Pooled optical screening and transcriptional measurements of cells comprising barcoded genetic perturbations

The method of 1, wherein the at least one round of FISH comprises at least one round of RNA FISH, wherein each round of RNA FISH is uniquely associated with at least one mRNA transcript from the at least one cell, wherein the at least one round of RNA FISH comprises:

• (i) contacting a plurality of mRNA transcripts comprising the at least one mRNA transcript with a plurality of 5′ loop probes, wherein each 5′ loop probe comprises a first target hybridization sequence complementary to a first portion of an mRNA transcript of the plurality of mRNA transcripts;
• (ii) contacting the plurality of mRNA transcripts comprising the at least one mRNA transcript with a plurality of 3′ probes, wherein each 3′ probe comprises a second target hybridization sequence complementary to a second portion of the mRNA transcript of the plurality of mRNA transcripts, wherein each 3′ probe is capable of specifically hybridizing with a 5′ loop probe, wherein hybridization of a 3′ probe with a corresponding 5′ loop probe forms a loop in the 5′ loop probe;
• (iii) connecting the ends of the loop in each 5′ loop probe to form a plurality of circular probes;
• (iv) amplifying a plurality of target sequences using the circular probes as templates to form a plurality of DNA amplicons;
• and
• (v) detecting the DNA amplicons by the at least one round of RNA FISH.

A method for analyzing known phenotypes or identifying new phenotypes of a plurality of cells, comprising:

• receiving a first image depicting the plurality of cells, wherein the first image indicates each cell of the plurality of cells by a boundary and associates each cell of the plurality of cells with a corresponding cell identifier;
• receiving a second image depicting locations of a plurality of barcode sequences, wherein the plurality of barcode sequences are associated with the plurality of cells after one or more in situ sequencing cycles are performed;
• aligning the first image and the second image;
• based on the alignment of the first image and the second image, identifying an association between each cell of the plurality of cells and a corresponding barcode sequence of the plurality of barcode sequences;
• receiving a FISH image of the plurality of cells after a FISH cycle is performed on the plurality of cells;
• aligning the first image and the FISH image;
• based on the alignment between the first image and the FISH image, resizing the FISH image;
• associating each cell of the plurality of cells with a portion of the resized FISH image, the corresponding barcode sequence, and the corresponding cell identifier;
• and
• analyzing known phenotypes or identifying new phenotypes of the plurality of cells.

A system for analyzing known phenotypes or identifying new phenotypes of a plurality of cells, comprising:

• one or more processors;
• a memory;
• and
• one or more programs, wherein the one or more programs are stored in the memory and configured to be executed by the one or more processors, the one or more programs including instructions for:
• receiving a first image depicting the plurality of cells, wherein the first image indicates each cell of the plurality of cells by a boundary and associates each cell of the plurality of cells with a corresponding cell identifier;
• receiving a second image depicting locations of a plurality of barcode sequences, wherein the plurality of barcode sequences are associated with the plurality of cells after one or more in situ sequencing cycles are performed;
• aligning the first image and the second image;
• based on the alignment of the first image and the second image, identifying an association between each cell of the plurality of cells and a corresponding barcode sequence of the plurality of barcode sequences;
• receiving a FISH image of the plurality of cells after a FISH cycle is performed on the plurality of cells;
• aligning the first image and the FISH image;
• based on the alignment between the first image and the FISH image, resizing the FISH image;
• associating each cell of the plurality of cells with a portion of the resized FISH image, the corresponding barcode sequence, and the corresponding cell identifier;
• and
• analyzing known phenotypes or identifying new phenotypes of the plurality of cells.