Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

Summary

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

Abstract

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Introduction

One approach to discover protein interactions is the yeast 2-hybrid (Y2H) assay, which exploits engineered yeast cells that grow only when a protein of interest binds to a fragment of an interacting partner¹. Detection of multiple Y2H interactions can now be done with the help of massive parallel high-throughput sequencing. Several formats have been described^2,3,4,5 including one that we developed where populations are grown in batch under conditions that select for yeast containing plasmids that produce a positive Y2....

Protocol

1. Mapping Fastq Files

NOTE: DEEPN software as well as many bioinformatics programs use DNA sequence data wherein each sequence read has been mapped for its position in reference DNA. A variety of mapping programs can be used for this including the MAPster interface here that uses the HISTAT2 program to produce .sam files used in subsequent steps.

1. Map the sequence data to the correct version of the genome. For Y2H libraries of mouse origin, use the UCSC mm10 genome; for those using human genes, use the UCSC hg38 reference genome, for Saccharomyces cerevisiae genes, use the UCSC SacCer3 reference genome.

Results

Mapping fastq data: the first step
In practically all NGS applications including DEEPN the initial output is a file of short sequence reads that must be mapped by alignment to genomic, transcriptomic, or other reference DNA⁸. Recently, the HISAT2 alignment program was developed that uses state-of-the-art indexing algorithms to dramatically increase the mapping speed^7,9. HISAT2 runs effi.......

Discussion

The software suite described here allows one to completely process and analyze high throughput DNA sequencing data from a DEEPN experiment. The first program used is MAPster, which takes the DNA sequence reads in standard fastq files and maps their position onto a reference DNA for downstream processing by a whole host of informatics programs including the DEEPN software. The utility of the MAPster interface and its ability to queue multiple jobs, combine input files, coveniently name output files, coupled with the speed.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mapster			https://github.com/emptyewer/MAPster/releases
DEEPN software			https://github.com/emptyewer/DEEPN/releases
Statmaker			https://github.com/emptyewer/DEEPN/releases
Minimum computer system	Apple		Mac Intel Core i5 or better
	-		4 Gb RAM or better
	-		500 Gb Disk spce or better
	-		OS 10.10 or higher
	Dell		Intel i5-7400 or better
	-		4 Gb RAM or better
	-		500 Gb Disk spce or better
	-		Windows 7 or higher