Methods and systems for analyzing nucleic acid sequences

Abstract

Methods of identifying changes in genomic DNA copy number are disclosed. This disclosure provides methods for detecting chromosomal aberrations in a subject using Hidden Markov modeling. In some cases, methods provided herein use de novo sequence assembly to detect chromosomal aberrations in a subject. The methods can be used to detect copy number changes in cancerous tissue compared to normal tissue. The methods can be used to diagnose cancer and other diseases associated with chromosomal anomalies.

1 - 25 . (canceled) 
     
     
         26 . A non-transitory computer-readable medium comprising machine executable code that, upon execution by one or more computer processors, implements a method of analyzing nucleic acid molecules from a biological sample of a subject of an organism, said method comprising:
 (a) obtaining sequence reads of the nucleic acid molecules from the biological sample of the subject;   (b) identifying a first genomic region in a reference genome of the organism with a mappability score below a threshold and a second genomic region in the reference genome of the subject with a mappability score above the threshold, wherein (i) the second genomic region is adjacent to one end of the first genomic region. (ii) the mappability score of a genomic region is expressed as the fraction of all possible reads of a specified length that map to the genomic region, and (iii) the specified length is a length of at least 10 nucleotides;   (c) performing local de novo assembly of a first subset of the sequence reads that align to the first genomic region and a second subset of the sequence reads that align to the second genomic region, thereby generating contigs, wherein at least one of the contigs extends over a portion of the first genomic region and a portion of the second genomic region;   (d) recovering sequence data corresponding to the contigs within the first genomic region, wherein the recovered genomic sequence data comprise at least a portion of the first subset of sequence read; and   (e) producing a consensus sequence for the at least one contig representing a nucleic acid corresponding to the at least one contig.   
     
     
         27 . The non-transitory computer-readable medium of  claim 26 , wherein the method further comprises identifying a third genomic region in the reference genome having a mappability score above the threshold, wherein the third genomic region is located on the other end of the first genomic region, wherein the contigs are generated by local de novo assembly of the first subset, the second subset, and the third subset of the sequence reads, and wherein at least one of the contigs extends over a portion of the first genomic region and a portion of the third genomic region. 
     
     
         28 . The non-transitory computer-readable medium of  claim 26 , wherein the reference genome comprises a plurality of genomic regions, and the method further comprises:
 (f) repeating steps (b)-(e) for each of the genomic regions having a mappability score below the threshold, thereby recovering portions of the sequence data from all of the genomic regions having mappability scores below the threshold.   
     
     
         29 . The non-transitory computer-readable medium of  claim 28 , wherein the method further comprises:
 combining the recovered sequence data from (f) with sequence data from genomic regions that have mappability scores above the threshold.   
     
     
         30 . The non-transitory computer-readable medium of  claim 29 , further comprising: normalizing the combined sequence data using a covariate of GC content. 
     
     
         31 . (canceled) 
     
     
         32 . The non-transitory computer-readable medium of  claim 26 , wherein the local de novo assembly is performed using an algorithm selected from the group consisting of a greedy algorithm assembler, graph method assembler, string graph assembler, De Bruijn graph assembler, Spades, Ray, ABySS, ALLPATHS-LG, and Trinity. 
     
     
         33 . The non-transitory computer-readable medium of  claim 26 , wherein the local de novo assembly comprises creation of a scaffold comprising the contigs, and wherein the recovered sequence data align to the scaffold. 
     
     
         34 . The non-transitory computer-readable medium of  claim 33 , wherein the sequence reads comprise paired-end sequence reads, and the scaffold is created by ordering the contigs based on paired-end information of the paired-end sequence reads. 
     
     
         35 . The non-transitory computer-readable medium of  claim 26 , wherein the recovered sequence data comprise sequence reads that align uniquely to the contigs within the first genomic region. 
     
     
         36 . (canceled) 
     
     
         37 . The non-transitory computer-readable medium of  claim 29 , wherein the method further comprises:
 segmenting the combined sequence data to identify a chromosomal aberration.   
     
     
         38 . The non-transitory computer-readable medium of  claim 37 , wherein the combined sequence data is segmented into a copy number state, the copy number state selected from the group consisting of a copy number loss state, a copy number gain state, and a copy number normal state. 
     
     
         39 . The non-transitory computer-readable medium of  claim 38 , wherein the copy number state comprises the copy number normal state, and wherein a variability of the copy number normal state is determined using at least one of a healthy reference sample and a tumor reference sample. 
     
     
         40 . The non-transitory computer-readable medium of  claim 37 , wherein the segmenting comprises using a-hidden Markov model statistical analysis. 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . The non-transitory computer-readable medium of  claim 37 , wherein said chromosomal aberration comprises a copy number aberration. 
     
     
         44 . The non-transitory computer-readable medium of  claim 43 , wherein the copy number aberration is from a tumor. 
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . (canceled) 
     
     
         48 . The non-transitory computer-readable medium of  claim 26 , wherein the mappability score of the first genomic region and the second genomic region is determined according to the following: partitioning the first genomic region into a first plurality of partitioned sequences and the second genomic region into a second plurality of partitioned sequences, and determining the mappability score of the first genomic region based, at least in part, on a first percentage of the first plurality of partitioned sequences that uniquely align to the first genomic region, and the mappability score of the second genomic region based, at least in part, on a second percentage of the second plurality of partitioned sequences that uniquely align to the second genomic region. 
     
     
         49 . (canceled) 
     
     
         50 . The non-transitory computer-readable medium of  claim 26 , wherein the nucleic acid molecules comprise cell-free nucleic acid molecules. 
     
     
         51 . A system for analyzing nucleic acid molecules from a biological sample of a subject of an organism, comprising:
 a database that stores a plurality of sequence reads generated upon sequencing said nucleic acid molecules; and   one or more computer processors operatively coupled to said database, wherein said one or more computer processors are individually or collectively programmed to execute general instructions for:
 (a) identifying a first genomic region in a reference genome of the organism with a mappability score below a threshold and a second genomic region in the reference genome of the subject with a mappability score above the threshold, wherein (i) the second genomic region is adjacent to one end of the first genomic region (ii) the mappability score of a genomic region is expressed as the fraction of all possible reads of a specified length that map to the genomic region, and (iii) the specified length is a length of at least 10 nucleotides; 
 (b) performing local de novo assembly of a first subset of the sequence reads that align to the first genomic region and a second subset of the sequence reads that align to the second genomic region, thereby generating contigs, wherein at least one of the contigs extends over a portion of the first genomic region and a portion of the second genomic region; and 
 (c) recovering sequence data corresponding to the contigs within the first genomic region, wherein the recovered genomic sequence data comprise at least a portion of the first subset of sequence read; and 
 d) producing a consensus sequence for the at least one contig representing a nucleic acid corresponding to the at least one contig. 
   
     
     
         52 . The system of  claim 51 , wherein the general instructions further comprise instructions for identifying a third genomic region in the reference genome having a mappability score above the threshold, wherein the third genomic region is located on the other end of the first genomic region, wherein the contigs are generated by local de novo assembly of the first subset, the second subset, and the third subset of the sequence reads, and wherein at least one of the contigs extends over a portion of the first genomic region and a portion of the third genomic region. 
     
     
         53 . The system of  claim 51 , wherein the reference genome comprises a plurality of genomic regions, and the general instructions further comprises instructions for:
 (e) repeating steps (a)-(d) for each of the genomic regions having a mappability score below the threshold, thereby recovering portions of the sequence data from all of the genomic regions having mappability scores below the threshold.   
     
     
         54 . The system of  claim 53 , wherein the general instructions further comprise instructions for:
 combining the recovered sequence data from (e) with sequence data from genomic regions that have mappability scores above the threshold.   
     
     
         55 . The system of  claim 54 , wherein the general instructions further comprise instructions for:
 normalizing the combined sequence data using a covariate of GC content.   
     
     
         56 . (canceled) 
     
     
         57 . The system of  claim 51 , wherein the local de novo assembly is performed using an algorithm selected from the group consisting of a greedy algorithm assembler, graph method assembler, string graph assembler, De Bruijn graph assembler, Spades, Ray, ABySS, ALLPATHS-LG, and Trinity. 
     
     
         58 . The system of  claim 51 , wherein the local de novo assembly comprises creation of a scaffold comprising the contigs, and wherein the recovered sequence data align to the scaffold. 
     
     
         59 . The system of  claim 58 , wherein the sequence reads comprise paired-end sequence reads, and the scaffold is created by ordering the contigs based on paired-end information of the paired-end sequence reads. 
     
     
         60 . The system of  claim 51 , wherein the recovered sequence data comprise sequence reads that align uniquely to the contigs within the first genomic region. 
     
     
         61 . (canceled) 
     
     
         62 . The system of  claim 54 , wherein the general instructions further comprise instructions for segmenting the combined sequence data to identify a chromosomal aberration. 
     
     
         63 . The system of  claim 62 , wherein the combined sequence data is segmented into a copy number state, the copy number state selected from the group consisting of a copy number loss state, a copy number gain state, and a copy number normal state. 
     
     
         64 . The system of  claim 63 , wherein the copy number state comprises the copy number normal state, and wherein a variability of the copy number normal state is determined using at least one of a healthy reference sample and a tumor reference sample. 
     
     
         65 . The system of  claim 62 , wherein the instructions for the segmenting comprise instructions for using hidden Markov model statistical analysis. 
     
     
         66 . (canceled) 
     
     
         67 . (canceled) 
     
     
         68 . The system of  claim 62 , wherein said chromosomal aberration comprises a copy number aberration. 
     
     
         69 . The system of  claim 68 , wherein the copy number aberration is from a tumor. 
     
     
         70 . (canceled) 
     
     
         71 . (canceled) 
     
     
         72 . The system of  claim 51 , wherein the mappability score of the first genomic region and the second genomic region is determined according to the following: partitioning the first genomic region into a first plurality of partitioned sequences and the second genomic region into a second plurality of partitioned sequences, and determining the mappability score of the first genomic region based, at least in part, on a first percentage of the first plurality of partitioned sequences that uniquely align to the first genomic region, and the mappability score of the second genomic region based, at least in part, on a second percentage of the second plurality of partitioned sequences that uniquely align to the second genomic region. 
     
     
         73 . (canceled) 
     
     
         74 . The system of  claim 51 , wherein the nucleic acid molecules comprise cell-free nucleic acid molecules.