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: Systems, apparatuses, and method are provided for determining the contributions of different tissues to a biological sample that includes a mixture of cell-free DNA molecules from various tissues types, e.g., as occurs in plasma or serum and other body fluids. Embodiments can analyze the methylation patterns of the DNA mixture (e.g., methylation levels at particular loci) for a particular haplotype and determine fractional contributions of various tissue types to the DNA mixture, e.g., of fetal tissue types or tissue types of specific organs that might have a tumor. Such fractional contributions determined for a haplotype can be used in a variety of ways.

Abstract: Disclosed herein are methods, systems, and apparatus for detecting microamplifications or microdeletions in the genome of a fetus. In some embodiments, the method comprises receiving sequence tags for each of a plurality of DNA fragments in a biological sample; determining genomic positions for the sequence tags; determining whether the density of DNA in each of a plurality of genomic regions is aberrantly high or low; identifying as a microamplification a set of consecutive genomic regions having aberrantly high density; and identifying as a microdeletion a set of consecutive genomic regions having aberrantly low density. The biological sample may be a blood sample obtained noninvasively from a female subject pregnant with the fetus.

Abstract: Disclosed herein are methods, systems, and apparatus for detecting microamplifications or microdeletions in the genome of a fetus. In some embodiments, the method comprises receiving sequence tags for each of a plurality of DNA fragments in a biological sample; determining genomic positions for the sequence tags; determining whether the density of DNA in each of a plurality of genomic regions is aberrantly high or low; identifying as a microamplification a set of consecutive genomic regions having aberrantly high density; and identifying as a microdeletion a set of consecutive genomic regions having aberrantly low density. The biological sample may be a blood sample obtained noninvasively from a female subject pregnant with the fetus.

Abstract: Various embodiments are directed to applications (e.g., classification of biological samples) of the analysis of the count, the fragmentation patterns, and size of cell-free nucleic acids, e.g., plasma DNA and serum DNA, including nucleic acids from pathogens, such as viruses. Embodiments of one application can determine if a subject has a particular condition. For example, a method of present disclosure can determine if a subject has cancer or a tumor, or other pathology. Embodiments of another application can be used to assess the stage of a condition, or the progression of a condition over time. For example, a method of the present disclosure may be used to determine a stage of cancer in a subject, or the progression of cancer in a subject over time (e.g., using samples obtained from a subject at different times).

Abstract: Methods, systems, and apparatus determine whether a first chromosomal region exhibits a deletion or an amplification associated with cancer in a sample from a subject (e.g., where the sample includes a mixture of cell-free DNA from tumor cells and non-malignant cells. Nucleic acid molecules of the biological sample are sequenced. Respective amounts of a clinically-relevant chromosomal region and of background chromosomal region(s) are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether first chromosomal region exhibits a deletion or an amplification associated with cancer.

Abstract: Whether a fetus has an aneuploidy associated with a first chromosome is detected using ratios of alleles detected in a maternal sample having a mixture of maternal and fetal DNA. DNA from the sample is enriched for target regions associated with polymorphic loci and then sequenced. Polymorphic loci (e.g., single nucleotide polymorphisms) in the target regions with fetal-specific alleles are identified on a first chromosome and on one or more reference chromosomes. A first ratio of the fetal-specific alleles and shared alleles is determined for the loci on the first chromosome. A second ratio of the fetal-specific alleles and shared alleles is determined for the loci on the reference chromosome(s). A third ratio of the first and second ratio can be compared to a cutoff to determine whether an aneuploidy is present, and whether the aneuploidy is maternally-derived or paternally-derived.

Abstract: Cell-free DNA fragments often include jagged ends, where one end of one strand of double-stranded DNA extends beyond the other end of the other strand. The length and amount of these jagged ends may be used to determine a level of a condition of an individual, a fractional concentration of clinically-relevant DNA in a biological sample, an age of individual, or a tissue type exhibiting cancer. The jagged end length and amount may be determined using various techniques described herein.

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: Embodiments of the present invention provide methods, systems, and apparatus for deducing the fetal DNA fraction in maternal plasma without using paternal or fetal genotypes. Maternal genotype information may be obtained from a maternal-only DNA sample or may be assumed from shallow-depth sequencing of a biological sample having both maternal and fetal DNA molecules. Because sequencing may be at shallow depths, a locus may have only few reads and may fail to exhibit a non-maternal allele even if a non-maternal allele is present. However, normalized parameters that characterize non-maternal alleles sequenced can be used to provide an accurate estimate of the fetal DNA fraction, even if the amount of non-maternal alleles is in error. Methods described herein may not need high-depth sequencing or enrichment of specific regions. As a result, these methods can be integrated into widely used non-invasive prenatal testing and other diagnostics.

Abstract: Factors affecting the fragmentation pattern of cell-free DNA (e.g., plasma DNA) and the applications, including those in molecular diagnostics, of the analysis of cell-free DNA fragmentation patterns are described. Various applications can use a property of a fragmentation pattern to determine a proportional contribution of a particular tissue type, to determine a genotype of a particular tissue type (e.g., fetal tissue in a maternal sample or tumor tissue in a sample from a cancer patient), and/or to identify preferred ending positions for a particular tissue type, which may then be used to determine a proportional contribution of a particular tissue type.

Abstract: Factors affecting the fragmentation pattern of cell-free DNA (e.g., plasma DNA) and the applications, including those in molecular diagnostics, of the analysis of cell-free DNA fragmentation patterns are described. Various applications can use a property of a fragmentation pattern to determine a proportional contribution of a particular tissue type, to determine a genotype of a particular tissue type (e.g., fetal tissue in a maternal sample or tumor tissue in a sample from a cancer patient), and/or to identify preferred ending positions for a particular tissue type, which may then be used to determine a proportional contribution of a particular tissue type.

Abstract: Provided herein are methods and systems for identifying chimeric nucleic acid fragments, e.g., organism-pathogen chimeric nucleic acid fragments and chromosomal rearrangement chimeric nucleic acid fragments. Also provided herein are methods and systems relating to determining a pathogen integration profile or a chromosomal rearrangement in a biological sample and determining a classification of pathology based at least in part on a pathogen integration profile or a chromosomal rearrangement in a biological sample. In certain aspects of the present disclosure, cell-free nucleic acid molecules from a biological sample are analyzed.

Abstract: Factors affecting the fragmentation pattern of cell-free DNA (e.g., plasma DNA) and the applications, including those in molecular diagnostics, of the analysis of cell-free DNA fragmentation patterns are described. Various applications can use a property of a fragmentation pattern to determine a proportional contribution of a particular tissue type, to determine a genotype of a particular tissue type (e.g., fetal tissue in a maternal sample or tumor tissue in a sample from a cancer patient), and/or to identify preferred ending positions for a particular tissue type, which may then be used to determine a proportional contribution of a particular tissue type.

Abstract: Provided herein are compositions comprising tissue-specific markers for identifying a tissue of origin of a cell-free nucleic acid, e.g., a cell-free DNA molecule. Also provided herein are methods, compositions, and systems for identifying a tissue of origin of a cell-free nucleic acid by determining an absolute amount of cell-free nucleic acids comprising the tissue-specific marker. Also provided herein are methods, compositions, and systems for detecting a cancer in a tissue of an organism by analyzing tissue-specific markers.

Abstract: Whether a fetus has an aneuploidy associated with a first chromosome is detected using ratios of alleles detected in a maternal sample having a mixture of maternal and fetal DNA. DNA from the sample is enriched for target regions associated with polymorphic loci and then sequenced. Polymorphic loci (e.g., single nucleotide polymorphisms) in the target regions with fetal-specific alleles are identified on a first chromosome and on one or more reference chromosomes. A first ratio of the fetal-specific alleles and shared alleles is determined for the loci on the first chromosome. A second ratio of the fetal-specific alleles and shared alleles is determined for the loci on the reference chromosome(s). A third ratio of the first and second ratio can be compared to a cutoff to determine whether an aneuploidy is present, and whether the aneuploidy is maternally-derived or paternally-derived.

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: Embodiments of the present invention provide methods, systems, and apparatus for deducing the fetal DNA fraction in maternal plasma without using paternal or fetal genotypes. Maternal genotype information may be obtained from a maternal-only DNA sample or may be assumed from shallow-depth sequencing of a biological sample having both maternal and fetal DNA molecules. Because sequencing may be at shallow depths, a locus may have only few reads and may fail to exhibit a non-maternal allele even if a non-maternal allele is present. However, normalized parameters that characterize non-maternal alleles sequenced can be used to provide an accurate estimate of the fetal DNA fraction, even if the amount of non-maternal alleles is in error. Methods described herein may not need high-depth sequencing or enrichment of specific regions. As a result, these methods can be integrated into widely used non-invasive prenatal testing and other diagnostics.

Abstract: Factors affecting the fragmentation pattern of cell-free DNA (e.g., plasma DNA) and the applications, including those in molecular diagnostics, of the analysis of cell-free DNA fragmentation patterns are described. Various applications can use a property of a fragmentation pattern to determine a proportional contribution of a particular tissue type, to determine a genotype of a particular tissue type (e.g., fetal tissue in a maternal sample or tumor tissue in a sample from a cancer patient), and/or to identify preferred ending positions for a particular tissue type, which may then be used to determine a proportional contribution of a particular tissue type.

Abstract: Analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for solid tumor assessment or cancer screening. However, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. Regarding the size profile of plasma DNA molecules, some studies reported increased integrity of tumor-derived plasma DNA while others reported shorter tumor-derived plasma DNA molecules. We performed an analysis of the size profiles of plasma DNA in patients with cancer using massively parallel sequencing at single base resolution and in a genomewide manner. Tumor-derived plasma DNA molecules were further identified using chromosome arm-level z-score analysis (CAZA). We showed that populations of aberrantly short and long DNA molecules co-existed in the plasma of patients with cancer. The short ones preferentially carried the tumor-associated copy number aberrations. These results show the ability to use plasma DNA as a molecular diagnostic tool.