family identifiers for more than one family by a computer system; (e) screening a set of common family identifiers by a computer system, wherein the common family identifier is a family identifier of a first sample that is identical or substantially identical to a family identifier of a second sample; (f) determining a quantitative measure of the set of common family identifiers by a computer system; and (g) classifying the first sample as contaminated by the second sample by the computer system if the quantitative measure of the set of common family identifiers is above a predetermined threshold, or classifying the first sample as not contaminated by the computer system if the quantitative measure of the common family identifiers is at or below a predetermined threshold.

[0107] On the other hand, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) obtaining sequence information comprising more than one sequencing read from the first sample and the second sample; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region, and (iii) a length of the sequence read, wherein each of the samples The family includes sequencing reads of progeny polynucleotides amplified from unique polynucleotides in the set of polynucleotides in the sample; (d) generating family identifiers for more than one family; (e) screening the set of common family identifiers, wherein the common family identifier is a family identifier of the first sample that is the same or substantially the same as the family identifier of the second sample; (f) determining a quantitative measure of the set of common family identifiers; and (g) if the quantitative measure of the set of common family identifiers is above a predetermined threshold, classifying the first sample as contaminated by the second sample, or if the quantitative measure of the common family identifiers is at or below a predetermined threshold, classifying the first sample as not contaminated.

[0108] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of polynucleotides from the sample to generate more than one sequencing read; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region, and (iii) a length of the polynucleotide, wherein each family in the sample The family includes sequencing reads of progeny polynucleotides amplified from unique polynucleotides in the set of polynucleotides in the sample; (d) generating family identifiers for more than one family; (e) screening the set of common family identifiers, wherein the common family identifier is a family identifier of the first sample that is the same or substantially the same as the family identifier of the second sample; (f) determining a quantitative measure of the set of common family identifiers; and (g) if the quantitative measure of the set of common family identifiers is above a predetermined threshold, classifying the first sample as contaminated by the second sample, or if the quantitative measure of the common family identifiers is at or below a predetermined threshold, classifying the first sample as not contaminated.

[0109] In some embodiments, prior to sequencing or prior to accessing / obtaining sequence information, a collection of polynucleotides is tagged to produce tagged polynucleotides, wherein each tagged polynucleotide comprises a tag and a polynucleotide. In these embodiments, for each sample, more than one sequencing read is grouped into more than one family based on a grouping feature comprising at least one of (i), (ii), (iii), and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) a length of the polynucleotide, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the collection of polynucleotides in the sample.

[0110] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a tagged polynucleotide or a collection of polynucleotides from a sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, the grouping feature comprising a tag, wherein each family in the sample comprises a tagged polynucleotide from the sample; (d) generating family identifiers for more than one family; (e) screening the set of shared family identifiers, wherein the shared family identifier is a family identifier for the first sample that is identical or substantially identical to a family identifier for the second sample; (f) determining a quantitative measure for the set of shared family identifiers; and (g) classifying the first sample as contaminated with the second sample if the quantitative measure for the shared family identifier is above a predetermined threshold, or classifying the first sample as not contaminated if the quantitative measure for the shared family identifier is at or below a predetermined threshold.

[0111] Figure 1 It is a flowchart representation of a method for detecting the presence or absence of contamination between two samples obtained from two different subjects according to an embodiment of the present disclosure. The grouping characteristics of the sequencing reads, and thus the grouping characteristics of the family, are used to determine the presence or absence of contamination between the two samples. The grouping characteristics of the sequencing reads generally include at least one of the following (i), (ii), (iii) and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) the length of the polynucleotide. In 101, a collection of polynucleotides from a sample (i.e., a first sample and a second sample) is sequenced to produce more than one sequencing read. In some embodiments, the first sample and the second sample are sequenced in the same flow cell. In some embodiments, the second sample is sequenced in a flow cell different from the first sample. In some embodiments, the first sample is processed at a different time from the second sample. For example, the second sample is processed at least 1 minute, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, or at least 4 hours after processing the first sample. In some embodiments, the first sample and the second sample are processed on different days. In some embodiments, the first sample and the second sample are in the same sample batch. In some embodiments, the second sample is processed with the same batch of reagents as the first sample. In some embodiments, the first sample and the second sample are processed by the same liquid handling robot. In some embodiments, the first sample and the second sample are processed by the same laboratory personnel.

[0112] In some embodiments, the first sample and the second sample are processed in different geographical locations. In some embodiments, the first sample is obtained from a body fluid of a subject, and the second sample is obtained from a body fluid of another subject. In some embodiments, the sample is blood. In some embodiments, the sample is plasma. In some embodiments, the sample is serum. In some embodiments, the polynucleotide is a cell-free polynucleotide. In some embodiments, the cell-free polynucleotide is a cell-free DNA. In some embodiments, at least one of the subjects suffers from a disease, such as cancer.

[0113] In some embodiments, the set of polynucleotides undergoes a series of library preparation steps before sequencing. The library preparation step includes end repair, connection of adapters (including tags-i.e., molecular barcodes), amplification of tagged polynucleotides and / or selective enrichment of at least a portion of amplified progeny polynucleotides from the genome or transcriptome region of the subject. In some embodiments, the first sample and the second sample are tagged with tags including molecular barcodes to produce a set of tagged polynucleotides. In some embodiments, the set of tagged polynucleotides of the sample is uniquely tagged. In some embodiments, the set of tagged polynucleotides of the sample is non-uniquely tagged. In some embodiments, the method also includes attaching one or more sample indexes to one or both ends of the amplified progeny polynucleotides before sequencing, wherein the sample index distinguishes the first sample and the second sample.

[0114] In order to determine the start region, the end region and / or the length of the polynucleotide, in 102, more than one sequencing read is typically aligned to a reference sequence. The reference sequence can be a human genome. In 103, more than one sequencing read in each sample is grouped into more than one family based on a grouping feature, the grouping feature comprising at least one of the following (i), (ii), (iii), and (iv): (i) a tag (if the polynucleotide is tagged), (ii) a start region, (iii) an end region, and (iv) the length of the polynucleotide, wherein each family in the sample comprises a sequencing read of a progeny polynucleotide amplified from a unique polynucleotide in a collection of polynucleotides in the sample or a tagged progeny polynucleotide (in the case where the polynucleotide is tagged with a molecular barcode). In some embodiments, the start region comprises a genomic start position of the sequencing read, at which the 5' end of the sequencing read is determined to start the alignment with the reference sequence, and the end region comprises a genomic end position of the sequencing read, at which the 3' end of the sequencing read is determined to end the alignment with the reference sequence. In some embodiments, the starting region includes the first 1, first 2, first 5, first 10, first 15, first 20, first 25, first 30 or at least the first 30 base positions of the 5' end of the sequencing read aligned with the reference sequence. In some embodiments, the ending region includes the last 1, last 2, last 5, last 10, last 15, last 20, last 25, last 30 or at least the last 30 base positions of the 3' end of the sequencing read aligned with the reference sequence. In some embodiments, the label includes one or more molecular barcodes attached to both ends of the polynucleotide molecule. In some embodiments, the length of one or more molecular barcodes is at least 2, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15 or at least 20 nucleotides. In some embodiments, the polynucleotides of a sample are tagged with at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 50,000, or at least 100,000 different tags / molecular barcodes.

[0115] At 104, family identifiers for more than one family are generated based on the grouping feature. At 105, a set of common family identifiers of the family identifiers is screened, wherein the common family identifier is a family identifier for a family in the first sample that is the same or substantially the same as a family identifier for a family in the second sample - that is, the grouping feature for the family in the first sample is the same or substantially the same as the grouping feature for the family in the second sample.

[0116] In 106, a quantitative measure of a set of shared family identifiers is determined to classify a sample as being contaminated by another sample or not. In some embodiments, the quantitative measure of a set of shared family identifiers is the number of shared family identifiers in the first sample. In some embodiments, the quantitative measure of a set of shared family identifiers includes the ratio of the number of shared family identifiers in the first sample to the total number of family identifiers in the first sample. In some embodiments, the quantitative measure of a set of shared family identifiers does not include the following shared family identifiers in the first sample: those shared family identifiers whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample. In some embodiments, the quantitative measure of a set of shared family identifiers in the first sample does not include shared family identifiers at over-represented genome start positions and genome termination positions. In some embodiments, the total number of family identifiers in the first sample does not include family identifiers at over-represented genome start positions and genome termination positions. In some embodiments, the over-represented genomic start position and genomic end position are determined by: (a) providing more than one sample, wherein the more than one sample includes a distribution of genomic start positions and genomic end positions that are identical or substantially identical to the first sample and / or the second sample; (b) determining the family identifier in more than one sample; (c) quantifying the number of family identifiers having a pair of genomic start positions and genomic end positions in more than one sample; and (d) if the number of family identifiers exceeds a set threshold, the pair of genomic start positions and genomic end positions are classified as over-represented. In some embodiments, wherein more than one sample does not include the first sample or the second sample. In some embodiments, more than one sample does not include the first sample and the second sample. In some embodiments, more than one sample includes a sample processed in the same flow cell as the first sample. In some embodiments, more than one sample includes a training sample. In some embodiments, the threshold is set at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 families. In some embodiments, the threshold is set at about 5 families. In some embodiments, the threshold is set at about 10 families. In some embodiments, the threshold is set at about 15 families. In some embodiments, the threshold is set at about 20 families. In some embodiments, the threshold is set at about 30 families. In some embodiments, the threshold is set at about 40 families. In some embodiments, the threshold is set at about 50 families. In some embodiments, the threshold can be at least 10 of the total families observed in more than one sample. -3 , at least 10-4 , at least 10 -5 , at least 10 -6 , at least 10 -7 , at least 10 -8 or at least 10 -9 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -4 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -5 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -6 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -7 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -8 .

[0117] In 107, if the quantitative measurement of the shared family identifier is above a predetermined threshold, the first sample is classified as being contaminated by the second sample, or if the quantitative measurement of the shared family identifier is at or below a predetermined threshold, the first sample is classified as being uncontaminated. In some embodiments, the predetermined threshold is at least 0.001%, at least 0.005%, at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, or at least 10% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.01% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.05% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.1% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.5% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 1% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 2% of the total number of families in the first sample.

[0118] In some embodiments, even if the first sample is classified as contaminated by the second sample, the method can also allow for reliable detection of at least one somatic variation in a polynucleotide of the first sample by excluding sequencing reads with a common family identifier of the first sample before detecting the somatic variation.

[0119] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of polynucleotides from the sample to generate more than one sequencing read; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on information from at least one of the following (i), (ii) and (iii): (i) the start region, (ii) the end region and (iii) the length of the polynucleotide. (d) screening more than one family to identify a set of shared families, wherein the shared family is a family of the first sample that is identical or substantially identical to a family of the second sample; (e) determining a quantitative measure of the set of shared families of the first sample; and (f) classifying the first sample as contaminated with the second sample if the quantitative measure of the shared family is above a predetermined threshold, or classifying the first sample as not contaminated if the quantitative measure of the shared family is at or below a predetermined threshold.

[0120] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of polynucleotides from the samples to generate more than one sequencing read; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) grouping the more than one sequencing reads of the two samples into more than one family based on a grouping feature, wherein the grouping feature comprises at least one of the following (i), (ii) and (iii): (i) a start region, (ii) an end region and (ii) a sequence of the first and second samples; (i) the length of a polynucleotide, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the sample; (d) screening more than one family to identify a set of common families, wherein the common family comprises sequencing reads from the first sample and the second sample; (e) determining a quantitative measure of the set derived from the common family; and (f) classifying the first sample as contaminated with the second sample if the quantitative measure of the common family is above a predetermined threshold, or classifying the first sample as not contaminated if the quantitative measure of the common family is at or below a predetermined threshold.

[0121] In some embodiments, prior to sequencing, the collection of polynucleotides can be tagged to produce tagged polynucleotides, wherein each tagged polynucleotide comprises a tag and a polynucleotide. In these embodiments, for each sample, more than one sequencing read is grouped into more than one family based on a grouping feature, the grouping feature comprising at least one of (i), (ii), (iii), and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) a length of the polynucleotide, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the collection of polynucleotides in the sample.

[0122] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of tagged polynucleotides from the sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, wherein the grouping feature comprises a tag, wherein each family in the sample The method comprises: (a) screening more than one family to identify a set of common families, wherein the common family is a family of the first sample that is the same or substantially the same as the family of the second sample; (b) determining a quantitative measure of the set of common families of the first sample; and (c) classifying the first sample as contaminated with the second sample if the quantitative measure of the common family is above a predetermined threshold, or classifying the first sample as not contaminated if the quantitative measure of the common family is at or below a predetermined threshold.

[0123] In another aspect, the present disclosure provides a method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of tagged polynucleotides from a sample to produce more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing read to a reference sequence, thereby determining a start region and an end region of the alignment; (c) grouping the more than one sequencing reads of the two samples into more than one families based on information from the tags, wherein each family in the sample comprises sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in the collection of tagged polynucleotides in the sample; (d) screening the more than one families to identify a collection of common families, wherein the common family comprises sequencing reads from the first sample and the second sample; (e) determining a quantitative measure of the collection derived from the common family; and (f) classifying the first sample as contaminated by the second sample if the quantitative measure of the common family is above a predetermined threshold, or classifying the first sample as not contaminated if the quantitative measure of the common family is at or below the predetermined threshold.

[0124] Figure 2 It is a flowchart representation of a method for detecting the presence or absence of contamination between two samples obtained from two different subjects according to an embodiment of the present disclosure. The grouping characteristics of the sequencing reads, and thus the grouping characteristics of the family, are used to determine the presence or absence of contamination between the two samples. The grouping characteristics of the sequencing reads generally include at least one of the following (i), (ii), (iii) and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) the length of the polynucleotide. In 201, a collection of polynucleotides from a sample (i.e., a first sample and a second sample) is sequenced to produce more than one sequencing read. In some embodiments, the first sample and the second sample are sequenced in the same flow cell. In some embodiments, the second sample is sequenced in a flow cell different from the first sample. In some embodiments, the first sample is processed at a different time from the second sample. For example, the second sample is processed at least 1 minute, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, or at least 4 hours after processing the first sample. In some embodiments, the first sample and the second sample are processed on different days. In some embodiments, the first sample and the second sample are in the same sample batch. In some embodiments, the second sample is processed with the same batch of reagents as the first sample.

[0125] In some embodiments, the first sample and the second sample are processed in different geographical locations. In some embodiments, the first sample is obtained from a body fluid of a subject, and the second sample is obtained from a body fluid of another subject. In some embodiments, the sample is blood. In some embodiments, the sample is plasma. In some embodiments, the sample is serum. In some embodiments, the polynucleotide is a cell-free polynucleotide. In some embodiments, the cell-free polynucleotide is a cell-free DNA. In some embodiments, at least one of the subjects suffers from a disease, such as cancer.

[0126] In some embodiments, the set of polynucleotides undergoes a series of library preparation steps before sequencing. The library preparation step includes end repair, connection of adapters (including tags-i.e., molecular barcodes), amplification of tagged polynucleotides and / or selective enrichment of at least a portion of amplified progeny polynucleotides from the genome or transcriptome region of the subject. In some embodiments, the first sample and the second sample are tagged with tags including molecular barcodes to produce a set of tagged polynucleotides. In some embodiments, the set of tagged polynucleotides of the sample is uniquely tagged. In some embodiments, the set of tagged polynucleotides of the sample is non-uniquely tagged. In some embodiments, the method also includes attaching one or more sample indexes to one or both ends of the amplified progeny polynucleotides before sequencing, wherein the sample index distinguishes the first sample and the second sample.

[0127] To determine the start region, the end region, and / or the length of the polynucleotide, in 202, more than one sequencing read is aligned to a reference sequence. The reference sequence can be a human genome (e.g., hg18, hg19). In 203, more than one sequencing read in each sample is grouped into more than one family based on a grouping feature, the grouping feature comprising at least one of the following (i), (ii), (iii), and (iv): (i) a tag (if the polynucleotide is tagged), (ii) a start region, (iii) an end region, and (iv) a length of the polynucleotide, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in a collection of polynucleotides in the sample or tagged progeny polynucleotides (in the case where the polynucleotide is tagged with a molecular barcode). In some embodiments, the starting region includes the genomic starting position of the sequencing read, at which the 5' end of the sequencing read is determined to start the comparison with the reference sequence, and the ending region includes the genomic termination position of the sequencing read, at which the 3' end of the sequencing read is determined to terminate the comparison with the reference sequence. In some embodiments, the starting region includes the first 1, first 2, first 5, first 10, first 15, first 20, first 25, first 30 or at least the first 30 base positions of the 5' end of the sequencing read aligned with the reference sequence. In some embodiments, the ending region includes the last 1, last 2, last 5, last 10, last 15, last 20, last 25, last 30 or at least the last 30 base positions of the 3' end of the sequencing read aligned with the reference sequence. In some embodiments, the label includes one or more molecular barcodes attached to both ends of the polynucleotide molecule. In some embodiments, one or more molecular barcodes are at least 2, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, or at least 20 nucleotides in length. In some embodiments, the polynucleotides of a sample are tagged with at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 50,000, or at least 100,000 different tags / molecular barcodes.

[0128] In 204, a set of common families of more than one family is screened out based on the grouping characteristics, wherein the common family is a family in the first sample that is the same or substantially the same as the family in the second sample, that is, the grouping characteristics of the family in the first sample are the same or substantially the same as the grouping characteristics of the family in the second sample.

[0129] In 205, determine the quantitative measure of the set of shared families, so that the sample is classified as whether it is contaminated by another sample. In some embodiments, the quantitative measure of the set of shared families is the number of shared families in the first sample. In some embodiments, the quantitative measure of the set of shared families includes the ratio of the number of shared families in the first sample to the total number of families in the first sample. In some embodiments, the quantitative measure of the set of shared families does not include the following shared families in the first sample: those shared families whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample. In some embodiments, the quantitative measure of the set of shared families in the first sample does not include the shared families at the over-represented genome starting position and genome termination position pairs. In some embodiments, the total number of families in the first sample does not include the families at the over-represented genome starting position and genome termination position pairs. In some embodiments, the over-represented genomic start position and genomic end position are determined by: (a) providing more than one sample, wherein the more than one sample includes a distribution of genomic start positions and genomic end positions that are identical or substantially identical to the first sample and / or the second sample; (b) determining the families in more than one sample; (c) quantifying the number of families with a pair of genomic start positions and genomic end positions in more than one sample; and (d) if the number of families exceeds a set threshold, the pair of genomic start positions and genomic end positions are classified as over-represented. In some embodiments, wherein more than one sample does not include the first sample or the second sample. In some embodiments, more than one sample does not include the first sample and the second sample. In some embodiments, more than one sample includes a sample processed in the same flow cell as the first sample. In some embodiments, more than one sample includes a training sample. In some embodiments, the threshold is set at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 families. In some embodiments, the threshold is set at about 5 families. In some embodiments, the threshold is set at about 10 families. In some embodiments, the threshold is set at about 15 families. In some embodiments, the threshold is set at about 20 families. In some embodiments, the threshold is set at about 30 families. In some embodiments, the threshold is set at about 40 families. In some embodiments, the threshold is set at about 50 families. In some embodiments, the threshold can be at least 10 of the total families observed in more than one sample. -3 , at least 10 -4 , at least 10 -5 , at least 10 -6, at least 10 -7 , at least 10 -8 or at least 10 -9 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -4 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -5 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -6 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -7 In some embodiments, the threshold may be set to about 10 of the total families observed in more than one sample. -8 .

[0130] In 206, if the quantitative measurement of the shared family identifier is higher than a predetermined threshold, the first sample is classified as being contaminated by the second sample, or if the quantitative measurement of the shared family identifier is at or below a predetermined threshold, the first sample is classified as being uncontaminated. In some embodiments, the predetermined threshold is at least 0.001%, at least 0.005%, at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, or at least 10% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.01% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.05% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.1% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 0.5% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 1% of the total number of families in the first sample. In some embodiments, the predetermined threshold is about 2% of the total number of families in the first sample.

[0131] In some embodiments, the method can detect at least one somatic genetic variation in a polynucleotide of the first sample by excluding sequencing reads of a common family of the first sample, even if the first sample is classified as contaminated by the second sample, wherein the first sample is classified as contaminated by the second sample.

[0132] Figure 3It is a schematic diagram illustrating the grouping of sequencing reads into families and the detection of the presence or absence of contamination between two samples (sample 1 and sample 2) according to an embodiment of the present disclosure. 301 represents a reference sequence (e.g., hG18 or hG19), and the sequencing reads of sample 1 and sample 2 are compared with it. For ease of explanation, reads 1 and 2 in the sequencing reads generated from the sequencer by paired end sequencing are shown as single paired end sequencing reads, wherein reads 1 and reads 2 sequence reads are merged together. The line with patterned filled boxes at both ends of the line represents paired end sequencing reads (reads 1 + reads 2). The box filled with patterns represents a molecular barcode, which has been attached to the two ends of the polynucleotide. Each different pattern represents a different molecular barcode sequence. Based on the grouping feature, the paired end sequencing reads are grouped into families. In this embodiment, the grouping feature is (i) a label (i.e., a molecular barcode); (ii) the starting position of the polynucleotide and (iii) the end position.

[0133] 302A, 303A, 304A and 305A are the total families of sample 1, because the grouping characteristics of these families are respectively the same or substantially the same as the grouping characteristics of family 302B, 303B, 304B and 305B of sample 2. Similarly, 302B, 303B, 304B and 305B are the total families of sample 2, because the grouping characteristics of these families are respectively the same or substantially the same as the grouping characteristics of family 302A, 303A, 304A and 305A of sample 1. 306 represents a pair of genome starting position and genome termination position. At 306, sample 1 has three families, and sample 2 has four families, and therefore the total number of the family at 306 is seven. In this embodiment, in order to determine whether a pair of specific genome starting position and genome termination position are overrepresented pairs, the threshold value is set to be 6. Due to the total number of the family at 306 (i.e. 7) being higher than the threshold value, 306 is the genome starting position and genome termination position pair of overrepresentation.

[0134] Scenario I : Determine whether sample 1 is contaminated by sample 2.

[0135] The number of shared families in sample 1 is four (302A, 303A, 304A and 305A), of which two families 302A and 303A are in the over-represented genome start position and genome end position pair. In this embodiment, in order to determine the quantitative measure of shared families in sample 1, the shared families in the over-represented genome start position and genome end position pair in sample 1 are not included. Since 306 is an over-represented pair, two families (302A and 303A) are not included when calculating the quantitative measure of shared families. Therefore, the quantitative measure of shared families of sample 1 is 2. In this embodiment, the quantitative measure also does not include the following shared families in sample 1: the shared families whose number of sequencing reads in the families of sample 1 is greater than the number of sequencing reads in the corresponding families of sample 2. In this embodiment, the common families (304A and 305A) of sample 1 each have three paired end sequencing reads (i.e., six sequencing reads), and the corresponding families (304B and 305B) of sample 2 each have one paired end sequencing read (i.e., two sequencing reads). Therefore, the common families 304A and 305A are not included in the calculation of the quantitative measure. Therefore, the quantitative measure of the common family in sample 1 is zero. In order to classify sample 1 as being contaminated by sample 2, the quantitative measure of the common family should be higher than a predetermined threshold. In this embodiment, the predetermined threshold is 0.5% of the total family. Since the quantitative measure (i.e., zero for the first sample) is lower than the predetermined threshold, sample 1 is determined to be not contaminated by sample 2.

[0136] Scenario II : Determine whether sample 2 is contaminated by sample 1

[0137] The number of shared families in sample 2 is four (302B, 303B, 304B and 305B), wherein two families 302B and 303B are in the genome starting position and genome termination position pairs of overrepresentation. In this embodiment, in order to determine the quantitative measure of shared families in sample 2, the shared families in the genome starting position and genome termination position pairs of overrepresentation in sample 2 are not included. Since 306 is an overrepresentation pair, two families (302B and 303B) are not included when determining the quantitative measure of shared families. Therefore, the quantitative measure of shared families of sample 2 is 2. In this embodiment, the quantitative measure also does not include the following shared families in sample 2: the shared families whose number of sequencing reads in the family of sample 2 is greater than the number of sequencing reads in the corresponding family of sample 1. In this embodiment, the shared families (304B and 305B) of sample 2 each have a paired end sequencing read (i.e., two sequencing reads), while the corresponding families (304A and 305A) in sample 1 each have three paired end sequencing reads (i.e., six sequencing reads). Therefore, shared families 304B and 305B are not excluded when calculating the quantitative measure. Therefore, the quantitative measure of shared families in sample 2 is 2. In order to classify sample 2 as being contaminated by sample 1, the quantitative measure of shared families of sample 2 should be higher than a predetermined threshold. In this embodiment, the predetermined threshold is 0.5% of the total family. For sample 2, the total number of families is 21. In this embodiment, the family at the genome starting position and genome starting position pair of over-representation is not included in the total number of families. The number of families at the genome starting position and genome termination position pair of over-representation is 4. Therefore, after excluding the family at the over-representation pair, the total number of families in sample 2 is 17. Furthermore, in this embodiment, the quantitative measure of the common family is the percentage of the common family in sample 2 to the total family, which is equal to 11.765% (100*2 / 17) and is above the predetermined threshold. Therefore, sample 2 is determined to be contaminated by sample 1.

[0138] The various steps of a method may be performed at the same or different times, in the same or different geographic locations (eg, countries), and by the same or different people or entities.

[0139] II. General characteristics of the method

[0140] A.Sample

[0141] Sample can be any biological sample separated from a subject.Sample can include body tissue, whole blood, platelets, serum, plasma, feces, red blood cells, white blood cells (white blood cell) or white blood cells (leucocyte), endothelial cells, tissue biopsy (e.g., biopsy from known or suspected solid tumors), cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial fluid or extracellular fluid (e.g., fluid from intercellular spaces), gingival fluid, gingival sulcus fluid, bone marrow, pleural effusion, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine.Sample is preferably body fluid, particularly blood and its fraction, and urine.Such sample includes nucleic acid shed from tumor.Nucleic acid can include DNA and RNA, and can be in double-stranded form and single-stranded form.Sample can be in the form initially separated from a subject, or can be subjected to further processing to remove or add components such as cells, enrich a component relative to another component, or convert a form of nucleic acid into another, such as converting RNA into DNA or converting single-stranded nucleic acid into double-stranded. Thus, for example, the body fluid for analysis is plasma or serum containing cell-free nucleic acids such as cell-free DNA (cfDNA). In some embodiments, the method includes obtaining a sample from a subject. Any sample type is optionally used. In certain embodiments, for example, the sample is tissue, blood, plasma, serum, sputum, urine, semen, vaginal fluid, feces, synovial fluid, spinal fluid, saliva and / or the like. Typically, the subject is a mammalian subject (e.g., a human subject). In some embodiments, the sample is blood. In some embodiments, the sample is plasma. In some embodiments, the sample is serum.

[0142] In some embodiments, the sample volume of the body fluid taken from the subject depends on the expected read depth of the sequencing region. Exemplary volumes are about 0.4ml-40ml, about 5ml-20ml, about 10ml-20ml. For example, the volume can be about 0.5ml, about 1ml, about 5ml, about 10ml, about 20ml, about 30ml, about 40ml or more milliliters. The volume of the sampled plasma is generally between about 5ml and about 20ml.

[0143] Samples can contain various amounts of nucleic acid. Typically, the amount of nucleic acid in a given sample is equivalent to a number of genome equivalents. For example, a sample of about 30 ng DNA may contain about 10,000 (10 4 ) haploid human genome equivalents, and in the case of cfDNA, can contain approximately 200 billion (2×10 11 ) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents, and in the case of cfDNA, can contain about 600 billion individual molecules.

[0144] In some embodiments, the sample includes nucleic acids from different sources, for example, from cell sources and from cell-free sources (e.g., blood samples, etc.). Typically, the sample includes nucleic acids carrying mutations. For example, the sample optionally includes DNA carrying germline mutations and / or somatic mutations. Typically, the sample includes DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations). In some embodiments, the sample includes cell-free DNA (i.e., cfDNA samples). In some embodiments, cfDNA samples include circulating tumor nucleic acids.

[0145] Exemplary amounts of cell-free nucleic acid in a sample prior to amplification generally range from about 1 femtogram (fg) to about 1 microgram (μg), for example, from about 1 picogram (pg) to about 200 nanograms (ng), from about 1 ng to about 100 ng, from about 10 ng to about 1000 ng. In some embodiments, the sample includes up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. Optionally, the amount is at least about 1 fg, at least about 10 fg, at least about 100 fg, at least about 1 pg, at least about 10 pg, at least about 100 pg, at least about 1 ng, at least about 10 ng, at least about 100 ng, at least about 150 ng, or at least about 200 ng of cell-free nucleic acid molecules. In certain embodiments, the amount is up to about 1 fg, about 10 fg, about 100 fg, about 1 pg, about 10 pg, about 100 pg, about 1 ng, about 10 ng, about 100 ng, about 150 ng, or about 200 ng of cell-free nucleic acid molecules. In some embodiments, the method includes obtaining between about 1 fg and about 200 ng of cell-free nucleic acid molecules from a sample. In certain embodiments, the method includes obtaining between about 5 ng and about 30 ng of cell-free nucleic acid molecules from a sample. In certain embodiments, the method includes obtaining between about 5 ng and about 100 ng of cell-free nucleic acid molecules from a sample. In certain embodiments, the method includes obtaining between about 5 ng and about 150 ng of cell-free nucleic acid molecules from a sample. In certain embodiments, the method includes obtaining between about 5 ng and about 200 ng of cell-free nucleic acid molecules from a sample. In some embodiments, the amount is up to about 100 ng of cell-free nucleic acid molecules from a sample. In some embodiments, the amount is up to about 150 ng of cell-free nucleic acid molecules from a sample. In some embodiments, the amount is up to about 200 ng of cell-free nucleic acid molecules from the sample. In some embodiments, the amount is up to about 250 ng of cell-free nucleic acid molecules from the sample. In some embodiments, the amount is up to about 300 ng of cell-free nucleic acid molecules from the sample. In some embodiments, the method comprises obtaining between about 1 fg and about 200 ng of cell-free nucleic acid molecules from the sample.

[0146] The cell-free nucleic acids typically have a size distribution between about 100 nucleotides in length and about 500 nucleotides in length, wherein molecules between about 110 nucleotides in length to about 230 nucleotides in length represent about 90% of the molecules in the sample, wherein the mode is about 168 nucleotides in length, and a secondary minor peak ranges between about 240 nucleotides in length to about 440 nucleotides in length. In certain embodiments, the cell-free nucleic acids are about 160 nucleotides in length to about 180 nucleotides, or about 320 nucleotides in length to about 360 nucleotides, or about 440 nucleotides in length to about 480 nucleotides in length.

[0147] In some embodiments, cell-free nucleic acid is separated from body fluid by a partitioning step, in which the cell-free nucleic acid found in the solution is separated from intact cells and other insoluble components in the body fluid. In some embodiments in these embodiments, the distribution includes techniques such as centrifugation or filtration. Optionally, the cells in the body fluid are lysed, and the cell-free nucleic acid and the cellular nucleic acid are processed together. Usually, after adding buffer and washing steps, the cell-free nucleic acid is precipitated with, for example, alcohol. In certain embodiments, other clean-up steps such as silica-based columns are used to remove pollutants or salts. For example, non-specific bulk carrier nucleic acids are optionally added throughout the reaction to optimize certain aspects of the exemplary procedure, such as yield. After such processing, the sample generally comprises various forms of nucleic acid, including double-stranded DNA, single-stranded DNA and / or single-stranded RNA. Optionally, single-stranded DNA and / or single-stranded RNA are converted into double-stranded forms so that they are included in subsequent processing and analysis steps.

[0148] B. Nucleic acid labeling

[0149] In some embodiments, nucleic acid molecules (from samples of polynucleotides) can be tagged with sample indexes and / or molecular barcodes (commonly referred to as "tags"). Tags can be incorporated into adapters or otherwise connected to adapters by chemical synthesis, connection (e.g., blunt end connection or sticky end connection) or overlap extension polymerase chain reaction (PCR) and other methods. Such adapters can ultimately be connected to target nucleic acid molecules. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) are typically applied to introduce sample indexes into nucleic acid molecules using conventional nucleic acid amplification methods. Amplification can be performed in one or more reaction mixtures (e.g., more than one microwell in an array). Molecular barcodes and / or sample indexes can be introduced simultaneously or in any order. In some embodiments, molecular barcodes and / or sample indexes are introduced before and / or after performing a sequence capture step. In some embodiments, only molecular barcodes are introduced before probe capture, and sample indexes are introduced after performing a sequence capture step. In some embodiments, both molecular barcodes and sample indexes are introduced before performing a probe-based capture step. In some embodiments, the sample index is introduced after the sequence capture step is performed. In some embodiments, the molecular barcode is incorporated into the nucleic acid molecule (e.g., cfDNA molecule) in the sample via a connection (e.g., a blunt end connection or a sticky end connection) through an adapter. In some embodiments, the sample index is incorporated into the nucleic acid molecule (e.g., cfDNA molecule) in the sample by overlap extension polymerase chain reaction (PCR). Typically, the sequence capture scheme involves the introduction of a single-stranded nucleic acid molecule complementary to a targeted nucleic acid sequence, such as a coding sequence of a genomic region, and mutations in such a region are associated with cancer types.

[0150] In some embodiments, the label can be located at one end or both ends of the sample nucleic acid molecule. In some embodiments, the label is a predetermined or random or semi-random sequence oligonucleotide. In some embodiments, the length of the label can be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide. The label can be randomly or non-randomly connected to the sample nucleic acid.

[0151] In some embodiments, each sample is uniquely labeled by a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or subsample is uniquely labeled by a molecular barcode or a combination of molecular barcodes. In other embodiments, more than one molecular barcode may be used so that the molecular barcodes are not necessarily unique to each other in the more than one molecular barcode (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are usually attached (e.g., by connection) to individual molecules so that the combination of molecular barcodes and sequences to which they can be attached produces a unique sequence that can be tracked individually. The detection of a combination of non-uniquely labeled molecular barcodes and endogenous sequence information (e.g., corresponding to the beginning (start) and / or end (termination) part of the original nucleic acid molecule sequence in the sample, a subsequence of a sequence read at one end or both ends, the length of the sequence read and / or the length of the original nucleic acid molecule in the sample) generally allows a unique identity to be assigned to a specific molecule. The length or number of base pairs of individual sequence reads is also optionally used to assign a unique identity to a given molecule. As described herein, fragments from a single strand of nucleic acid that have been assigned a unique identity can thereby allow for subsequent identification of fragments from the parental strand and / or the complementary strand.

[0152] In some embodiments, molecular barcodes are introduced with a ratio of a set of expected identifiers (e.g., a combination of unique molecular barcodes or non-unique molecular barcodes) to molecules in the sample. An exemplary form uses about 2 to about 1,000,000 different molecular barcodes, or about 5 to about 150 different molecular barcodes, or about 20 to about 50 different molecular barcodes connected to both ends of the target molecule. Alternatively, about 25 to about 1,000,000 different molecular barcodes can be used. For example, 20-50 × 20-50 molecular barcodes can be used. In some embodiments, 20-50 different molecular barcodes can be used. In some embodiments, 5-100 different molecular barcodes can be used. In some embodiments, 5-150 molecular barcodes can be used. In some embodiments, 5-200 different molecular barcodes can be used. Such a number of identifiers is generally sufficient to provide different molecules with the same start and end points with a high probability (e.g., at least 94%, 99.5%, 99.99%, or 99.999%) of receiving different identifier combinations. In some embodiments, about 80%, about 90%, about 95%, or about 99% of the molecules have the same molecular barcode combination.

[0153] In some embodiments, the assignment of unique or non-unique molecular barcodes in the reaction is performed using methods and systems described in, for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is hereby incorporated by reference in its entirety. Alternatively, in some embodiments, only endogenous sequence information (e.g., start position and / or end position, subsequence of one or both ends of the sequence, and / or length) can be used to identify different nucleic acid molecules of a sample.

[0154] C. Amplification

[0155] Sample nucleic acids flanked by adapters are typically amplified by PCR and other amplification methods using nucleic acid primers that bind to primer binding sites in the adapters flanking the DNA molecule to be amplified. In some embodiments, the amplification method involves cycles of extension, denaturation, and annealing generated by thermal cycling, or can be isothermal, for example, in transcription-mediated amplification. Other exemplary amplification methods that are optionally used include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and autonomous continuous sequence-based replication, as well as other methods.

[0156] One or more amplification cycles are usually applied to introduce molecular barcodes and / or sample indexes into nucleic acid molecules using conventional nucleic acid amplification methods. Amplification is usually carried out in one or more reaction mixtures. Molecular barcodes and sample indexes are optionally introduced simultaneously or in any order. In some embodiments, molecular barcodes and sample indexes are introduced before and / or after the sequence capture step. In some embodiments, only molecular barcodes are introduced before probe capture, and sample indexes are introduced after the sequence capture step. In certain embodiments, both molecular barcodes and sample indexes are introduced before the probe-based capture step. In some embodiments, sample indexes are introduced after the sequence capture step. Typically, sequence capture schemes involve the introduction of single-stranded nucleic acid molecules complementary to targeted nucleic acid sequences, such as coding sequences of genomic regions, and mutations in such regions are associated with cancer types. Typically, the amplification reaction produces more than one non-uniquely or uniquely tagged nucleic acid amplicon with a molecular barcode and a sample index ranging in size from about 200 nucleotides (nt) to about 700 nt, 250 nt to about 350 nt, or about 320 nt to about 550 nt. In some embodiments, the amplicon has a size of about 300 nt. In some embodiments, the amplicon has a size of about 500 nt.

[0157] D. Enrichment

[0158] Sequences can be enriched before sequencing. Specific target regions can be enriched or non-specifically enriched ("target sequences"). In some embodiments, the target regions of interest can be enriched using differential tiling and capture schemes with capture probes ("baits") selected for one or more bait set panels. Differential tiling and capture schemes use bait sets of different relative concentrations to differentially tile (e.g., with different "resolutions") across genomic regions associated with the baits, subject to a set of restrictions (e.g., sequencer restrictions, such as sequencing load, the utility of each bait, etc.), and capture them at the level desired for downstream sequencing. These targeted genomic regions of interest can include natural nucleotide sequences or synthetic nucleotide sequences of nucleic acid constructs. In some embodiments, biotin-labeled beads with probes for one or more regions of interest can be used to capture target sequences, optionally followed by amplification of these regions to enrich the regions of interest.

[0159] Sequence capture can include using oligonucleotide probes that hybridize with the target sequence. The probe set strategy can involve tiling the probe on the region of interest. Such a probe can be, for example, about 60 to 120 bases long. The group can have a depth of about 2x, 3x, 4x, 5x, 6x, 8x, 9x, 10x, 15x, 20x, 50x or more than 50x. The effectiveness of sequence capture depends in part on the length of the sequence complementary (or almost complementary) to the sequence of the probe in the target molecule.

[0160] In some embodiments, more than one genomic region is included in the genetic variation found in COSMIC, Cancer Genome Atlas (TCGA) or Exome Aggregation Consortium (ExAC). In some cases, the genetic variation may belong to a set of predefined clinically actionable variations (clinically actionable variants). For example, such variation can be found in various variation databases, and the presence of these variation in the sample of the subject has been shown to be associated with a disease or condition (e.g., cancer) in the subject or indicates the disease or condition. Such variation databases may include, for example, Catalogue of Somatic Mutations in Cancer, COSMIC, Cancer Genome Atlas (TCGA) and Exome Aggregation Consortium (ExAC). A set of predefined such classified variation can be designated for further bioinformatics analysis due to its relevance to clinical decision making (e.g., diagnosis, prognosis, treatment selection, targeted therapy, treatment monitoring, recurrence monitoring, etc.). Such predefined groups can be determined based on, for example, analysis of clinical samples (eg, clinical samples from pairs of patients known to be present or absent a disease or condition) and annotation information from public databases and clinical literature.

[0161] E. Sequencing

[0162] The sample nucleic acid with adapters, with or without pre-amplification on the flank can be subjected to sequencing.Sequencing methods include, for example, Sanger sequencing, high-throughput sequencing, pyrophosphate sequencing, synthesis sequencing, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, connection sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule synthesis sequencing (SMSS) (Helicos), large-scale parallel sequencing, cloned single-molecule array (colonal singlemolceule array) (Solexa), shotgun sequencing, Ion Torrent, Oxford nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, using PacBio, SOLiD, Ion Torrent or nanopore platform sequencing.Sequencing reaction can be carried out in many kinds of sample processing units, and the sample processing unit can include multiple lanes (multiplelane), multichannel, multiporous or other devices (means) of substantially processing multiple sample groups simultaneously.The sample processing unit can also include multiple sample chambers to realize processing multiple operations simultaneously.

[0163] Sequencing reaction can be carried out to one or more nucleic acid fragment types or regions of the marker known to contain cancer or other diseases. Sequencing reaction can also be carried out to any nucleic acid fragment present in the sample. Sequencing reaction can be carried out to at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 100% of the genome. In other cases, sequencing reaction can be carried out to less than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 100% of the genome.

[0164] Multiple sequencing techniques can be used to perform simultaneous sequencing reactions. In some cases, the cell-free polynucleotides can be sequenced with at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. In other cases, the cell-free polynucleotides can be sequenced with less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. The sequencing reactions can be performed sequentially or simultaneously. Subsequent data analysis can be performed on all or a portion of the sequencing reactions. In some cases, data analysis can be performed on at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. In other cases, data analysis can be performed on less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. An exemplary read depth is 1000-50000 reads / locus (bases). In some embodiments, the read depth can be greater than 50000 reads / locus (bases).

[0165] F. Analysis

[0166] Sequencing according to an embodiment of the present invention produces more than one sequencing read or read. Sequencing reads or reads according to the present invention generally include sequences of nucleotide data having a length less than about 150 bases or a length less than about 90 bases. In certain embodiments, the length of the read is between about 80 bases and about 90 bases, such as about 85 bases. In some embodiments, the method of the present invention is applied to very short reads, i.e., a length less than about 50 bases or about 30 bases. Sequencing read data may include sequence data and meta information. Sequence read data may be stored in any suitable file format, including, for example, VCF files, FASTA files, or FASTQ files.

[0167] FASTA was originally a computer program for searching sequence databases, and the name FASTA also refers to a standard file format. See Pearson & Lipman, 1988, Improved tools for biological sequence comparison, PNAS 85: 2444-2448. A sequence in FASTA format begins with a single-line description, followed by a sequence data row. The description row is distinguished from the sequence data by the greater than (">") symbol in the first column. The word after the ">" symbol is the identifier of the sequence, and the rest of the row is a description (both are optional). There should be no space between the ">" and the first letter of the identifier. It is recommended that all lines of text be shorter than 80 characters. If another line starting with ">" appears, the sequence ends; This indicates the beginning of another sequence.

[0168] The FASTQ format is a text-based format for storing biological sequences (usually nucleotide sequences) and their corresponding quality scores. The FASTQ format is similar to the FASTA format, but has a quality score after the sequence data. For simplicity, both sequence letters and quality scores are encoded using a single ASCII character. The FASTQ format is a conventional standard for storing the output of high-throughput sequencing instruments such as Illumina genome analyzers, such as Cock et al. ("The Sanger FASTQ file format for sequences with quality scores, and the Solexa / Illumina FASTQ variants," Nucleic Acids Res 38 (6): 1767-1771, 2009) described, which is incorporated herein by reference in its entirety.

[0169] For FASTA and FASTQ files, meta information includes description lines but does not include sequence data lines. In some embodiments, for FASTQ files, meta information includes quality scores. For FASTA and FASTQ files, sequence data begins after the description line and is typically presented using some subsets of IUPAC fuzzy codes optionally with "-". In preferred embodiments, sequence data will use A, T, C, G, and N characters, optionally including "-" or including U (e.g., to represent a gap or uracil) as needed.

[0170] In some embodiments, at least one of the master sequence read file and the output file is stored as a plain text file (e.g., using an encoding such as ASCII; ISO / IEC 646; EBCDIC; UTF-8; or UTF-16). The computer system provided by the present invention may include a text editor program capable of opening a plain text file. A text editor program may refer to a computer program capable of presenting the contents of a text file (such as a plain text file) on a computer screen, allowing a person to edit the text (e.g., using a screen, keyboard, and mouse). Exemplary text editors include, but are not limited to, Microsoft Word, emacs, pico, vi, BBEdit, and TextWrangler. Preferably, the text editor program is capable of displaying a plain text file on a computer screen, displaying meta-information and sequence reads in a human-readable format (e.g., not binary encoding but using alphanumeric characters, as alphanumeric characters can be used to print human writing).

[0171] Although the methods have been discussed with reference to FASTA or FASTQ files, the methods and systems of the present invention can be used to compress any suitable sequence file format, including, for example, files in Variant Call Format (VCF) format. A typical VCF file includes a header portion and a data portion. The header contains any number of meta-information rows, each beginning with the characters '##', and TAB-delimited field definition rows beginning with a single '#' character. The field definition row names eight required columns, and the body portion contains data rows that fill in the columns defined by the field definition row. The VCF format is described by Danecek et al. ("The variant call format and VCFtools," Bioinformatics 27(15):2156-2158, 2011), which is hereby incorporated by reference in its entirety. The header portion can be viewed as meta-information to be written to the compressed file, and the data portion can be viewed as rows, each of which is stored in the master file only if it is unique.

[0172] Certain embodiments of the present invention provide for the assembly of sequencing reads. For example, in an assembly by alignment, sequencing reads are aligned to each other or to a reference sequence. By aligning each read, and then to a reference genome, all reads are positioned relative to each other to produce an assembly. In addition, alignment of sequencing reads to a reference sequence or mapping to a reference sequence can also be used to identify variant sequences in sequencing reads. Identification of variant sequences can be used in combination with the methods and systems described herein to further aid in the diagnosis or prognosis of a disease or condition or to guide treatment decisions.

[0173] In some embodiments, any or all steps are automated. Alternatively, the method of the present invention can be implemented in whole or in part in one or more special programs, for example, each special program is optionally written in a compiled language such as C++, and then compiled and distributed in binary. The method of the present invention can be implemented in whole or in part as a module in an existing sequence analysis platform or by calling the function in an existing sequence analysis platform. In certain embodiments, the method of the present invention includes a plurality of steps that are automatically called in response to a single starting signal (starting cue) (for example, an event or event combination in a triggering event derived from human activity, another computer program or machine). Therefore, the present invention provides a method in which any one of the steps or any combination of steps can automatically occur in response to a signal. Automatically generally means not intervening in human input, influence or interaction (i.e., only in response to original or pre-prompted human activity (pre-cue humanactivity)).

[0174] The system also includes various forms of output, which includes accurate and sensitive interpretation of the subject nucleic acid. The output of the retrieval can be provided in the format of a computer file. In certain embodiments, the output is a FASTA file, a FASTQ file or a VCF file. The output can be processed to generate a text file or XML file containing sequence data (such as a sequence of a nucleic acid aligned with a reference genome). In other embodiments, the processing generates an output comprising coordinates or strings describing one or more mutations in the subject nucleic acid relative to the reference genome. The alignment string can include Simple UnGapped Alignment Report (SUGAR), Verbose Useful Labeled Gapped Alignment Report (VALGAR) and Compact Idiosyncratic Gapped Alignment Report (CIGAR) (Ning et al., Genome Research 11 (10): 1725-9, 2001, the document is hereby incorporated by reference in its entirety). These strings are, for example, implemented in the Exonerate sequence alignment software from the European Bioinformatics Institute (Hinxton, UK).

[0175] In some embodiments, a sequence alignment containing CIGAR strings is generated—such as, for example, a sequence alignment map (SAM) or a binary alignment map (BAM) file (the SAM format is described, for example, by Li et al., "The Sequence Alignment / Map format and SAMtools," Bioinformatics, 25(16): 2078-9, 2009, which is incorporated herein by reference in its entirety). In some embodiments, CIGAR displays or includes one gapped alignment per line. CIGAR is a compressed pairwise alignment format reported as CIGAR strings. CIGAR strings can be used to present long (e.g., genomic) pairwise alignments. CIGAR strings are used in the SAM format to represent the alignment of a read segment to a reference genomic sequence.

[0176] CIGAR word strings follow established motifs. Each character is preceded by a number giving the base count of the event. The characters used may include M, I, D, N, and S (M = match; I = insertion; D = deletion; N = gap; S = substitution). CIGAR word strings define sequences of matches / mismatches and deletions (or gaps). For example, the CIGAR word string 2MD3M2D2M would mean that the alignment contains 2 matches, 1 deletion (the number 1 is omitted to save some space), 3 matches, 2 deletions, and 2 matches.

[0177] In some embodiments, a nucleic acid colony for sequencing is prepared by enzymatically forming a flat end on a double-stranded nucleic acid with a single-stranded overhang at one or both ends. In these embodiments, in the presence of nucleotides (e.g., A, C, G and T or U) in the form of dNTPs, the colony is usually treated with an enzyme with 5'-3' DNA polymerase activity and 3'-5' exonuclease activity. The exemplary enzyme or its catalytic fragment optionally used includes Klenow large fragment and T4 polymerase. At 5' overhang, the enzyme usually extends the 3' end of the depression on the relative chain until it is flush with the 5' end to produce a flat end. At 3' overhang, the enzyme is usually digested from the 3' end, reaches the 5' end of the relative chain and sometimes exceeds the 5' end of the relative chain. If the digestion advances beyond the 5' end of the relative chain, the vacancy can be filled by having an enzyme with the same polymerase activity used for 5' overhang. The formation of the flat end on the double-stranded nucleic acid is conducive to the attachment of, for example, adapters and subsequent amplification.

[0178] In some embodiments, the nucleic acid population is subjected to additional processing, such as converting single-stranded nucleic acids into double-stranded nucleic acids and / or converting RNA into DNA. These forms of nucleic acids are also optionally ligated to adapters and amplified.

[0179] The nucleic acid subjected to the above-described flat-end treatment, with or without prior amplification, and optionally other nucleic acids in the sample, can be sequenced to produce sequenced nucleic acid. Sequenced nucleic acid can refer to the sequence (i.e., sequence information) of a nucleic acid or a nucleic acid whose sequence has been determined. Sequencing can be performed to provide sequence data of individual nucleic acid molecules in a sample directly or indirectly from the consensus sequence of the amplification products of the individual nucleic acid molecules in the sample.

[0180] In some embodiments, after the double-stranded nucleic acid with single-stranded overhang in the sample is formed at the flat end, it is connected to the adapter containing the molecular barcode at both ends, and the sequencing determines the nucleic acid sequence and the molecular barcode introduced by the adapter. The flat-ended DNA molecule is optionally connected to the flat end of the at least partially double-stranded adapter (e.g., Y-shaped adapter or bell-shaped adapter). Alternatively, the flat ends of the sample nucleic acid and the adapter can be tailed with complementary nucleotides to facilitate connection (e.g., sticky end connection).

[0181] The nucleic acid sample is typically contacted with a sufficient number of adapters so that the probability that any two copies of the same nucleic acid receive the same adapter barcode combination from the adapters connected at the two ends is low (e.g., less than <1% or <0.1%). Using adapters in this manner allows the identification of a family of nucleic acid sequences that have the same start and end points on a reference nucleic acid and are connected to the same molecular barcode combination. Such a family represents the sequence of the amplified product of the nucleic acid in the sample before amplification. The sequences of the family members can be compiled to obtain one or more common nucleotides or a complete common sequence of the nucleic acid molecules in the original sample, which are modified by blunt end formation and adapter attachment. In other words, the nucleotides occupying a specific position of the nucleic acid in the sample are determined to be the common nucleotides of the nucleotides occupying the corresponding position in the family member sequence. The family can include the sequence of one or two chains of a double-stranded nucleic acid. If a member of the family includes sequences from two chains of a double-stranded nucleic acid, the sequence of one chain can be converted into their complementary sequences for the purpose of compiling all sequences to obtain one or more common nucleotides or sequences. Some families contain only a single member sequence. In this case, the sequence can be regarded as the sequence of the nucleic acid in the sample before amplification. Alternatively, families with only a single member sequence can be excluded from subsequent analysis.

[0182] By comparing the nucleic acid through sequencing with the reference sequence, the nucleotide variation in the nucleic acid through sequencing can be determined. The reference sequence is usually a known sequence, for example, from a known genome sequence of all or part of a subject (for example, a full genome sequence of a human subject). The reference sequence can be, for example, hG19 or hG38. As described above, the nucleic acid through sequencing can represent the sequence of the nucleic acid in the sample directly determined or the consensus sequence of the amplification product of such nucleic acid. Comparison can be made at one or more designated positions on the reference sequence. When the corresponding sequence is aligned to the greatest extent, a subset of the nucleic acid through sequencing can be identified, which subset includes a position corresponding to the designated position of the reference sequence. In such a subset, it can be determined which (if any) nucleic acid through sequencing includes nucleotide variation at the designated position, and optionally which (if any) includes reference nucleotides (that is, the same as in the reference sequence). If the number of nucleic acids through sequencing including nucleotide variation in the subset exceeds a selected threshold value, the variant nucleotides can be identified at the designated position. The threshold can be a simple number, such as at least 1, 2, 3, 4, 5, 6, 7, 9, or 10 sequenced nucleic acids in the subset that include nucleotide variations, or the threshold can be a ratio, such as at least 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, or 20% of the sequenced nucleic acids in the subset that include nucleotide variations, as well as other possibilities. Comparisons can be repeated for any specified position of interest in the reference sequence. Sometimes comparisons can be made for specified positions that occupy at least about 20, 100, 200, or 300 consecutive positions on the reference sequence, for example, about 20-500 or about 50-300 consecutive positions.

[0183] Additional details about nucleic acid sequencing, including the formats and applications described herein, are also provided in the following references: for example, Levy et al., Annual Review of Genomics and Human Genetics, 17:95-115 (2016); Liu et al., J. of Biomedicine and Biotechnology, 2012, Article ID 251364:1-11 (2012); Voelkerding et al., Clinical Chem., 55:641-658 (2009); MacLean et al., Nature Rev. Microbiol., 7:287-296 (2009); Astier et al., J Am Chem. Soc., 128(5):1705-10(2006); U.S. Patent No. 6,210,891, U.S. Patent No. 6,258,568, U.S. Patent No. 6,833,246, U.S. Patent No. 7,115,400, U.S. Patent No. 6,969,488, U.S. Patent No. 5,912,148, U.S. Patent No. 6,130,073, U.S. Patent No. 7,169,560, U.S. Patent No. 7,282,337, U.S. Patent No. No. 7,482,120, U.S. Pat. No. 7,501,245, U.S. Pat. No. 6,818,395, U.S. Pat. No. 6,911,345, U.S. Pat. No. 7,501,245, U.S. Pat. No. 7,329,492, U.S. Pat. No. 7,170,050, U.S. Pat. No. 7,302,146, U.S. Pat. No. 7,313,308, and U.S. Pat. No. 7,476,503, each of which is incorporated herein by reference in its entirety.

[0184] III. Computer System

[0185] The method of the present disclosure may be implemented using or with the aid of a computer system. For example, such a method may include (a) obtaining more than one sequencing read of a set of tagged polynucleotides from a first sample and a second sample generated by a nucleic acid sequencer, wherein the sequencing read includes a tag sequence and a sequence derived from the polynucleotide; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, wherein the grouping feature includes at least one of the following (i), (ii), (iii) and (iv): (i) a tag, (ii) a start region, (iii) an end region, and (iv) a length of the polynucleotide, wherein each family in the sample The method can be performed using a computer processor and comprising: (a) a method of generating a family identifier for more than one family, wherein the family identifier is a family identifier for a first sample that is the same as or substantially the same as a family identifier for a second sample; (b) determining a quantitative measure for the family identifier; and (c) classifying the first sample as contaminated by the second sample if the quantitative measure for the family identifier is above a predetermined threshold, or classifying the first sample as not contaminated if the quantitative measure for the family identifier is at or below a predetermined threshold.

[0186] Figure 4 A computer system 401 that is programmed or otherwise configured to implement the methods of the present disclosure is shown. The computer system 401 can regulate various aspects of sample preparation, sequencing, and / or analysis. In some instances, the computer system 401 is configured to perform sample preparation and sample analysis, including nucleic acid sequencing.

[0187] The computer system 401 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 405, which may be a single-core processor or a multi-core processor or more than one processor for parallel processing. The computer system 401 also includes a memory or memory location 410 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 415 (e.g., a hard disk), a communication interface 420 (e.g., a network adapter) for communicating with one or more other systems, and peripherals 425, such as cache, other memory, data storage, and / or an electronic display adapter. The memory 410, storage unit 415, interface 420, and peripherals 425 communicate with the CPU 405 via a communication network or bus (real wires) such as a motherboard. The storage unit 415 may be a data storage unit (or data repository) for storing data. The computer system 401 may be operably coupled to a computer network 430 by means of the communication interface 420. Computer network 430 can be the Internet, an intranet and / or an extranet, or an intranet and / or an extranet that communicates with the Internet. In some cases, computer network 430 is a telecommunications and / or data network. Computer network 430 may include one or more computer servers, which may enable distributed computing, such as cloud computing. In some cases, with the aid of computer system 401, computer network 430 may implement a peer-to-peer network that enables devices coupled to computer system 401 to operate as clients or servers.

[0188] The CPU 405 may execute a series of machine-readable instructions, which may be implemented in a program or software. The instructions may be stored in a memory location, such as the memory 410. Examples of operations performed by the CPU 405 may include reading, decoding, executing, and writing back.

[0189] Storage unit 415 can store files, such as drivers, libraries, and programs that are saved. Storage unit 415 can store user-generated programs and recorded sessions and one or more outputs associated with the program. Storage unit 415 can store user data, such as user preferences and user programs. In some cases, computer system 401 may include one or more additional data storage units, which are external to computer system 401, such as being located on a remote server that communicates with computer system 401 via an intranet or the Internet. Data can be transferred from one location to another using, for example, a communication network or a physical data transfer (e.g., using a hard drive, a thumb drive, or other data storage mechanisms).

[0190] Computer system 401 can communicate with one or more remote computer systems via network 430. For example, computer system 401 can communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include personal computers (e.g., portable PCs), slate or tablet PCs (e.g., iPad, Galaxy Tab), phones, smartphones (such as iPhone, Android supported devices, ) or a personal digital assistant. A user can access the computer system 401 via the network 430.

[0191] The methods as described herein may be implemented by means of machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 401, such as, for example, the memory 410 or the electronic storage unit 415. The machine executable code or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 405. In some cases, the code may be retrieved from the storage unit 415 and stored on the memory 410 for immediate access by the processor 405. In some cases, the electronic storage unit 415 may not be included, and the machine executable instructions may be stored on the memory 410.

[0192] In one aspect, the present disclosure provides a non-transitory computer-readable medium comprising computer-executable instructions, which when executed by at least one electronic processor, performs a method, the method comprising: (a) obtaining more than one sequencing read of a set of tagged polynucleotides from a first sample and a second sample generated by a nucleic acid sequencer, wherein the sequencing read comprises a tag sequence and a sequence derived from the polynucleotide; (b) aligning the more than one sequencing read with a reference sequence, thereby determining a start region and an end region of the alignment; (c) for each sample, grouping the more than one sequencing read into more than one family based on a grouping feature, wherein the grouping feature comprises at least one of the following (i), (ii), (iii), and (iv): (i) a tag, (ii) a sequence derived from a polynucleotide; (iii) a sequence derived from a polynucleotide; (iv) a sequence derived from a polynucleotide; (v ... ) a starting region, (iii) an ending region, and (iv) a length of a polynucleotide, wherein each family in the sample comprises sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the sample; (d) generating family identifiers for more than one family; (e) screening a set of shared family identifiers, wherein the shared family identifier is a family identifier for the first sample that is identical or substantially identical to a family identifier for the second sample; (f) determining a quantitative measure for the set of shared family identifiers; and (g) classifying the first sample as contaminated by the second sample if the quantitative measure for the shared family identifier is above a predetermined threshold, or classifying the first sample as not contaminated if the quantitative measure for the shared family identifier is at or below a predetermined threshold.

[0193] The code may be precompiled and configured for use with a machine having a processor suitable for executing the code or may be compiled during runtime. The code may be provided in a programming language that may be selected so that the code can be executed in a precompiled or as-compiled manner.

[0194] Aspects of the systems and methods provided herein, such as computer system 401, can be implemented in programming. Various aspects of the technology can be considered to be "products" or "articles of manufacture" in the form of machine (or processor) executable code and / or related data, which are generally carried on a type of machine-readable medium or implemented in a type of machine-readable medium. The machine executable code can be stored in an electronic storage unit such as a memory (e.g., a read-only memory, a random access memory, a flash memory) or a hard disk. "Storage" type media can include computers, processors, etc. or their related modules, any or all tangible memories such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-temporary storage for software programming at any time.

[0195] All or part of the software can sometimes communicate through the Internet or various other telecommunication networks. For example, such communication can realize loading the software from one computer or processor to another computer or processor, for example, from a management server or mainframe to a computer platform of an application server. Therefore, another type of medium that can carry software elements includes optical waves, electric waves, and electromagnetic waves such as those used across physical interfaces between local devices, through wired and optical fiber landline networks, and on various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered as media that carry software. As used herein, unless limited to non-temporary, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0196] Thus, machine-readable media, such as computer executable code, may 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 or magnetic disks, such as any storage device shown in the accompanying drawings in any computer, etc., such as may be used to implement a database, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and optical fiber, including the wires that make up a bus within a computer system. Carrier transmission media may take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and an EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave that transmits data or instructions, a cable or link that transmits such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer-readable media can participate in carrying one or more strings of one or more instructions to a processor for execution.

[0197] Computer system 401 may include or communicate with an electronic display including a user interface (UI) to provide, for example, one or more results of a sample analysis. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0198] Additional details about computer systems and networks, databases, and computer program products are also provided in the following documents: for example, Peterson, Computer Networks: A Systems Approach, Morgan Kaufmann, 5th Edition (2011); Kurose, Computer Networking: A Top-Down Approach, Pearson, 7th Edition (2016); Elmasri, Fundamentals of Database Systems, Addison Wesley, 6th Edition (2010); Coronel, Database Systems: Design, Implementation, & Management, Cengage Learning, 11th Edition (2014); Tucker, Programming Languages, McGraw-Hill Science / Engineering / Math, 2nd Edition (2006); and Rhoton, Cloud Computing Architected: Solution Design Handbook, Recursive Press (2011), each of which is hereby incorporated by reference in its entirety.

[0199] application

[0200] Cancer and other diseases

[0201] Typically, the disease under consideration is a type of cancer. Non-limiting examples of such cancers include biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, hepatoepithelial carcinoma (liver carcinoma), hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary tumor, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, stomach cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer or uterine sarcoma.

[0202] Non-limiting examples of other genetically based diseases, disorders, or conditions that are optionally assessed using the methods and systems disclosed herein include achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth (CMT), Cry-a-Cat syndrome, Crohn's disease, cystic fibrosis, Dercum disease, Down syndrome, Duane syndrome, Duchenne muscular dystrophy, Factor V Leiden's thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency (scid), sickle cell disease, spinal muscular atrophy, Tay-Sachs, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, Wilson's disease, etc.

[0203] Although this description has been described with reference to specific embodiments thereof, these specific embodiments are merely illustrative and not restrictive. The concepts shown in the embodiments can be applied to other embodiments and implementations.

[0204] As liquid biopsy assays are altered (e.g., changes in sequencing depth and common SNP panels), the methods and systems of the present disclosure can be retrained as needed to obtain an applicable set of thresholds (e.g., one or more criteria / thresholds to detect the presence or absence of contamination in a sample). Example

[0205] Example 1: Determining contamination of a sample according to an embodiment of the present disclosure

[0206] A group of patient samples were analyzed at Guardant Health (Redwood City, CA, USA) using blood-based cfDNA assay. In order to check the quality of the assay performance and determine whether there is any contamination of the sample, the group of samples was analyzed according to the embodiment of the present disclosure. The analysis of two samples (sample 1 and sample 2) in the group of samples is described in the present embodiment. The total number of families in sample 1 and sample 2 is 7,811,148 and 7,141,008, respectively. In this embodiment, the family at the genome start position and genome end position pair of overrepresentation is not included in the analysis, and the threshold value for classifying a pair of genome start position and genome end position as overrepresentation is 10 families. Therefore, the total number of families in sample 1 and sample 2 is 6,452,057 and 6,039,099, respectively.

[0207] I: Determine whether sample 1 is contaminated by sample 2

[0208] Of the 6,452,057 families of sample 1, 54,212 families are shared families (shared with sample 2). Of the 54,212 shared families: (i) 9362 shared families have the same number of sequencing reads in the families of both sample 1 and sample 2; and (ii) 1647 shared families have a greater number of sequencing reads in the families of sample 1 than in the corresponding families of sample 2. In this embodiment, when determining the quantitative measure of shared families, shared families having a greater number of sequencing reads in the families of sample 1 than in the corresponding families of sample 2 are not included. In addition, in this embodiment, the quantitative measure of shared families is the percentage of shared families in sample 1 to the total families, which is equal to 0.815% (100*(54212-1647) / 6452057). In this embodiment, the predetermined threshold for classifying a sample as contaminated is 0.5%. Since the quantitative measure of the shared family of sample 1 was greater than 0.5%, sample 1 was determined to be contaminated by sample 2.

[0209] II: Determine whether sample 2 is contaminated by sample 1

[0210] Of the 6,039,099 families of sample 2, 54,212 families are shared families (shared with sample 1). Of the 54,212 shared families: (i) 9362 shared families have the same number of sequencing reads in the families of both sample 1 and sample 2; and (ii) 43,203 shared families have a greater number of sequencing reads in the families of sample 2 than in the corresponding families of sample 1. Excluding shared families having a greater number of sequencing reads in the families of sample 2 than in the corresponding families of sample 1, the quantitative measure of shared families of sample 2 is equal to 0.182% (100*(54212-43203) / 6039099). Since the quantitative measure of shared families of sample 2 is below the predetermined threshold (0.5%), sample 2 is determined to be uncontaminated by sample 1.

[0211] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only by way of example. It is not intended that the present invention be limited to the specific examples provided in this specification. Although the present invention has been described with reference to the description mentioned above, the description and illustration of the embodiments herein are not intended to be interpreted in a restrictive sense. Those skilled in the art will now think of many variations, changes and substitutions without departing from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions depending on various conditions and variables set forth herein. It should be understood that various alternatives of the embodiments of the present invention described herein can be adopted when practicing the present invention. It is therefore contemplated that the present invention should also encompass any such alternatives, modifications, variations or equivalents. The appended claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

[0212] Although the foregoing disclosure has been described in some detail by way of illustration and example for the purpose of clarity and understanding, it will be clear to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the actual scope of the disclosure and may be practiced within the scope of the appended claims. For example, all methods, systems, computer-readable media and / or component features, steps, elements or other aspects may be used in various combinations.

[0213] All patents, patent applications, websites, other publications or documents, accession numbers, etc. cited herein are incorporated by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, it is meant to refer to the version associated with the accession number on the effective filing date of the present application. If applicable, the effective filing date means the earlier of the actual filing date or the filing date of the priority application that mentions the accession number. Similarly, if different versions of a publication, website, etc. are published at different times, it is meant to refer to the version that was published most recently on the effective filing date of the present application, unless otherwise indicated.

Claims

1. A method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of polynucleotides from the first sample and the second sample to generate more than one sequencing read; (b) aligning the more than one sequencing reads with a reference sequence, thereby determining a start region and an end region of the alignment, wherein the start region includes a genomic start position of the sequencing read, at which the 5' end of the sequencing read is determined to start the alignment with the reference sequence, and the end region includes a genomic stop position of the sequencing read, at which the 3' end of the sequencing read is determined to stop the alignment with the reference sequence; (c) for each sample, grouping the more than one sequencing reads into more than one family based on a grouping feature, the grouping feature comprising at least one of (i) the start region, (ii) the end region, and (iii) the length of the polynucleotide, wherein each family in the first sample and the second sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the first sample and the second sample; (d) generating family identifiers for the more than one families; (e) screening a set of common family identifiers, wherein a given common family identifier is a family identifier of the first sample that is identical or at least 95% identical to a family identifier of the second sample; (f) determining a quantitative measure of the set of consensus family identifiers, wherein the quantitative measure comprises one or more of: the number of common family identifiers in the first sample, the ratio of the number of common family identifiers in the first sample to the total number of family identifiers in the first sample, excluding those common family identifiers in the first sample whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample, and excluding common family identifiers at over-represented genomic start position and genomic end position pairs, wherein an over-represented genomic start position and genomic end position pair refers to a genomic start position and genomic end position pair in which the number or frequency of families sharing the genomic start position and genomic end position pair in more than one sample exceeds a set threshold; and (g) classifying the first sample as being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is at or below the predetermined threshold.

2. A method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) accessing, by a computer system, sequence information comprising more than one sequencing read from the first sample and the second sample; (b) aligning the more than one sequencing reads with a reference sequence by the computer system, thereby determining a start region and an end region of the alignment, wherein the start region includes a genomic start position of the sequencing read, at which the 5' end of the sequencing read is determined to start the alignment with the reference sequence, and the end region includes a genomic end position of the sequencing read, at which the 3' end of the sequencing read is determined to end the alignment with the reference sequence; (c) for each sample, grouping, by the computer system, the more than one sequencing reads into more than one family based on a grouping feature, the grouping feature comprising at least one of (i) the start region, (ii) the end region, and (iii) the length of the polynucleotide, wherein each family in the first sample and the second sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the first sample and the second sample; (d) generating, by the computer system, family identifiers for the more than one family; (e) screening a set of common family identifiers by the computer system, wherein a given common family identifier is a family identifier of the first sample that is identical or at least 95% identical to a family identifier of the second sample; (f) determining, by the computer system, a quantitative measure of the set of consensus family identifiers, wherein the quantitative measure comprises one or more of: the number of common family identifiers in the first sample, the ratio of the number of common family identifiers in the first sample to the total number of family identifiers in the first sample, excluding those common family identifiers in the first sample whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample, and excluding common family identifiers at over-represented genomic start position and genomic end position pairs, wherein an over-represented genomic start position and genomic end position pair refers to a genomic start position and genomic end position pair in which the number or frequency of families sharing the genomic start position and genomic end position pair in more than one sample exceeds a set threshold; and (g) classifying, by the computer system, the first sample as being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or classifying, by the computer system, the first sample as being not contaminated by the second sample if the quantitative measure of the set of shared family identifiers is at or below the predetermined threshold.

3. The method according to any one of claims 1-2, further comprising, before (a), tagging the collection of polynucleotides to produce tagged polynucleotides, wherein each tagged polynucleotide comprises a tag and a polynucleotide.

4. A method according to claim 3, wherein for each sample, the more than one sequencing reads are grouped into more than one family based on grouping features, and the grouping features include at least one of the following (i), (ii), (iii) and (iv): (i) the tag, (ii) the starting region, (iii) the ending region and (iv) the length of the polynucleotide, wherein each family in the first sample and the second sample includes sequencing reads of progeny polynucleotides amplified from unique polynucleotides in the set of polynucleotides in the first sample and the second sample.

5. A method for detecting the presence or absence of contamination of a first sample by a second sample, the method comprising: (a) sequencing a collection of tagged polynucleotides from the first sample and the second sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing reads with a reference sequence, thereby determining a start region and an end region of the alignment, wherein the start region includes a genomic start position of the sequencing read, at which the 5' end of the sequencing read is determined to start the alignment with the reference sequence, and the end region includes a genomic stop position of the sequencing read, at which the 3' end of the sequencing read is determined to stop the alignment with the reference sequence; (c) for each sample, grouping the more than one sequencing reads into more than one family based on a grouping feature that includes the tag, wherein each family in the first sample and the second sample includes sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in the set of tagged polynucleotides in the first sample and the second sample; (d) generating family identifiers for the more than one families; (e) screening a set of common family identifiers, wherein a given common family identifier is a family identifier of the first sample that is identical or at least 95% identical to a family identifier of the second sample; (f) determining a quantitative measure of the set of consensus family identifiers, wherein the quantitative measure comprises one or more of: the number of common family identifiers in the first sample, the ratio of the number of common family identifiers in the first sample to the total number of family identifiers in the first sample, excluding those common family identifiers in the first sample whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample, and excluding common family identifiers at over-represented genomic start position and genomic end position pairs, wherein an over-represented genomic start position and genomic end position pair refers to a genomic start position and genomic end position pair in which the number or frequency of families sharing the genomic start position and genomic end position pair in more than one sample exceeds a set threshold; and (g) classifying the first sample as being contaminated by the second sample if the quantitative measure of the common family identifier is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of common family identifiers is at or below the predetermined threshold.

6. The method of claim 1, 2 or 5, wherein the over-represented genomic start position and genomic end position pair is determined by: (a) providing more than one sample, wherein the more than one sample comprises a distribution of genomic starting positions and genomic ending positions that are the same or substantially the same as the first sample and / or the second sample, wherein substantially the same means that the starting region and the ending region of more than one sample are different within 25bp; (b) determining family identifiers in said more than one samples; (c) quantifying the number of family identifiers that share a pair of genomic start positions and genomic end positions in the more than one samples; and (d) classifying the genomic start position and genomic end position pair as over-represented if the number of family identifiers exceeds a set threshold. The method of claim 6 , wherein the more than one samples do not include the first sample or the second sample. The method of claim 6 , wherein the more than one samples do not include the first sample and the second sample.

9. The method of claim 6, wherein the more than one samples include samples processed in the same flow cell as the first sample.

10. The method of claim 6, wherein the more than one samples comprise training samples. The method according to claim 6 , wherein the set threshold is at least 2 families.

12. The method of claim 6, wherein the set threshold is at least 10 families.

13. The method of claim 6, wherein the set threshold is at least 20 families.

14. The method of claim 6, wherein the set threshold is at least 30 families.

15. The method of claim 6, wherein the set threshold is at least 40 families.

16. The method of claim 6, wherein the set threshold is at least 50 families.

17. The method of claim 6, wherein the set threshold is at least 60 families.

18. The method of claim 5, further comprising, prior to the sequencing, tagging the collection of polynucleotides to produce tagged polynucleotides, wherein each tagged polynucleotide comprises a tag and a polynucleotide.

19. A method according to claim 18, wherein for each sample, the more than one sequencing reads are grouped into more than one family based on grouping features, and the grouping features include at least one of the following (i), (ii), (iii) and (iv): (i) the tag, (ii) the starting region, (iii) the ending region and (iv) the length of the polynucleotide, wherein each family in the first sample and the second sample includes sequencing reads of progeny polynucleotides amplified from unique polynucleotides in the set of polynucleotides in the first sample and the second sample.

20. The method of any one of claims 1-2, 4-5, and 7-19, wherein the starting region includes the first 30 base positions of the 5' end of the sequencing read aligned to the reference sequence.

21. The method of claim 20, wherein the starting region comprises the first 20 base positions of the 5' end of the sequencing read aligned to the reference sequence.

22. The method of claim 20, wherein the starting region comprises the first 10 base positions of the 5' end of the sequencing read aligned to the reference sequence.

23. The method of claim 20, wherein the starting region comprises the first 2 base positions of the 5' end of the sequencing read aligned to the reference sequence.

24. The method of any one of claims 1-2, 4-5, 7-19 and 21-23, wherein the end region comprises the last 30 base positions of the 3' end of the sequencing read aligned to the reference sequence.

25. The method of claim 24, wherein the end region comprises the last 20 base positions of the 3' end of the sequencing reads aligned to the reference sequence.

26. The method of claim 24, wherein the end region comprises the last 10 base positions of the 3' end of the sequencing reads aligned to the reference sequence.

27. The method of claim 24, wherein the end region comprises the last 2 base positions of the 3' end of the sequencing read aligned to the reference sequence.

28. The method of claim 3, wherein the tag comprises one or more molecular barcodes attached to a terminus of the polynucleotide.

29. The method of claim 28, wherein the one or more molecular barcodes are at least 2 nucleotides in length.

30. The method of claim 28, wherein the one or more molecular barcodes are at least 10 nucleotides in length.

31. The method of claim 28, wherein the one or more molecular barcodes are at least 20 nucleotides in length.

32. The method of claim 28, wherein the one or more molecular barcodes attached to the polynucleotides of the first sample are different from the one or more molecular barcodes attached to the polynucleotides of the second sample.

33. The method of claim 5, wherein the tag comprises one or more molecular barcodes attached to the ends of the polynucleotides.

34. The method of claim 33, wherein the one or more molecular barcodes are at least 2 nucleotides in length.

35. The method of claim 33, wherein the one or more molecular barcodes are at least 10 nucleotides in length.

36. The method of claim 33, wherein the one or more molecular barcodes are at least 20 nucleotides in length.

37. The method of claim 33, wherein the one or more molecular barcodes attached to the polynucleotides of the first sample are different from the one or more molecular barcodes attached to the polynucleotides of the second sample.

38. The method of any one of claims 1-2, 4-5, 7-19, 21-23, and 25-37, wherein the polynucleotides of the first sample and the second sample are tagged with at least 5 different molecular barcodes.

39. The method of claim 38, wherein the polynucleotides of the first sample and the second sample are tagged with at least 10 different molecular barcodes.

40. The method of claim 38, wherein the polynucleotides of the first sample and the second sample are tagged with at least 20 different molecular barcodes.

41. The method of claim 38, wherein the polynucleotides of the first sample and the second sample are tagged with at least 50 different molecular barcodes.

42. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, and 39-41, wherein the first sample and the second sample are sequenced in the same flow cell.

43. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, and 39-41, wherein the second sample is sequenced in a different flow cell than the first sample.

44. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, and 39-41, wherein the second sample is processed on the same day as the first sample but at a different time than the first sample.

45. The method of claim 44, wherein the second sample is processed at least 1 minute after processing the first sample.

46. ​​The method of claim 44, wherein the second sample is processed at least 1 hour after processing the first sample.

47. The method of claim 44, wherein the second sample is processed at least 2 hours after processing the first sample.

48. The method of claim 44, wherein the second sample is processed at least 3 hours after processing the first sample.

49. The method of claim 44, wherein the second sample is processed at least 4 hours after processing the first sample.

50. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, and 39-41, wherein the first sample and the second sample are processed on different days.

51. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, 39-41, and 45-49, wherein the first sample and the second sample are in the same sample batch.

52. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, 39-41, and 45-49, wherein the second sample is processed with the same batch of reagents as the first sample.

53. The method of claim 52, wherein the first sample and the second sample are processed at different geographic locations.

54. The method of claim 3, wherein the collections of tagged polynucleotides of the first sample and the second sample are uniquely tagged.

55. The method of claim 5, wherein the collections of tagged polynucleotides of the first sample and the second sample are uniquely tagged.

56. The method of claim 3, wherein the collections of tagged polynucleotides of the first sample and the second sample are non-uniquely tagged.

57. The method of claim 5, wherein the collections of tagged polynucleotides of the first sample and the second sample are non-uniquely tagged.

58. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, 39-41, 45-49, and 53-57, wherein the first sample is obtained from a body fluid of one subject and the second sample is obtained from a body fluid of another subject.

59. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, 39-41, 45-49, and 53-57, wherein the polynucleotide is a cell-free polynucleotide.

60. The method of claim 59, wherein the cell-free polynucleotide is cell-free DNA.

61. The method of claim 58, wherein at least one of the subjects has a disease.

62. The method of claim 61, wherein the disease is cancer.

63. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, 39-41, 45-49, 53-57, and 60-62, wherein the collection of polynucleotides of the first sample and the second sample are amplified prior to sequencing, thereby generating amplified progeny polynucleotides.

64. The method of claim 63, further comprising selectively enriching at least a portion of the amplified progeny polynucleotides from a region of the subject's genome or transcriptome prior to the sequencing.

65. The method of claim 64, further comprising attaching one or more sample indexes to one or both ends of the amplified progeny polynucleotides prior to sequencing, wherein the sample index distinguishes the first sample from the second sample.

66. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, 39-41, 45-49, 53-57, 60-62, and 64-65, wherein the predetermined threshold is at least 0.001% of the total number of families in the first sample.

67. The method of claim 66, wherein the predetermined threshold is at least 0.01% of the total number of families in the first sample.

68. The method of claim 66, wherein the predetermined threshold is at least 0.1% of the total number of families in the first sample.

69. The method of claim 66, wherein the predetermined threshold is at least 1% of the total number of families in the first sample.

70. The method of claim 66, wherein the predetermined threshold is at least 2% of the total number of families in the first sample.

71. The method of claim 66, wherein the predetermined threshold is at least 10% of the total number of families in the first sample.

72. The method of any one of claims 1-2, 4-5, 7-19, 21-23, 25-37, 39-41, 45-49, 53-57, 60-62, 64-65, and 67-71, further comprising generating a report.

73. A method according to claim 72, wherein the report includes information about the contamination status of the sample and / or information derived from the contamination status of the sample.

74. The method of claim 72, further comprising transmitting the report to a third party.

75. The method of claim 74, wherein the third party is a subject from whom the sample originated or a health care practitioner.

76. A system for detecting the presence or absence of contamination of a first sample by a second sample, the system comprising: a communication interface that receives, over a communication network, more than one sequencing read of a set of tagged polynucleotides from a sample generated by a nucleic acid sequencer, wherein the sequencing read includes a tag sequence and a sequence derived from the polynucleotide; and A computer in communication with a communication interface, wherein the computer includes one or more computer processors and a computer readable medium containing machine executable code, the machine executable code when executed by the one or more computer processors implements a method comprising: (a) receiving, via the communication network, the more than one sequencing reads of the set of tagged polynucleotides from the first sample and the second sample generated by the nucleic acid sequencer; (b) aligning the more than one sequencing reads with a reference sequence, thereby determining a start region and an end region of the alignment, wherein the start region includes a genomic start position of the sequencing read, at which the 5' end of the sequencing read is determined to start the alignment with the reference sequence, and the end region includes a genomic stop position of the sequencing read, at which the 3' end of the sequencing read is determined to stop the alignment with the reference sequence; (c) for each sample, grouping the more than one sequencing reads into more than one family based on a grouping feature comprising at least one of (i), (ii), (iii), and (iv) (i) the tag, (ii) the start region, (iii) the end region, and (iv) the length of the polynucleotide, wherein each family in the first sample and the second sample comprises sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the first sample and the second sample; (d) generating family identifiers for the more than one families; (e) screening a set of common family identifiers, wherein a given common family identifier is a family identifier of the first sample that is identical or at least 95% identical to a family identifier of the second sample; (f) determining a quantitative measure of the set of consensus family identifiers, wherein the quantitative measure comprises one or more of: the number of common family identifiers in the first sample, the ratio of the number of common family identifiers in the first sample to the total number of family identifiers in the first sample, excluding those common family identifiers in the first sample whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample, and excluding common family identifiers at over-represented genomic start position and genomic end position pairs, wherein an over-represented genomic start position and genomic end position pair refers to a genomic start position and genomic end position pair in which the number or frequency of families sharing the genomic start position and genomic end position pair in more than one sample exceeds a set threshold; and (g) classifying the first sample as being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is at or below the predetermined threshold.

77. A system comprising a controller comprising or having access to a computer readable medium containing non-transitory computer executable instructions that when executed by at least one electronic processor perform a method comprising: (a) sequencing a collection of polynucleotides from a first sample and a second sample to generate more than one sequencing read; (b) aligning the more than one sequencing reads with a reference sequence, thereby determining a start region and an end region of the alignment, wherein the start region includes a genomic start position of the sequencing read, at which the 5' end of the sequencing read is determined to start the alignment with the reference sequence, and the end region includes a genomic stop position of the sequencing read, at which the 3' end of the sequencing read is determined to stop the alignment with the reference sequence; (c) for each sample, grouping the more than one sequencing reads into more than one family based on a grouping feature, the grouping feature comprising at least one of (i) the start region, (ii) the end region, and (iii) the length of the polynucleotide, wherein each family in the first sample and the second sample comprises sequencing reads of progeny polynucleotides amplified from a unique polynucleotide in the set of polynucleotides in the first sample and the second sample; (d) generating family identifiers for the more than one families; (e) screening a set of common family identifiers, wherein a given common family identifier is a family identifier of the first sample that is identical or at least 95% identical to a family identifier of the second sample; (f) determining a quantitative measure of the set of consensus family identifiers, wherein the quantitative measure comprises one or more of: the number of common family identifiers in the first sample, the ratio of the number of common family identifiers in the first sample to the total number of family identifiers in the first sample, excluding those common family identifiers in the first sample whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample, and excluding common family identifiers at over-represented genomic start position and genomic end position pairs, wherein an over-represented genomic start position and genomic end position pair refers to a genomic start position and genomic end position pair in which the number or frequency of families sharing the genomic start position and genomic end position pair in more than one sample exceeds a set threshold; and (g) classifying the first sample as being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is at or below the predetermined threshold.

78. A system comprising a controller comprising or having access to a computer readable medium containing non-transitory computer executable instructions that when executed by at least one electronic processor perform a method comprising: (a) sequencing a collection of tagged polynucleotides from a first sample and a second sample to generate more than one sequencing read, wherein each tagged polynucleotide comprises a tag and a polynucleotide; (b) aligning the more than one sequencing reads with a reference sequence, thereby determining a start region and an end region of the alignment, wherein the start region includes a genomic start position of the sequencing read, at which the 5' end of the sequencing read is determined to start the alignment with the reference sequence, and the end region includes a genomic stop position of the sequencing read, at which the 3' end of the sequencing read is determined to stop the alignment with the reference sequence; (c) for each sample, grouping the more than one sequencing reads into more than one family based on a grouping feature that includes the tag, wherein each family in the first sample and the second sample includes sequencing reads of tagged progeny polynucleotides amplified from a unique polynucleotide in the set of tagged polynucleotides in the first sample and the second sample; (d) selecting a set of common family identifiers, wherein a given common family identifier is a family identifier of a first sample that is identical or at least 95% identical to a family identifier of a second sample; (e) determining a quantitative measure of the set of consensus family identifiers, wherein the quantitative measure comprises one or more of: the number of common family identifiers in the first sample, the ratio of the number of common family identifiers in the first sample to the total number of family identifiers in the first sample, excluding those common family identifiers in the first sample whose number of sequencing reads in the family of the first sample is greater than the number of sequencing reads in the corresponding family of the second sample, and excluding common family identifiers at over-represented genomic start position and genomic end position pairs, wherein an over-represented genomic start position and genomic end position pair refers to a genomic start position and genomic end position pair in which the number or frequency of families sharing the genomic start position and genomic end position pair in more than one sample exceeds a set threshold; and (f) classifying the first sample as being contaminated by the second sample if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or classifying the first sample as not being contaminated by the second sample if the quantitative measure of the shared family identifiers is at or below the predetermined threshold.

79. A non-transitory computer-readable medium containing non-transitory computer-executable instructions that, when executed by at least one electronic processor, perform the method of any one of claims 1-75.

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