Method for analyzing the ability of target nucleic acid sequences to impact gene expression

A method for analyzing an ability of target nucleic acid sequences to impact gene expression, the method comprising:

• cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids;
• sequencing the plurality of plasmids to provide long-read sequencing information based on a target nucleic acid sequence of the target nucleic acid sequences and an associated barcode nucleic acid sequence within a plasmid of the plurality of plasmids;
• associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the long-read sequencing information;
• transducing the plurality of plasmids into a plurality of cells;
• extracting DNA, total mRNA, and polysome-bound mRNA from the plurality of cells;
• sequencing the barcode nucleic acid sequences in the extracted DNA, total mRNA, and polysome-bound mRNA to provide short-read sequencing information;
• analyzing the target nucleic acid sequences by comparing the long-read sequencing information and the short-read sequencing information;
• determining a number of target nucleic sequences, a number of RNA molecules translated from the target nucleic acid sequences, and a number of polysome-bound mRNA molecules from the long-read nucleic acid sequencing information and the short-read sequencing information;
• quantitating mRNA transcript levels by determining a ratio of the number of RNA molecules translated from the target nucleic acid sequences to the number of target nucleic sequences;
• and
• comparing mRNA transcript levels of a wild-type target nucleic acid sequence to mRNA transcript levels of a mutant target nucleic acid sequence

A method for analyzing an ability of target nucleic acid sequences to impact gene expression, the method comprising:

• cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids;
• sequencing the plurality of plasmids to provide long-read sequencing information based on a target nucleic acid sequence of the target nucleic acid sequences and an associated barcode nucleic acid sequence within a plasmid of the plurality of plasmids;
• associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the long-read sequencing information;
• transducing the plurality of plasmids into a plurality of cells;
• extracting DNA, total mRNA, and polysome-bound mRNA from the plurality of cells;
• sequencing the barcode nucleic acid sequences in the extracted DNA, total mRNA, and polysome-bound mRNA to provide short-read sequencing information;
• analyzing the target nucleic acid sequences by comparing the long-read sequencing information and the short-read sequencing information;
• determining a number of target nucleic sequences, a number of RNA molecules translated from the target nucleic acid sequences, and a number of polysome-bound mRNA molecules from the long-read nucleic acid sequencing information and the short-read sequencing information;
• quantitating mRNA translation levels by determining a ratio of the number of polysome-bound mRNA molecules to the number of RNA molecules translated from the target nucleic acid sequences;
• and
• comparing mRNA translation levels of a mutant target nucleic acid sequence to mRNA translation levels of a wild-type target nucleic acid sequence.