Method of determining clonotypes and clonotype profiles

Abstract

The invention is directed so methods for determining clonotypes and clonotype profiles in assays for analyzing immune repertoires by high throughput nucleic acid sequencing of somatically recombined immune molecules. In one aspect the invention comprises generating a clonotype profile from an individual by generating sequence reads from a sample of recombined immune molecules; forming from the sequence reads a sequence free representing candidate clonotypes each having a frequency; coalescing with a highest frequency candidate clonotype any lesser frequency candidate clonotypes whenever such lesser frequency is below a predetermined value and whenever a sequence difference therebetween is below a predetermined value to form a clonotype. After such coalescence, the candidate clonotypes is removed from the sequence tree and the process is repeated. This approach permits rapid and efficient differentiation of candidate clonotypes with genuine sequence differences from those with experimental or measurement errors, such as sequencing errors.

1 - 29 . (canceled) 
     
     
         30 . A method for forming a clonotype profile comprising the steps of:
 obtaining from a subject a sample comprising T-cells and/or B-cells;   spatially isolating on a solid surface individual molecules of recombined nucleic acids encoding T cell receptor molecules or immunoglobulin molecules from the T-cells and/or B-cells of the sample;   sequencing the spatially isolated individual molecules of recombined nucleic acids to generate a set of sequence reads, the molecules of recombined nucleic acid each having a V region, an NDN region and a J region, wherein clonotypes are formed from the set of sequence reads by:   (a) constructing from sequence reads encompassing at least a portion of an NDN region a data structure having elements representing candidate clonotypes, each element and its corresponding candidate clonotype having a frequency,   (b) coalescing with a highest frequency candidate clonotype any lesser frequency candidate clonotypes whenever such lesser frequency is below a predetermined frequency value and a sequence difference therebetween is below a predetermined difference value to form a clonotype having a sequence of the highest frequency candidate clonotype and having associated sequence reads summed from the highest frequency candidate clonotype and the lesser frequency candidate clonotype,   (c) removing the coalesced candidate clonotype from the data structure or thereafter disregarding the coalesced candidate clonotype in the data structure, and   (d) repeating steps (b) and (c) until a clonotype profile is formed.   
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 30  wherein said predetermined difference value is a predetermined Hamming distance. 
     
     
         33 . The method of  claim 30  wherein said sequence difference between said highest frequency candidate clonotype and said lesser frequency candidate clonotype comprises differences at one or more nucleotide locations, and wherein said highest frequency candidate clonotype is coalesced with any said lesser frequency candidate clonotypes to form said clonotype whenever a function depending on frequencies of said highest frequency candidate clonotype, said lesser frequency candidate clonotype, said sequence difference and quality scores of the one or more nucleotide locations is greater than a predetermined coalescence value, wherein the function (i) monotonically increases with increasing ratio of frequency of said highest frequency candidate clonotype and frequency of said lesser frequency candidate clonotype, (ii) monotonically decreases with increasing sequence difference between said highest frequency candidate clonotype and said lesser frequency candidate clonotype, and (iii) monotonically decreases with increasing quality scores of the one or more nucleotide locations. 
     
     
         34 . The method of  claim 33  wherein said function monotonically decreases with an average of quality scores of said one or more nucleotide locations. 
     
     
         35 . The method of  claim 33  wherein said sequence difference includes differences due to nucleotide substitutions, insertions and deletions. 
     
     
         36 . The method of  claim 30  further comprising a step of amplifying said molecules of recombined nucleic acids from said sample. 
     
     
         37 . The method of  claim 36  wherein said sample comprises at least 10,000 T cells and wherein said recombined nucleic acids encode a TCRβ chain or a portion thereof. 
     
     
         38 . The method of  claim 36  wherein said sample comprises at least 10,000 B cells and wherein said recombined nucleic acids encode an IgH chain or a portion thereof. 
     
     
         39 . The method of  claim 36  wherein said sequence reads each have a length in a range of from 20 to 400 nucleotides. 
     
     
         40 . The method of  claim 36  wherein said sample is a blood sample. 
     
     
         41 . The method of  claim 36  wherein said sequence reads each have a length less than 300 bp. 
     
     
         42 . The method of  claim 30  wherein said steps of (b) and (c) are repeated until clonotypes have been constructed from all non-singleton lesser frequency candidate clonotypes, thereby generating said clonotype profile. 
     
     
         43 . The method of  claim 30  wherein said steps of (b) and (c) are repeated until a highest frequency of a lesser frequency candidate clonotype is below a predetermined stopping value.