Adaptive Biotechnologies Corporation

Sequenta, Inc.

Martin Moorhead

Malek Faham

Thomas Willis

Jianbiao Zheng

Thomas Asbury

Li Weng

Tobias Wittkop

Kieran Hervold

Chitra Kotwaliwale

Stephen C Macevicz

Victoria Carlton

Francois Pepin

The invention is directed to sequence-based profiling of populations of nucleic acids by multiplex amplification and attachment of one or more sequence tags to target nucleic acids and/or copies thereof followed by high-throughput sequencing of the amplification product. In some embodiments, the invention includes successive steps of primer extension, removal of unextended primers and addition of new primers either for amplification (for example by PCR) or for additional primer extensions. Some embodiments of the invention are directed to minimal residual disease (MRD) analysis of patients being treated for cancer. Sequence tags incorporated into sequence reads provide an efficient means for determining clonotypes and at the same time provide a convenient means for detecting carry-over contamination from other samples of the same patient or from samples of a different patient which were tested in the same laboratory.

Method for genotyping clonotype profiles using sequence tags

The invention is directed to sequence-based profiling of populations of nucleic acids by multiplex amplification and attachment of one or more sequence tags to target nucleic acids anchor copies thereof followed by high-throughput sequencing of the amplification product. In some embodiments, the invention includes successive steps of primer extension, removal of unextended primers and addition of new primers either for amplification (for example by PCR) or for additional primer extension. Some embodiments of the invention are directed to minimal residual disease (MRD) analysis of patients being treated for cancer. Sequence tags incorporated into sequence reads provide an efficient means for determining clonotypes and at the same time provide a convenient means for detecting carry-over contamination from other samples of the same patient or from samples of a different patient which were tested in the same laboratory.

Method for generating clonotype profiles using sequence tags

The invention is directed to a method of selecting disease-correlated clonotypes that have a reduced likelihood of producing a false positive signal of relapse when, used to monitor minimal residual disease. In accordance with the invention, candidate correlating clonotype are obtained from a patient, the rarity of each is determined either by comparison with a clonotype database or a clonotype model, and one or more of the rarest of such clonotypes are used to monitor the minimal residual disease.

Rare clonotypes and uses thereof

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.

Sequence analysis of complex amplicons

The invention is directed to the use of sequence tags to improve sequence determination of amplicons of related sequences, particularly large and complex amplicons, such as those comprising recombined nucleic acids encoding immune receptor molecules. In one aspect, sequence reads having the same sequence tags are aligned after which final base calls are determined from a (possibly weighted) average base call from sequence read base calls at each position. Similarly, in another aspect, sequence reads comprising series of incorporation signals are aligned by common sequence tags and base calls in homopolymer regions are made as a function incorporation signal values at each 'flow' position.

Method of sequence determination using sequence tags

The invention is directed to methods for detecting and quantifying nucleic acid contamination in a tissue sample of an individual containing T cells and/or B cells, which is used for generating a sequence-based clonotype profile. In one aspect, the invention is implemented by measuring the presence and/or level of an endogenous or exogenous nucleic acid tag by which nucleic acid from an intended individual can be distinguished from that of unintended individuals. Endogenous tags include genetic identity markers, such as short tandem repeats, rare clonotypes or the like, and exogenous tags include sequence tags employed to determine clonotype sequences from sequence reads.

Detection and quantification of sample contamination in immune repertoire analysis

The invention is directed to 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 tree 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.

Method of determining clonotypes and clonotype profiles

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 dispite 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.

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San Mateo, CA, United States of America

Martin Moorhead