Methods and compositions for in situ analysis of V(D)J sequences

Inventors

KÜHNEMUND, Malte

Assignees

10X Genomics Inc

Abstract

The present disclosure relates in some aspects to methods for analyzing antigen receptor transcripts in a biological sample. In some aspects, nucleic acid molecules are generated from V(D)J transcripts in situ in the biological sample to enrich molecules comprising V(D)J joins. In some aspects, the presence, amount, and/or identity of a plurality of V(D)J transcripts are analyzed in situ. Also provided are oligonucleotides, sets of oligonucleotides, compositions, and kits for use in accordance with the methods.

Core Innovation

The document relates to analyzing a biological sample by generating nucleic acid molecules from an antigen receptor transcript at a location in the biological sample. Reverse transcription is performed using a polymerase having strand displacement activity and a plurality of primers that specifically target different sequences of the antigen receptor transcript, and the resulting nucleic acid molecules include a V(D)J join comprising a V segment, a J segment, and optionally a D segment between the V and J segments.

At least a nucleic acid molecule is immobilized at the location, and the nucleic acid molecule or a circularizable probe or probe set hybridized to the nucleic acid molecule is circularized to generate a circularized molecule. The circularized molecule is then used to generate a rolling circle amplification product, and a signal associated with the RCA product is detected to thereby detect the antigen receptor transcript or a sequence thereof at the location in the biological sample.

The disclosure further supports in situ detection of V(D)J information from RCA products using detectable probes, optically detectable moieties, and sequential hybridization. Signal code sequences are read by imaging to detect a corresponding antigen receptor transcript at a location, including approaches that detect a V(D)J join sequence in a unit sequence of an RCA product, with probe-region designs for V and J targeting and barcode regions on circularizable probes or probe sets for encoding and decoding.

Claims Coverage

The document includes one independent claim directed to a location-preserving workflow with five core stages: strand-displacement reverse transcription using a plurality of primers targeting different sequences of an antigen receptor transcript, immobilization of nucleic acids at the location, circularization of the nucleic acid or a hybridized circularizable probe or probe set, rolling circle amplification of the circularized molecule, and signal detection associated with the RCA product to detect the antigen receptor transcript or a sequence thereof at the location. The inventive features emphasized across dependents include primer targeting within constant-region sequences, strand-displacement constraints, circularizable probe or probe-set barcoding, in situ or matrix-embedded RCA generation, and signal-code decoding by imaging with sequential hybridization.

Overall, the claimed approach centers on strand-displacement reverse transcription with a plurality of primers that target different sequences, followed by immobilization, circularization of the nucleic acid or a circularizable probe or probe set, rolling circle amplification, and detection of an RCA-associated signal at the sample location. Dependent features further specify primer targeting to adjacent constant-region sequences with constraints on adjacent, non-overlapping binding and displacement behavior, support in situ or matrix-embedded RCA generation, add barcode regions on circularizable probes or probe sets, and refine optical detection and decoding through sequential hybridization with signal-code imaging from RCA unit-sequence copies.

Stated Advantages

Documented Applications