Chemical genomic profiling via barcode sequencing to predict compound mode of action

Abstract

Chemical genomics is an unbiased, whole-cell approach to characterizing novel compounds to determine mode of action and cellular target. Our version of this technique is built upon barcoded deletion mutants of Saccharomyces cerevisiae and has been adapted to a high-throughput methodology using next-generation sequencing. Here we describe the steps to generate a chemical genomic profile from a compound of interest, and how to use this information to predict molecular mechanism and targets of bioactive compounds.

Figure 1

Overview of the steps of chemical genomic profiling by barcode sequencing

Figure 2

PCR amplicon design and gel extraction of the amplified barcodes. The indexed primer design and components of the PCR amplicon used in sequencing (A). Using a 2 % agarose gel with Syber Safe of Ethidium Bromide, run the pooled PCR product for 45 min using a 1 kb ladder for reference and visualize under UV. Three bands will be apparent, a lower band of unused primers, the middle band at 267 bp, and an upper band of amplification artifacts. Excise the center band for gel extraction (B). The other bands contain the Illumina regions of the primers and if run on the sequencing flowcell, with for clusters but will not provide usable reads (loss of read depth).

Figure 3

Distribution of Barcodes based on pooling method. We constructed 2 pools of 300 strains using 2 different pooling methods: agar scraping and mixing of liquid cultures. We then grew the pools in our chemical genomics assay. While the pools performed similarly, we found that the liquid pool had better distribution of strains, as determined by fraction of individual strains in the pool. In this figure, a straighter line indicates a more even strain distribution.