Methods for assessing sample quality prior to spatial analysis using templated ligation

The method of any one of the preceding claims, wherein the plurality of nucleotides comprise one or more of a dATP, a dTTP, a dGTP, a dUTP, and a dCTP.

The method of any one of the preceding claims, wherein the labelled nucleotides comprise one or more of a labelled dATP, a labelled dTTP, a labeled dUTP, a labelled dGTP, or a labelled dCTP.

The method of any one of the preceding claims, wherein labelled nucleotides comprise a label selected from the group consisting of:

• a radiolabel, a fluorescent label, a chemiluminescent label, a bioluminescent label, a calorimetric label, and a colorimetric label.

The method of claim 16, wherein the fluorescent label comprises a fluorophore selected from the group consisting of:

• Cy3, Cy5, Cy5.5, Cy7, TAMRA, 5-ROX, TYE 653, HEX, TEX 615, TYE 665, and TYE 705.

The method of any one of the preceding claims, wherein the target nucleic acid comprises RNA.

The method of any one of the preceding claims, wherein the first probe comprises a sequence that is at least 80% identical to SEQ ID NO:

• 1.

The method of any one of the preceding claims, wherein the second probe comprises a sequence that is at least 80% identical to SEQ ID NO:

• 2.

The method of any one of the preceding claims, wherein the first probe further comprises a functional sequence, wherein the functional sequence is a primer sequence.

The method of any one of the preceding claims, wherein the biological sample is contacted with the first probe and the second probe at a total concentration of about 25 nM to about 2500 nM.

The method of any one of the preceding claims, wherein the first probe and/or the second probe is a DNA probe.

The method of any one of the preceding claims, further comprising contacting the biological sample with one or more additional probe pairs, wherein a probe pair comprises an additional first probe and an additional second probe.

The method of any one of the preceding claims, wherein generating a ligation product comprises ligating the first probe to the second probe, wherein the enzymatic ligation utilizes a ligase.

The method of any one of the preceding claims, further comprising providing a capture probe capture domain blocking moiety that interacts with the capture probe capture domain.

The method of any one of the preceding claims, wherein the capture probe capture domain comprises a poly-adenylated (poly(A)) sequence or a complement thereof.

The method of any one of the preceding claims, wherein the capture probe capture domain comprises a sequence that is complementary to all or a portion of the capture domain of the capture probe.

The method of any one of the preceding claims, wherein the capture probe capture domain blocking moiety is a DNA probe.

The method of any one of the preceding claims, further comprising removing the biological sample from the substrate.

The method of any one of the preceding claims, wherein the signal corresponding to the bound ligation product comprises the signal from the labeled dNTPs.

The method of any one of the preceding claims, wherein the detecting step comprises obtaining an image corresponding to the signal corresponding to the bound ligation product on the substrate.

The method of any one of the preceding claims, further comprising analyzing a signal corresponding to the bound ligation product on the substrate.

The method of any one of the preceding claims, further comprising identifying, based on the signal analysis, the quality of the biological sample.

The method of any one of the preceding claims, wherein the biological sample is a tissue sample.

The method of any one of the preceding claims, wherein the biological sample was previously stained.

The method of any one of the preceding claims, further comprising selecting, based on the detected signal, a second tissue section of the biological sample for the detection of one or more additional target nucleic acids.

The method of any one of the preceding claims, further comprising analyzing the second portion of the biological sample for the detection of one or more additional target nucleic acids, wherein analyzing the second portion comprises determining a location and abundance of one or more target nucleic acids in the second portion of the biological sample.

The method of any one of the preceding claims, further comprising a releasing step that comprises removing the ligation product from the nucleic acid prior to hybridizing the ligation product to the capture domain.