Computational Methods for Translating a Sequence of Multi-Base Color Calls to a Sequence of Bases

Abstract

Disclosed are systems and methods for resequencing using color calls. A DNA sample is encoded and sequenced according to a multi-base code producing a string of read color calls for a fragment of the sample. A reference sequence is obtained. The string of read color calls is mapped to the reference sequence. A base sequence is extracted from the reference sequence. The base sequence is encoded as a string of reference color codes according to the multi-base code. The string of read color calls is aligned with the string of reference color codes and mismatches in the alignment are detected. One or more mismatches of the string of read color calls are annotated as inconsistent. The one or more inconsistent mismatches of the string of read color calls are corrected. The string of corrected read color calls is decoded to bases producing a read sequence.

1 . A system for resequencing using color calls, comprising:
 a processor configured to:
 obtain a string of read color calls from the DNA sequencer, the string of read color calls encoded with a multi-base code, 
 obtain a reference sequence, 
 map the string of read color calls to the reference sequence, 
 extract a base sequence from the reference sequence, 
 encode the base sequence as a string of reference color codes according to the multi-base code, 
 align the string of read color calls with the string of reference color codes string of reference color codes and detects mismatches in the alignment; 
 annotate one or more mismatches of the string of read color calls as inconsistent, 
 correct the one or more mismatches of the string of read color calls, and 
 decode the string of read color calls to bases producing a read sequence. 
   
     
     
         2 . The system of  claim 1 , further comprising the DNA sequencer in communication with the processor, wherein the DNA sequencer is configured to interrogate two or more nucleotides at a time from a DNA sample that is encoded with color dyes according to a multi-base code and produce the string of read color calls for a fragment from the DNA sample. 
     
     
         3 . The system of  claim 1 , wherein if the mapping of the string of read color calls to the reference sequence includes an ambiguity code, the processor extracts the base sequence from the reference sequence by replacing the ambiguity code with a missing base code and encodes the base sequence as a string of reference color codes by replacing the missing base code combined with any other base code with a missing color code. 
     
     
         4 . (canceled) 
     
     
         5 . The system of  claim 1 , wherein the processor corrects the one or more mismatches of the string of read color calls by replacing each of the one or more mismatches with its corresponding color call from the string of reference color codes. 
     
     
         6 . (canceled) 
     
     
         7 . The system of  claim 1 , wherein the processor corrects the one or more mismatches of the string of read color calls by replacing each mismatch of the one or more mismatches with its corresponding color call from the string of reference color codes if the each mismatch is not adjacent to another mismatch and with the missing color call if the each mismatch is adjacent to another mismatch. 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The system of  claim 1 , wherein the processor decodes the string of read color calls to bases producing a read sequence by
 (a) selecting a last base of the primer of the string of read color calls as the first base of the read sequence,   (b) obtaining a first two color calls from the string of read color calls,   (c) pairing the first base of the primer of the string of read color calls with a first color call of the first two color calls to produce a base color pair,   (d) if the first color call of the first two color calls is a missing color call and a second color call of the first two color calls is not a missing color call, copying a corresponding reference base from the base sequence as a next base of the read sequence, otherwise determining the next base of the read sequence by using the base color pair as an input base color pair to a decode function that decodes the input base color pair to an output base defined by the multi-base code,   (e) obtaining a next two color calls from the string of read color calls,   (f) pairing the next base of the read sequence with a first color call of the next two color calls of the string of read color calls to produce the base color pair,   (g) if the first color call of the next two color calls is a missing color call and a second color call of the next two color calls is not a missing color call, copying a corresponding reference base from the base sequence as the next base of the read sequence, otherwise determining the next base of the read sequence by using the base color pair as the input base color pair to the decode function that decodes, and   (h) performing steps (e)-(h) until all color calls of the string of read color calls have been used to determine the next base.   
     
     
         13 . The system of  claim 12 , wherein the decode function decodes A0 to A, A1 to C, A2 to G, A3 to T, A. to X, C0 to C, C1 to A, C2 to T, C3 to G, C. to X, G0 to G, G1 to T, G2 to A, G3 to C, G. to X, T0 to T, T1 to G, T2 to C, T3 to A, T. to X, X0 to X, X1 to X, X2 to X, X3 to X, and X. to X. 
     
     
         14 . The system of  claim 1 , wherein the multi-base code is a dibase code. 
     
     
         15 . A method for resequencing using color calls, comprising:
 obtaining the string of read color calls from the DNA sequencer using a processor, the string of read color calls encoded according to a multi-base code;   obtaining a reference sequence using the processor;   mapping the string of read color calls to the reference sequence using the processor;   extracting a base sequence from the reference sequence using the processor;   encoding the base sequence as a string of reference color codes according to the multi-base code using the processor;   aligning the string of read color calls with the string of reference color codes and detecting mismatches in the alignment using the processor;   annotating one or more mismatches of the string of read color calls as inconsistent using the processor;   correcting the one or more mismatches of the string of read color calls using the processor; and   decoding the string of read color calls to bases producing a read sequence using the processor.   
     
     
         16 . The method of  claim 15 , further comprising interrogating two nucleotides at a time from a DNA sample that is encoded with color dyes according to the multi-base code and producing the string of read color calls for a fragment from the DNA sample using a DNA sequencer. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 15 , further comprising extracting a base sequence from the reference sequence and encoding the base sequence as a string of reference color codes when the mapping of the string of read color calls to the reference sequence includes one or more ambiguity codes by
 extracting two or more base sequences from the reference sequence by replacing the one or more ambiguity codes with different possible bases,   encoding each of the two or more base sequences according to the multi-base code producing two or more strings of base color calls,   comparing each of the two or more strings of base color calls to the string of read color calls, and   selecting a string of reference color codes that has a minimum number of mismatches with the string of read color calls as the string of reference color codes of the base sequence.   
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 15 , further comprising correcting the one or more mismatches of the string of read color calls by replacing each of the one or more mismatches with a missing color call. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 15 , further comprising correcting the one or more mismatches of the string of read color calls by
 grouping the one or more mismatches into one or more blocks of mismatches, wherein each block of the one or more blocks of mismatches includes one or more adjacent mismatches and two non-mismatches adjacent to and on either side of the one or more adjacent mismatches,   selecting a color call for correction in the each block that has a lowest quality value,   defining an addition operator based on the multi-base code,   adding the color call for correction with a sum of base color calls from a portion of the string of reference color codes corresponding to the each block and a sum of read color calls in the each block using the addition operator to produce a replacement color call, and   replacing the color call for correction with the replacement color in the each block.   
     
     
         23 . The method of  claim 22 , wherein the addition operator defines the sum of color call 0 and color call 0 as 0, color call 0 and color call 1 as 1, color call 0 and color call 2 as 2, color call 0 and color call 3 as 3, color call 1 and color call 0 as 1, color call 1 and color call 1 as 0, color call 1 and color call 2 as 3, color call 1 and color call 3 as 2, color call 2 and color call 0 as 2, color call 2 and color call 1 as 3, color call 2 and color call 2 as 0, color call 2 and color call 3 as 1, color call 3 and color call 0 as 3, color call 3 and color call 1 as 2, color call 3 and color call 2 as 1, and color call 3 and color call 3 as 0. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 15 , wherein the decode function decodes A0 to A, A1 to C, A2 to G, A3 to T, A. to X, C0 to C, C1 to A, C2 to T, C3 to G, C. to X, G0 to G, G1 to T, G2 to A, G3 to C, G. to X, T0 to T, T1 to G, T2 to C, T3 to A, T. to X, X0 to X, X1 to X, X2 to X, X3 to X, and X. to X. 
     
     
         26 . The method of  claim 15 , further comprising decoding the string of read color calls to bases producing a read sequence by
 (a) selecting a last base of the primer of the string of read color calls as the first base of the read sequence,   (b) obtaining a first two color calls from the string of read color calls,   (c) pairing the first base of the primer of the string of read color calls with a first color call of the first two color calls to produce a base color pair,   (d) if the first color call of the first two color calls is a missing color call and a second color call of the first two color calls is not a missing color call, copying a corresponding reference base from the base sequence as a next base of the read sequence, otherwise determining the next base of the read sequence by using the base color pair as an input base color pair to a decode function that decodes the input base color pair to an output base defined by the multi-base code,   (e) obtaining a next two color calls from the string of read color calls,   (f) pairing the next base of the read sequence with a first color call of the next two color calls of the string of read color calls to produce the base color pair,   (g) if the first color call of the next two color calls is a missing color call and a second color call of the next two color calls is not a missing color call, copying a corresponding reference base from the base sequence as the next base of the read sequence, otherwise determining the next base of the read sequence by using the base color pair as the input base color pair to the decode function that decodes, and   (h) performing steps (e)-(h) until all color calls of the string of read color calls have been used to determine the next base.   
     
     
         27 . The method of  claim 26 , wherein the decode function decodes A0 to A, A1 to C, A2 to G, A3 to T, A. to X, C0 to C, C1 to A, C2 to T, C3 to G, C. to X, G0 to G, G1 to T, G2 to A, G3 to C, G. to X, T0 to T, T1 to G, T2 to C, T3 to A, T. to X, X0 to X, X1 to X, X2 to X, X3 to X, and X. to X. 
     
     
         28 . The method of  claim 15 , wherein the multi-base code is a dibase code. 
     
     
         29 . A computer program product, comprising a tangible computer readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method for resequencing using color calls, comprising:
 providing a system, wherein the system comprises distinct software modules, and wherein the distinct software modules comprise a sequencing module, an extraction module, an encoding module, an alignment module, an annotation module, a correction module, and a decoding module;   obtaining a string of read color calls from a DNA sequencer that interrogates two nucleotides at a time from a DNA sample that is encoded with color dyes according to a multi-base code and that produces the string of read color calls for a fragment from the DNA sample using the sequencing module;   obtaining a reference sequence using the extraction module;   mapping the string of read color calls to the reference sequence using the extraction module;   extracting a base sequence from the reference sequence using the extraction module;   encoding the base sequence as a string of reference color codes according to the multi-base code using the encoding module;   aligning the string of read color calls with the string of reference color codes and detecting mismatches in the alignment using the alignment module;   annotating one or more mismatches of the string of read color calls as inconsistent using the annotation module;   correcting the one or more mismatches of the string of read color calls using the correction module; and   decoding the string of read color calls to bases producing a read sequence using the decoding module.   
     
     
         30 . The computer program product of  claim 29 , wherein the multi-base code is a dibase code.