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Method of determining clonotypes and clonotype profiles

  • US 9,043,160 B1
  • Filed: 08/02/2011
  • Issued: 05/26/2015
  • Est. Priority Date: 11/09/2009
  • Status: Active Grant
First Claim
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1. A method of determining clonotypes of an immune repertoire, said method comprising the steps:

  • 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 said T-cells and/or B-cells of said sample;

    sequencing by synthesis using reversibly terminated labeled nucleotides said spatially isolated individual molecules of recombined nucleic acids to generate a set of sequence reads, said molecules of recombined nucleic acids each having a V region, an NDN region and a J region, wherein clonotypes are formed from said set of sequence reads by;

    (a) constructing from sequence reads encompassing at least a portion of an NDN region a sequence tree having leaves representing candidate clonotypes, each leaf and its corresponding candidate clonotype having a frequency;

    (b) coalescing with a highest frequency candidate clonotype any lesser frequency candidate clonotype whenever a lesser frequency of said lesser frequency candidate clonotype 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 said highest frequency candidate clonotype and having associated sequence reads summed from said highest frequency candidate clonotype and said lesser frequency candidate clonotype;

    (c) removing leaves corresponding to said coalesced candidate clonotypes from said sequence tree; and

    (d) repeating steps (b) and (c) until a highest frequency of a lesser frequency candidate clonotype is below a predetermined stopping value, thereby determining clonotypes from said sample.

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