Size-based genomic analysis

Abstract

Systems, methods, and apparatuses for performing a prenatal diagnosis of a sequence imbalance are provided. A shift (e.g., to a smaller size distribution) can signify an imbalance in certain circumstances. For example, a size distribution of fragments of nucleic acids from an at-risk chromosome can be used to determine a fetal chromosomal aneuploidy. A size ranking of different chromosomes can be used to determine changes of a rank of an at-risk chromosome from an expected ranking. Also, a difference between a statistical size value for one chromosome can be compared to a statistical size value of another chromosome to identify a significant shift in size. A genotype and haplotype of the fetus may also be determined using a size distribution to determine whether a sequence imbalance occurs in a maternal sample relative to a genotypes or haplotype of the mother, thereby providing a genotype or haplotype of the fetus.

What is claimed is: 
     
         1 . A method for performing prenatal diagnosis of a sequence imbalance in a biological sample obtained from a female subject pregnant with a fetus, wherein the biological sample includes a mixture of cell-free DNA molecules that are part of DNA sequences of a human genome, the biological sample including DNA molecules from the fetus and the female subject, the method comprising:
 for each of a plurality of the DNA molecules in the biological sample:
 measuring a size of the DNA molecule, wherein measuring the size of the DNA molecule comprises:
 performing single-molecule sequencing of the DNA molecule to obtain a complete read of the molecule; 
 
 identifying which nucleic acid sequence in the human genome the DNA molecule is derived from, wherein identifying which DNA sequence the DNA molecule is derived from includes:
 aligning, by a computer system, a portion of the complete read to the human genome; 
 
   calculating, by the computer system, a first statistical value from the sizes of DNA molecules from a first sequence;   comparing the first statistical value to a threshold value; and   determining a classification of whether a sequence imbalance exists for the first sequence based on a comparison of the first statistical value to the threshold value.   
     
     
         2 . The method of  claim 1 , wherein the single-molecule sequencing comprises nanopore sequencing. 
     
     
         3 . The method of  claim 1 , wherein the single-molecule sequencing comprises single-molecule, real time sequencing. 
     
     
         4 . The method of  claim 1 , further comprising extracting the mixture of cell-free DNA to obtain the biological sample. 
     
     
         5 . The method of  claim 1 , wherein the threshold value is determined using a reference haplotype. 
     
     
         6 . The method of  claim 1 , wherein the threshold value is determined using a euploid sample. 
     
     
         7 . The method of  claim 1 , wherein comparing the first statistical value to the threshold value comprises:
 calculating, by the computer system, a second statistical value from the sizes of DNA molecules from one or more reference sequences; and   determining the threshold value using the second statistical value.   
     
     
         8 . The method of  claim 1 , wherein the first statistical value includes the median or average size of the measured sizes for the DNA molecules from the first sequence. 
     
     
         9 . The method of  claim 1 , wherein the first sequence is a chromosome and the sequence imbalance is a fetal chromosomal aneuploidy. 
     
     
         10 . The method of  claim 9 , wherein the threshold value is determined using one or more reference sequences, and the one or more reference sequences comprise one chromosome. 
     
     
         11 . The method of  claim 9 , wherein the threshold value is determined using one or more reference sequences, and the one or more reference sequences are a plurality of chromosomes. 
     
     
         12 . The method of  claim 1 , wherein the biological sample includes blood, plasma, serum, maternal blood containing fetal cells, fetal cells obtained from maternal blood, urine, saliva, or uterine lavage fluid. 
     
     
         13 . The method of  claim 1 , wherein the plurality of the DNA molecules includes at least one million DNA molecules. 
     
     
         14 . The method of  claim 1 , further comprising:
 collecting a blood sample from the female subject, and   extracting plasma from the blood sample to obtain the biological sample.   
     
     
         15 . The method of  claim 1 , further comprising displaying, by the computer system, the classification of whether the sequence imbalance exists for the first sequence. 
     
     
         16 . The method of  claim 1 , further comprising:
 determining a first amount of sequences identified as aligning to the first sequence of the human genome;   determining a second amount of sequences identified as aligning to one or more second sequences;   using the first amount and the second amount to determine another parameter;   comparing the other parameter to one or more second cutoff values to determine another classification of whether the sequence imbalance exists for the first sequence.   
     
     
         17 . The method of  claim 16 , further comprising:
 comparing the classification determined using the first statistical value and the threshold value to the other classification determined using the first amount and the second amount.   
     
     
         18 . The method of  claim 1 , wherein the threshold value is determined using one or more reference sequences, and the one or more reference sequences have a GC content that is similar to a GC content of the first sequence. 
     
     
         19 . The method of  claim 18 , further comprising:
 calculating the GC content of the one or more reference sequences; and   calculating the GC content of the first sequence.   
     
     
         20 . The method of  claim 18 , wherein the GC content of the one or more reference sequences and the GC content of the first sequence are obtained for a particular sequencing platform.