SEQUENCE ANALYSIS OF COMPLEX AMPLICONS

摘要

The invention is directed to methods of generating sequence profiles of populations of nucleic acids, whose member nucleic acids contain regions of high variability, such as populations of nucleic acids encoding T cell receptors or B cell receptors. In one aspect, the invention provides pluralities of sets of primers for generating nested sets of templates from nucleic acids in such populations, thereby insuring the production of at least one template from which sequence reads are generated, despite such variability, or despite limited lengths or quality of sequence reads. In another aspect, members of such populations are bidirectionally sequenced so that further sequence information is obtained by analyzing overlapping sequence reads in the zones of highest variability.

主权项

1. A method for determining a clonotype profile of T cell receptors and/or B cell receptors of an individual, the method comprising the steps of:
a) obtaining a nucleic acid sample from T-cells and/or B-cells of the individual; b) spatially isolating individual molecules derived from such nucleic acid sample, the individual molecules comprising nested sets of templates each generated from a nucleic acid in the sample and each containing a somatically rearranged region or a portion thereof, each nested set comprising a plurality of overlapping templates such that every template of the plurality has a common end and a different end and each nested set being capable of producing a plurality of sequence reads each extending in the same direction and each starting from a different position on the nucleic acid from which the nested set was generated; c) sequencing said spatially isolated individual molecules to provide a plurality of sequence reads for each nested set; d) combining information from the plurality of sequence reads from each nested set to form a clonotype; and e) determining abundances of each of the clonotypes to generate the clonotype profile.