Abstract: Techniques are provided for using a nucleosome signal pattern of fragmentation at positions around target site(s) for various purposes. For example, the nucleosome signal pattern can be used to determine a methylation level of a target site (e.g., a CpG site). Signals can be associated with nucleosomal patterns of cfDNA molecules within genomic region(s) that are differentially methylated in a target tissue type by having a different methylation level (or multiple levels, e.g., as a pattern) relative to one or more other tissue types (e.g., blood cells). The nucleosome signal pattern can be compared to one or more reference patterns having a known methylation level. Another example approach can determine a level of pathology in a subject. Another example can determine a fractional concentration of DNA of a particular tissue type.

Abstract: Fragmentation of cell-free DNA molecules is measured and used for various purposes, including determining methylation, e.g., at a particular site of a DNA molecule, at a particular genomic site in a reference genome for a biological sample (e.g., plasma, serum, urine, saliva) of cell-free DNA of a subject, or for a particular region in the reference genome for the biological sample (also just referred to as a sample). Various types of fragmentation measurements can be used, e.g., end motifs and cleavage profiles. Another purpose is determining a fractional concentration of DNA of a particular tissue type (e.g., clinically-relevant DNA). Another purpose is determining a pathology of a subject using a biological sample including cell-free DNA. The cell-free DNA can be of the subject or of a pathogen (e.g., a virus) in the subject's sample. Sites/regions that are hypermethylated, hypomethylated, 5hmC-enriched, and 5hmC-depleted for a particular tissue type can be used.

Abstract: Various applications can use fragmentation patterns related of cell-free DNA, e.g., plasma DNA and serum DNA. For example, the end positions of DNA fragments can be used for various applications. The fragmentation patterns of short and long DNA molecules can be associated with different preferred DNA end positions, referred to as size-tagged preferred ends. In another example, the fragmentation patterns relating to tissue-specific open chromatin regions were analyzed. A classification of a proportional contribution of a particular tissue type can be determined in a mixture of cell-free DNA from different tissue types. Additionally, a property of a particular tissue type can be determined, e.g., whether a sequence imbalance exists in a particular region for a tissue type or whether a pathology exists for the tissue type.

Abstract: Methods and systems described herein involve using long cell-free DNA fragments to analyze a biological sample from a pregnant subject. The status of methylated CpG sites and single nucleotide polymorphisms (SNPs) is often used to analyze DNA fragments of a biological sample. A CpG site and a SNP are typically separated from the nearest CpG site or SNP by hundreds or thousands of base pairs. Finding two or more consecutive CpG sites or SNPs on most cell-free DNA fragments is improbable or impossible. Cell-free DNA fragments longer than 600 bp may include multiple CpG sites and/or SNPs. The presence of multiple CpG sites and/or SNPs on long cell-free DNA fragments may allow for analysis than with short cell-free DNA fragments alone. The long cell-free DNA fragments can be used to identify a tissue of origin and/or to provide information on a fetus in a pregnant female.

Abstract: Size-band analysis is used to determine whether a chromosomal region exhibits a copy number aberration or an epigenetic alteration. Multiple size ranges may be analyzed instead of focusing on specific sizes. By using multiple size ranges instead of specific sizes, methods may analyze more sequence reads and may be able to determine whether a chromosomal region exhibits a copy number aberration even when clinically-relevant DNA may be a low fraction of the biological sample. Using multiple ranges may allow for the use of all sequence reads from a genomic region, rather than a selected subset of reads in the genomic region. The accuracy of analysis may be increased with higher sensitivity at similar or higher specificity. Analysis may include fewer sequencing reads to achieve the same accuracy, resulting in a more efficient process.

Abstract: Methods and systems are described herein that include using sequence reads of linear DNA molecules naturally present in a biological sample to classify a set of the linear DNA molecules that are eccDNA remnants, i.e., linear DNA molecules resulting from in vivo opening of eccDNA molecules. In various embodiments, characteristics of the classified eccDNA remnants can be analyzed to determine a property of the biological sample or of the subject from whom the biological sample was obtained. Examples of properties that can be determined include a classification of a pathology, e.g., a level of a cancer, or an inferred age of the subject.

Abstract: Cell-free DNA molecules in a mixture of a biological sample can be analyzed to detect viral DNA. Methylation of viral DNA molecules at one or more sites in the viral genome can be determined. Mixture methylation level(s) can be measured based on one or more amounts of the plurality of cell-free DNA molecules methylated at a set of site(s) of the particular viral genome. The mixture methylation level(s) can be determined in various ways, e.g., as a density of cell-free DNA molecules that are methylated at a site or across multiple sites or regions. The mixture methylation level(s) can be compared to reference methylation level(s), e.g., determined from at least two cohorts of other subjects. The cohorts can have different classifications (including the first condition) associated with the particular viral genome. A first classification of whether the subject has the first condition can be determined based on the comparing.

Abstract: Methods, systems, and apparatus are provided for determining whether a nucleic acid sequence imbalance exists within a biological sample. One or more cutoff values for determining an imbalance of, for example, the ratio of the two sequences (or sets of sequences) are chosen. The cutoff value may be determined based at least in part on the percentage of fetal DNA in a sample, such as maternal plasma, containing a background of maternal nucleic acid sequences. The percentage of fetal DNA can be calculated from the same or different data used to determine the cutoff value, and can use a locus where the mother is homozygous and the fetus is heterozygous. The cutoff value may be determined using many different types of methods, such as sequential probability ratio testing (SPRT).

Abstract: An aberration in a fetal genome can be identified by analyzing a sample of fetal and maternal DNA. Classifications of whether an aberration (amplification or deletion) exists in a subchromosomal region are determined using count-based and size-based methods. The count classification and the size classification can be used in combination to determine whether only the fetus or only the mother, or both, have the aberration in the subchromosomal region, thereby avoiding false positives when the mother has the aberration and the fetus does not.

Abstract: An aberration in a fetal genome can be identified by analyzing a sample of fetal and maternal DNA. Classifications of whether an aberration (amplification or deletion) exists in a subchromosomal region are determined using count-based and size-based methods. The count classification and the size classification can be used in combination to determine whether only the fetus or only the mother, or both, have the aberration in the subchromosomal region, thereby avoiding false positives when the mother has the aberration and the fetus does not.

Abstract: Systems, apparatus, and methods are provided for determining aberrations in a biological sample from an organism. Biological samples including cell-free DNA fragments are analyzed to identify imbalances in chromosomal regions, e.g., due to deletions and/or amplifications in a tumor. Multiple loci are used for each chromosomal region. Imbalances can be used to diagnose a patient for cancer, prognosticate a patient with cancer, or to detect the presence or monitor progress of a premalignant condition. The severity of an imbalance as well as the number of regions exhibiting an imbalance can be used. A systematic analysis of non-overlapping segments of a genome can provide a general screening tool for a sample. Additionally, a patient can be tested over time to track severity of each of one or more chromosomal regions and a number of chromosomal regions to enable screening and prognosticating, as well as monitoring of progress (e.g. after treatment).

Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

Abstract: Methods, apparatuses, and system are provided for analyzing a maternal sample to determine whether a male fetus of a pregnant female has inherited an X-linked mutation from the mother. A percentage of fetal DNA in the sample is obtained, and cutoff values for the two possibilities (fetus inherits mutant or normal allele) are determined. A proportion of mutant alleles relative to a normal allele on the X-chromosome can then be compared to the cutoff values to make a classification of which allele is inherited. Alternatively, a number of alleles from a target region on the X-chromosome can be compared to a number of alleles from a reference region on the X-chromosome to identify a deletion or amplification. The fetal DNA percentage can be computed by counting reactions with a fetal-specific allele, and correcting the number to account for a statistical distribution among the reactions.

Abstract: Techniques are provided for analyzing circular DNA in a biological sample (e.g., including cell-free DNA, such as plasma). For example, to measure circular DNA, cleaving can be performed to linearize the circular DNA so that they may be sequenced. Example cleaving techniques include restriction enzymes and transposases. Then, one or more criteria can be used to identify linearized DNA molecules, e.g., so as to differentiate from linear DNA molecules. An example criterion is mapping a pair of reversed end sequences to a reference genome. Another example criterion is identification of a cutting tag, e.g., associated with a restriction enzyme or an adapter sequence added by a transposase. Once circular DNA molecules (e.g., eccDNA and circular mitochondrial DNA) are identified, they may be analyzed (e.g., to determine a count, size profile, and/or methylation) to measure a property of the biological sample, including genetic properties and level of a disease.

Abstract: Methods and systems are described herein that include using multiplexed digital amplification reactions to measure the sizes of a plurality of nucleic acid molecules. In various embodiments, the digital amplification reactions use multiple pairs of separate forward and reverse primer pairs to amplify targeted regions of the nucleic acid molecules, where the targeted regions are separated by a specified number of base pairs. In other embodiments, the digital amplification reactions use multiple primer pairs that share a common pair to amplify targeted regions that overlap with one another. The methods and systems are particularly useful for determining the size distributions of the nucleic acid molecules, or classifying a pathology of a subject from whom the nucleic acid molecules are sampled. Advantageously, the methods and systems can measure the sizes of long nucleic acid molecules without relying on inefficient amplification of very long amplicons.

Abstract: The present disclosure describes techniques for measuring quantities (e.g., relative frequencies) of sequence end motifs of cell-free DNA fragments in a biological sample of an organism for measuring a property of the sample (e.g., fractional concentration of clinically-relevant DNA) and/or determining a condition of the organism based on such measurements. Different tissue types exhibit different patterns for the relative frequencies of the sequence end motifs. The present disclosure provides various uses for measures of the relative frequencies of sequence end motifs of cell-free DNA, e.g., in mixtures of cell-free DNA from various tissues. DNA from one of such tissue may be referred to as clinically-relevant DNA.

Abstract: Methods and systems described herein involve using long cell-free DNA fragments to analyze a biological sample from a pregnant subject. The status of methylated CpG sites and single nucleotide polymorphisms (SNPs) is often used to analyze DNA fragments of a biological sample. A CpG site and a SNP are typically separated from the nearest CpG site or SNP by hundreds or thousands of base pairs. Finding two or more consecutive CpG sites or SNPs on most cell-free DNA fragments is improbable or impossible. Cell-free DNA fragments longer than 600 bp may include multiple CpG sites and/or SNPs. The presence of multiple CpG sites and/or SNPs on long cell-free DNA fragments may allow for analysis than with short cell-free DNA fragments alone. The long cell-free DNA fragments can be used to identify a tissue of origin and/or to provide information on a fetus in a pregnant female.

Abstract: Techniques are provided for analyzing circular DNA in a biological sample (e.g., including cell-free DNA, such as plasma). For example, to measure circular DNA, cleaving can be performed to linearize the circular DNA so that they may be sequenced. Example cleaving techniques include restriction enzymes and transposases. Then, one or more criteria can be used to identify linearized DNA molecules, e.g., so as to differentiate from linear DNA molecules. An example criterion is mapping a pair of reversed end sequences to a reference genome. Another example criterion is identification of a cutting tag, e.g., associated with a restriction enzyme or an adapter sequence added by a transposase. Once circular DNA molecules (e.g., eccDNA and circular mitochondrial DNA) are identified, they may be analyzed (e.g., to determine a count, size profile, and/or methylation) to measure a property of the biological sample, including genetic properties and level of a disease.

Abstract: The contributions of different tissues to a DNA mixture are determined using methylation levels at particular genomic sites. Tissue-specific methylation levels of M tissue types can be used to deconvolve mixture methylation levels measured in the DNA mixture, to determine fraction contributions of each of the M tissue types. Various types of genomic sites can be chosen to have particular properties across tissue types and across individuals, so as to provide increased accuracy in determining contributions of the various tissue types. The fractional contributions can be used to detect abnormal contributions of a particular tissue, indicating a disease state for the tissue. A differential in fractional contributions for different sizes of DNA fragments can also be used to identify a diseased state of a particular tissue. A sequence imbalance for a particular chromosomal region can be detected in a particular tissue, e.g., identifying a location of a tumor.

Abstract: The fragmentomic feature of which strand (3? or 5?), if any, overhangs the other at one or both ends double-stranded cell-free DNA fragments can be used to analyze a biological sample. The amount of fragments with the 3? strand overhanging the 5? strand, the 5? strand overhanging the 3? strand, and/or strands being even (blunt) at one or both ends can be used to determine the type of DNA or a level of condition, including cancer and nuclease activity deficiencies. Embodiments described herein allow for determining the amount of these different end modalities, unlike prior techniques. The end modality information can be paired with end motifs to further analyze biological samples. Related systems are also described.