Determination of base modifications of nucleic acids

Abstract

Systems and methods for using determination of base modification in analyzing nucleic acid molecules and acquiring data for analysis of nucleic acid molecules are described herein. Base modifications may include methylations. Methods to determine base modifications may include using features derived from sequencing. These features may include the pulse width of an optical signal from sequencing bases, the interpulse duration of bases, and the identity of the bases. Machine learning models can be trained to detect the base modifications using these features. The relative modification or methylation levels between haplotypes may indicate a disorder. Modification or methylation statuses may also be used to detect chimeric molecules.

What is claimed is: 
     
         1 . A sequencing system for detecting a methylation of a nucleotide in a nucleic acid molecule, the methylation being a 5mC (5-methylcytosine) methylation at a CpG site, the sequencing system configured to perform single molecule, real-time sequencing, the sequencing system comprising:
 a sample holder, the sample holder configured to receive a biological sample comprising the nucleic acid molecule;   a detector configured to:
 measure pulses in an optical signal corresponding to nucleotides of the nucleic acid molecule, 
 create a data signal from the measured pulses, and 
 send the data signal to a logic system; and 
   the logic system comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control the logic system to:
 receive the data signal, 
 create an input data structure using the data signal, the input data structure comprising a window of the nucleotides sequenced in the nucleic acid molecule, wherein the window comprises five nucleotides, wherein the input data structure comprises values for the following properties:
 for each nucleotide within the window:
 an identity of the nucleotide, 
 a position of the nucleotide with respect to a target position within the respective window, 
 a width of a pulse corresponding to the nucleotide, and 
 an interpulse duration representing a time between the pulse corresponding to the nucleotide and a pulse corresponding to a neighboring nucleotide; 
 
 
 input the input data structure into a model, the model trained by:
 receiving a first plurality of first data structures, each first data structure of the first plurality of first data structures corresponding to a respective window of nucleotides sequenced in a respective nucleic acid molecule of a plurality of first nucleic acid molecules, wherein each of the first nucleic acid molecules is sequenced by measuring pulses in a signal corresponding to the nucleotides, wherein the methylation has a known first state in a nucleotide at a target position in each window of each first nucleic acid molecule, each first data structure comprising values for the same properties as the input data structure, 
 storing a plurality of first training samples, each including one of the first plurality of first data structures and a first label indicating the first state of the nucleotide at the target position, and 
 optimizing, using the plurality of first training samples, parameters of the model based on outputs of the model matching or not matching corresponding labels of the first labels when the first plurality of first data structures is input to the model, wherein an output of the model specifies whether the nucleotide at the target position in the respective window has the methylation, and 
 
 determine, using the model, whether the methylation is present in a nucleotide at the target position within the window in the input data structure. 
   
     
     
         2 . The sequencing system of  claim 1 , wherein the model is a convolutional neural network. 
     
     
         3 . The sequencing system of  claim 1 , wherein the optical signal is a fluorescence intensity signal. 
     
     
         4 . The sequencing system of  claim 1 , wherein the sample holder comprises the biological sample. 
     
     
         5 . The sequencing system of  claim 4 , wherein the sample holder further comprises a primer and a polymerase. 
     
     
         6 . The sequencing system of  claim 1 , wherein the sample holder comprises a zero-mode waveguide. 
     
     
         7 . The sequencing system of  claim 1 , wherein the window comprises five consecutive nucleotides upstream of a nucleotide at a target position within the window. 
     
     
         8 . The sequencing system of  claim 1 , wherein the window comprises five consecutive nucleotides downstream of a nucleotide at a target position within the window. 
     
     
         9 . The sequencing system of  claim 1 , wherein the window comprises 11 consecutive nucleotides on a first strand of the nucleic acid molecule. 
     
     
         10 . The sequencing system of  claim 1 , wherein the window consists of:
 5 to 10 consecutive nucleotides upstream of a nucleotide at a target position within the window, and   5 to 10 consecutive nucleotides downstream of the nucleotide at the target position within the window.   
     
     
         11 . The sequencing system of  claim 1 , wherein the window of the input data structure comprises 16 consecutive nucleotides on a first strand of the nucleic acid molecule. 
     
     
         12 . The sequencing system of  claim 1 , wherein the window of the input data structure comprises:
 six consecutive nucleotides upstream of a nucleotide at a target position within the window, and   six consecutive nucleotides downstream of the nucleotide at the target position.   
     
     
         13 . The sequencing system of  claim 1 , wherein the window of the input data structure has a different number of consecutive nucleotides upstream of the nucleotide at the target position than the number of consecutive nucleotides downstream of the nucleotide at the target position. 
     
     
         14 . The sequencing system of  claim 1 , wherein the window of the input data structure comprises:
 10 consecutive nucleotides upstream of the nucleotide at the target position, and   10 consecutive nucleotides downstream of the nucleotide at the target position.   
     
     
         15 . The sequencing system of  claim 1 , wherein the window of the input data structure comprises:
 21 consecutive nucleotides upstream of the nucleotide at the target position, and   21 consecutive nucleotides downstream of the nucleotide at the target position.   
     
     
         16 . The sequencing system of  claim 1 , wherein the identity of the nucleotide, the width of the pulse corresponding to the nucleotide, and the interpulse duration for each nucleotide within the window of the input data structure are determined by aligning subreads to a circular consensus sequence and without alignment of the subreads to a reference genome. 
     
     
         17 . The sequencing system of  claim 1 , wherein the optical signal is a fluorescence signal from a dye-labeled nucleotide. 
     
     
         18 . A sequencing system for detecting a methylation of a nucleotide in a nucleic acid molecule, the methylation being a 5mC (5-methylcytosine) methylation at a CpG site, the sequencing system configured to perform single molecule, real-time sequencing, the sequencing system comprising:
 a sample holder, the sample holder configured to receive a biological sample comprising the nucleic acid molecule;   a detector configured to:
 measure pulses in an optical signal corresponding to nucleotides of the nucleic acid molecule, 
 create a data signal from the measured pulses, and 
 send the data signal to a logic system; and 
   the logic system comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control the logic system to:
 receive the data signal, 
 create an input data structure using the data signal, the input data structure comprising a window of the nucleotides sequenced in the nucleic acid molecule, wherein the window comprises five nucleotides, wherein the input data structure comprises values for the following properties:
 for each nucleotide within the window:
 an identity of the nucleotide, 
 a position of the nucleotide with respect to a target position within the respective window, 
 a width of the pulse corresponding to the nucleotide, and 
 an interpulse duration representing a time between the pulse corresponding to the nucleotide and a pulse corresponding to a neighboring nucleotide; and 
 
 
 determine whether the methylation is present in a nucleotide at the target position within the window using:
 for each nucleotide within the window:
 the identity of the nucleotide, 
 the position of the nucleotide with respect to the target position, 
 the width of the pulse corresponding to the nucleotide, and 
 the interpulse duration.