Nucleic acid rearrangement and integration analysis

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.

1 .- 124 . (canceled) 
     
     
         125 . A method of analyzing a biological sample of an organism to detect a chromosomal rearrangement, the method comprising:
 identifying sequence read pairs of cell-free nucleic acid molecules from the biological sample comprising a same potential chromosomal rearrangement;   determining a strand orientation of a first sequence read and a second sequence read of each of the sequence read pairs;   filtering out a sequence read pair comprising a strand orientation of the first sequence read and the second sequence read inconsistent with a strand orientation of the first sequence read and the second sequence read of a majority of the sequence read pairs; and   after the filtering out, detecting the chromosomal rearrangement in the organism based on the sequence read pairs.   
     
     
         126 . The method of  claim 125 , wherein the organism is a human. 
     
     
         127 . The method of  claim 125 , wherein the chromosomal rearrangement comprises a chromosome translocation, chromosome deletion, chromosome inversion, or chromosome amplification. 
     
     
         128 . The method of  claim 125 , wherein the identifying the sequence read pairs comprises:
 identifying chromosomal chimeric sequence read pairs generated from paired-end sequencing of the cell-free nucleic acid molecules from the biological sample that comprise a first sequence read aligning to a first genomic region of a reference genome of the organism, and a second sequence read aligning to a second genomic region of the reference genome, thereby identifying Type A chromosomal chimeric sequence read pairs, wherein a relative positioning of the first genomic region and second genomic region in the reference genome is indicative of the potential chromosomal rearrangement;   grouping, from the Type A chromosomal chimeric sequence read pairs, Type A chromosomal chimeric sequence read pairs comprising first sequence reads that are overlapping or separated within a predetermined distance in the first genomic region, and second sequence reads that are overlapping or separated within a predetermined distance in the second genomic region, thereby identifying a candidate rearrangement region in the first genomic region and the second genomic region; and   identifying chromosomal chimeric sequence read pairs generated from paired-end sequencing of the cell-free nucleic acid molecules from the biological sample that comprise a first sequence read aligning to the candidate rearrangement region and a second sequence read comprising a first sequence aligning to the first genomic region and a second sequence aligning to the second genomic region, thereby identifying Type B chromosomal chimeric sequence read pairs.   
     
     
         129 . The method of  claim 128 , wherein the filtering out comprises: filtering out Type B chromosomal chimeric sequence read pairs that have a strand orientation of the first sequence read and the second sequence read inconsistent with the strand orientation of the first sequence read and the second sequence read of a majority of the Type A chromosomal chimeric sequence read pairs within the candidate rearrangement region. 
     
     
         130 . The method of  claim 128 , wherein a distance between the first genomic region and the second genomic region is at least 140 bases, at least 180 bases, at least 250 bases, at least 350 bases, at least 450 bases, at least 550 bases, at least 750 bases, at least 900 bases, at least 1100 bases, at least 1250 bases, at least 1800 bases, at least 2500 bases, at least 3500 bases, at least 5500 bases, at least 7500 bases, at least 9000 bases, or at least 10 4  bases. 
     
     
         131 . The method of  claim 128 , wherein a relative 5′ to 3′ relationship of the first genomic region and the second genomic region in the reference genome is opposite to a relative 5′ to 3′ relationship of the first sequence read and the second sequence read in the respective cell-free nucleic acid molecule of each of the Type A chromosomal chimeric sequence read pairs. 
     
     
         132 . A method comprising determining a classification of pathology based at least in part on the chromosomal rearrangement that is determined by the method of  claim 125 . 
     
     
         133 . A computer system comprising one or more processors and a non-transitory computer readable medium comprising instructions operable, when executed by the one or more computer processors, to cause the computer system to perform the method of  claim 125 . 
     
     
         134 . A non-transitory computer-readable medium comprising instructions operable, when executed by one or more processors of a computer system, to cause the computer system to perform the method of  claim 125 . 
     
     
         135 . A method of analyzing a biological sample of an organism to detect a chromosomal rearrangement, the method comprising:
 (a) sequencing cell-free nucleic acid fragments from a biological sample of the organism to produce paired-end sequencing reads;   (b) aligning the paired-end sequencing reads to a reference genome of the organism, wherein the aligning uses an alignment algorithm;   (c) identifying one or more chimeric read pairs, wherein a chimeric read pair comprises (i) a first read that aligns to a first genomic region of the reference genome and (ii) a second read that aligns to a second genomic region; and   (d) identifying a genomic location in the genome of the organism within 500 nucleotides of the second read of the one or more chimeric read pairs as a breakpoint position of the chromosomal rearrangement;   wherein position or orientation of the first genomic region relative to the second genomic region is inconsistent with the corresponding sequenced cell-free nucleic acid fragment.   
     
     
         136 . A method of analyzing a biological sample of an organism to detect a chromosomal rearrangement, the method comprising:
 (a) obtaining a plurality of paired-end sequencing reads for cell-free nucleic acid molecules from a biological sample of the organism;   (b) performing a first alignment of the plurality of paired end sequencing reads with a first alignment algorithm, wherein the first alignment identifies one or more Type A fragments comprising (i) a first sequence read of a read pair that aligns to a first genomic region of a reference genome of the organism, and (ii) a second sequence read of the read pair that aligns to a second genomic region of the reference genome; and further wherein position or orientation of the first genomic region relative to the second genomic region is inconsistent with the corresponding cell-free nucleic acid molecules;   (c) identifying one or more candidate rearrangement regions in the reference genome of the organism, wherein a candidate rearrangement region is identified as being within 500 bases of where one of the first sequence reads of a Type A fragment aligns;   (d) performing a second alignment of a subset of the plurality of paired end sequencing reads against the one or more candidate rearrangement regions with a second alignment algorithm, wherein the second alignment identifies one or more Type B fragments comprising (i) a first sequence read of a read pair that aligns to the reference genome, and (ii) a second sequence read comprising a first portion that aligns to the first genomic region of one of the Type A fragments and a second portion that aligns to the corresponding second genomic region of the respective Type A fragment; and   (e) identifying a chromosomal rearrangement breakpoint position at a junction formed by the first portion and the second portion of the second sequence read of one of the one or more Type B fragments.   
     
     
         137 . The method of  claim 135 , wherein step (c) further comprises identifying a plurality of different chimeric read pairs of cell-free nucleic acid molecules from the biological sample comprising a same potential chromosomal rearrangement breakpoint. 
     
     
         138 . The method of  claim 137 , wherein step (d) further comprises:
 determining a strand orientation of a first sequence read and a second sequence read of each of the plurality of different chimeric read pairs;   filtering out a chimeric read pair comprising a strand orientation of the first sequence read and the second sequence read inconsistent with a strand orientation of the first sequence read and the second sequence read of a majority of the chimeric read pairs; and   after the filtering out, detecting the breakpoint position of the chromosomal rearrangement based on the remaining chimeric read pairs.   
     
     
         139 . The method of  claim 137 , wherein step (d) further comprises assessing a variability in lengths of sequences of sequence reads of the sequence read pairs aligning to a genomic region flanking the potential chromosomal rearrangement breakpoint. 
     
     
         140 . The method of  claim 137 , wherein the identifying the plurality of different chimeric read pairs comprises:
 identifying Type A chimeric sequence read pairs generated from paired-end sequencing of the cell-free nucleic acid molecules from the biological sample that comprise (i) a first sequence read aligning to a first genomic region of the reference genome, and (ii) a second sequence read aligning to a second genomic region of the reference genome; wherein position or orientation of the first genomic region relative to the second genomic region is inconsistent with the corresponding cell-free nucleic acid fragment   grouping Type A chimeric sequence read pairs comprising first sequence reads that are overlapping or separated within a predetermined distance in the reference genome, and second sequence reads that are overlapping or separated within a predetermined distance in the reference genome, thereby identifying a candidate rearrangement region in the reference genome; and   identifying Type B chimeric sequence read pairs generated from paired-end sequencing of the cell-free nucleic acid molecules from the biological sample that comprise (i) a first sequence read aligning to the reference genome, and (ii) a second sequence read comprising a first portion aligning to the first genomic region of one of the Type A chimeric sequence read pairs and a second portion that aligns to the corresponding second genomic region of the respective Type A chimeric sequence read pair.   
     
     
         141 . The method of  claim 140 , wherein identifying the breakpoint position of the chromosomal rearrangement comprises:
 determining a strand orientation of the first sequence read and the second sequence read of each of the Type A chimeric sequence read pairs and the Type B chimeric sequence read pairs;   filtering out Type B chimeric sequence read pairs that have a strand orientation of the first sequence read and the second sequence read inconsistent with the strand orientation of the first sequence read and the second sequence read of a majority of the Type A chimeric sequence read pairs within the candidate rearrangement region; and   after the filtering out, detecting the integration breakpoint based on the Type B chimeric sequence read pairs.   
     
     
         142 . The method of  claim 136 , wherein identifying the one or more candidate rearrangement regions further comprises performing a local alignment of unmapped reads from the first alignment. 
     
     
         143 . The method of  claim 136 , further comprising sequencing the cell-free nucleic acid molecules from the biological sample to produce the paired-end sequencing reads. 
     
     
         144 . The method of  claim 142 , wherein the local alignment uses a first sequence stretch length, and the second alignment uses a second sequence stretch length that is shorter than the first sequence stretch length.