Systems and methods for mitochondrial analysis

Abstract

The invention provides methods of analyzing an individual's mtDNA by transforming available reference sequences into a directed graph that compactly represents all the information without duplication and comparing sequence reads from the mtDNA to the graph to identify the individual or describe their mtDNA. A directed graph can represent all of the genetic variation found among the mitochondrial genomes across all of a number of reference organisms while providing a single article to which sequence reads can be aligned or compared. Thus any sequence read or other sequence fragment can be compared, in a single operation, to the article that represents all of the reference mitochondrial sequences.

1 - 20 . (canceled) 
     
     
         21 . A system, comprising:
 at least one processor, and   at least one non-transitory memory storing processor-executable instructions that, when executed by the at least one processor, cause the at least one processor to perform a method for analyzing a mitochondrial genome comprising a plurality of mitochondrial sequences, the method comprising:
 generating, in at least one non-transitory memory, a mitochondrial DNA (mtDNA) reference graph representing at least some of the plurality of mitochondrial sequences, the mtDNA reference graph comprising nodes and edges connecting the nodes, the nodes including a first node and a second node, wherein:
 the first node is stored as a first object in the at least one non-transitory memory, 
 the second node is stored as a second object in the at least one non-transitory memory, and 
 a first edge of the edges is stored as a first pointer from the first object to the second object in the at least one non-transitory memory, 
 
 obtaining a plurality of sequence reads from a biological sample previously obtained from a subject; 
 aligning one or more sequence reads of the plurality of sequence reads to the mtDNA reference graph in the at least one non-transitory memory at least in part by determining alignment scores between the one or more sequence reads and symbol strings associated with the nodes in the mtDNA reference graph, the symbol strings representing sequences of one or more nucleotides of the mitochondrial genome; and 
 identifying, based on results of the aligning, one or more mitochondrial sequences of the plurality of mitochondrial sequences to which the one or more sequence reads align. 
   
     
     
         22 . The system of  claim 21 , further comprising identifying mitochondrial heteroplasmy in the subject based on the identified one or more mitochondrial sequences to which the one or more sequence reads align. 
     
     
         23 . The system of  claim 21 , wherein the plurality of sequence reads correspond to at least a portion of a D-loop of mitochondria of the subject. 
     
     
         24 . The system of  claim 21 , wherein the plurality of mitochondrial sequences is obtained from relatives of the subject. 
     
     
         25 . The system of  claim 21 , wherein the subject is an unknown subject, and wherein the method further comprises:
 determining an identity of the unknown subject based on the identified one or more mitochondrial sequences to which the one or more sequence reads align.   
     
     
         26 . The system of  claim 25 , wherein the mtDNA reference graph represents variations in a mitochondrial genome of a maternal-line individual related to the unknown subject, variations in a hyper-variable region of the mitochondrial genome, or a combination thereof. 
     
     
         27 . The system of  claim 21 ,
 wherein the first object comprises a first symbol string representing a first sequence of one or more nucleotides of the mitochondrial genome,   wherein the second object comprises a second symbol string representing a second sequence of one or more nucleotides of the mitochondrial genome, and   wherein determining an alignment score for the second node comprises, for a first symbol in the second symbol string associated with the second node, determining the alignment score for the second node based on an alignment score associated with the first node.   
     
     
         28 . The system of  claim 27 , wherein aligning a second read of the one or more sequence reads against the mtDNA reference graph comprises:
 determining a first alignment score between a portion of the sequence read and a portion of the mtDNA reference graph preceding and including a symbol in the second symbol string.   
     
     
         29 . The system of  claim 28 , wherein determining the first alignment score between the portion of the sequence read and the portion of the mtDNA reference graph preceding and including the symbol in the second symbol string comprises:
 determining the first alignment score based on an alignment score associated with the first node, if and only if the symbol comprises the first symbol of the second symbol string.   
     
     
         30 . The system of  claim 21 ,
 wherein the nodes of the mtDNA reference graph further comprise a third node,   wherein the third node is stored as a third object in the at least one non-transitory memory, and   wherein a second edge of the edges is stored as a second pointer from the third object to the second object in the at least one non-transitory memory.   
     
     
         31 . The system of  claim 21 , wherein the mtDNA reference graph is a directed acyclic graph (DAG). 
     
     
         32 . At least one non-transitory memory storing processor-executable instructions that, when executed by at least one processor, cause the at least one processor to perform a method for analyzing a mitochondrial genome comprising a plurality of mitochondrial sequences, the method comprising:
 generating, in at least one non-transitory memory, a mitochondrial DNA (mtDNA) reference graph representing at least some of the plurality of mitochondrial sequences, the mtDNA reference graph comprising nodes and edges connecting the nodes, the nodes including a first node and a second node, wherein:
 the first node is stored as a first object in the at least one non-transitory memory, 
 the second node is stored as a second object in the at least one non-transitory memory, and 
 a first edge of the edges is stored as a first pointer from the first object to the second object in the at least one non-transitory memory, 
   obtaining a plurality of sequence reads from a biological sample previously obtained from a subject;   aligning one or more sequence reads of the plurality of sequence reads to the mtDNA reference graph in the at least one non-transitory memory at least in part by determining alignment scores between the one or more sequence reads and symbol strings associated with the nodes in the mtDNA reference graph, the symbol strings representing sequences of one or more nucleotides of the mitochondrial genome; and   identifying, based on results of the aligning, one or more mitochondrial sequences of the plurality of mitochondrial sequences to which the one or more sequence reads align.   
     
     
         33 . The at least one non-transitory memory of  claim 32 , further comprising identifying mitochondrial heteroplasmy in the subject based on the identified one or more mitochondrial sequences to which the one or more sequence reads align. 
     
     
         34 . The at least one non-transitory memory of  claim 32 , wherein the plurality of sequence reads correspond to at least a portion of a D-loop of mitochondria of the subject. 
     
     
         35 . The at least one non-transitory memory of  claim 32 , wherein the plurality of mitochondrial sequences is obtained from relatives of the subject. 
     
     
         36 . The at least one non-transitory memory of  claim 32 , wherein the subject is an unknown subject, and wherein the method further comprises:
 determining an identity of the unknown subject based on the identified one or more mitochondrial sequences to which the one or more sequence reads align.   
     
     
         37 . The at least one non-transitory memory of  claim 36 , wherein the mtDNA reference graph represents variations in a mitochondrial genome of a maternal-line individual related to the unknown subject, variations in a hyper-variable region of the mitochondrial genome, or a combination thereof. 
     
     
         38 . The at least one non-transitory memory of  claim 32 ,
 wherein the first object comprises a first symbol string representing a first sequence of one or more nucleotides of the mitochondrial genome,   wherein the second object comprises a second symbol string representing a second sequence of one or more nucleotides of the mitochondrial genome, and   wherein determining an alignment score for the second node comprises, for a first symbol in the second symbol string associated with the second node, determining the alignment score for the second node based on an alignment score associated with the first node.   
     
     
         39 . The at least one non-transitory memory of  claim 38 , wherein aligning a second read of the one or more sequence reads against the mtDNA reference graph comprises:
 determining a first alignment score between a portion of the sequence read and a portion of the mtDNA reference graph preceding and including a symbol in the second symbol string.   
     
     
         40 . The at least one non-transitory memory of  claim 39 , wherein determining the first alignment score between the portion of the sequence read and the portion of the mtDNA reference graph preceding and including the symbol in the second symbol string comprises:
 determining the first alignment score based on an alignment score associated with the first node, if and only if the symbol comprises the first symbol of the second symbol string.