Hybdecon

Description
Hybdecon was developed to reduce the complexity of the multi-dimensional pooling analysis. It is a stand-alone Java and Perl application that provides an easy-to-use interface for manually calling of hits on a pooled array and deconvolution of those hits. The program generates an ACE file for loading markers into FPC. The software also provides image loading and processing functions to aid in manual calling and users can stop, save and resume their work at any time.

The graphical user-interface is derived from HybSweeper (Lazo et al. Biotechniques 39:320, 2005) a web-based screening tool. The Perl deconvolution script was developed entirely at CUGI.

Usefulness
Hybridization screening methods can be used to map genetic markers to BACs and in turn anchor those markers onto a physical map, creating an integrated physical and genetic map. The integrated map can be used to assist with the design of sequencing strategies and assembly of complete genomes. Multi-dimensional hybridization pooling strategies have been employed to reduce the time and cost of hybridizations, but require a deconvolution step to tease out one-to-one mappings.

Download

• Hybdecon-0.1.jar
• The source code can be downloaded here

How to Install and Launch Hybdecon

• There is no need to install Hybdecon. Simply download the file and double click on the program.
• Alternatively, the following command can be issued to start the program:

  java -jar Hybdecon-0.1.jar

Experiment Setup
CUGI offers an online tool for assistance with setup of the pooling experiment. You can upload you probes and indicate the number of dimensions and pools you desire. The tool will generate all files necessary for the deconvolution as well as provide files for the lab that indicate which probes should be placed in each pool. the link for this tool can be found here: Pooling Experiment Setup.

Requirements
Hybdecon should run smoothly under the following specifications:

• 1.0 GHz CPU or higher
• 512 Mb RAM or more
• Video card: 1024x768 or capable of higher resolution.
• Operating System: Windows, Mac, Linux, Solaris, or any operating systems that support Java and Perl.
• Java: Java Runtime Environment (JRE) is required for launching Hybdecon user interface. The
  recommended and tested version is JRE 6. Older versions may or may not work. For more information, go to http://www.java.com
• Perl: Perl 5.10 or above is required for running deconvolution. The older versions might work but were not tested. Perl should be added to the PATH environment variable. Go to http://www.activestate.com/activeperl for more information.

Test Dataset
HybDecon was used in a Chinese Chestnut (Castanea mollissima) hybridization experiment with 3 dimensions and 125 overgos. This work was funded by the NSF grant Genomic Tool Development for the Fagaceae (NSF-PGRP 0605135). Probes were individually labeled and compiled into 15 pools. Four filters were required for a single copy of two Chinese Chestnut BAC libraries. With a set of filters hybridized for each pool, a total of 120 filter images were created and individually called with HybDecon. Hybridizations were performed at 60 degree overnight, and BAC filters were washed with 0.1% SDS and 1X SSC at 60 degrees. Images of the hybridizations were recorded by phosphor screens and read by a Typhoon 9400 imager (GE Healthcare, Piscataway, NJ, USA). A total of 124 probes were successfully associated with at least one BAC clone by the deconvolution function of HybDecon.

Pools

• Pools.gz - a zip file of 120 individual pool files produced by calling hits with HybDecon

Probe List

• probes.txt - the list of probes used as input for the experimental setup program.
• probelist.txt - the resulting probe list file for deconvolution.

Main output files from deconvolution:

• cc_example.qualityReport.txt - The main deconvolution report.
• cc_example.ace - The ace file output by HybDecon for upload to FPC.
• cc_example.successBAC.txt - The list of successfully hybridized BACs.
• cc_example.successProbe.txt - The list of successfully hybridized probes and the number of BACs each hybridized to.

Deconvolution output files with contigs that were unable to be deconvoluted:

• cc_example.clones1Hits.txt - A list of contigs with a hybridization hit on a single filter, and the clones that might have caused this hit.
• cc_example.clones2Hits.txt - A list of contigs with a hybridization hits on 2 filters, and the clones that might have caused these hits.
• cc_example.clones3Hits.txt - A list of contigs with a hybridization hits on 3 filters, and the clones that might have caused these hits.
• cc_example.clones4Hits.txt - A list of contigs with a hybridization hits on 4 filters, and the clones that might have caused these hits.
• cc_example.clones5Hits.txt - A list of contigs with a hybridization hits on 5 filters, and the clones that might have caused these hits.
• cc_example.clones6Hits.txt - A list of contigs with a hybridization hits on 6 filters, and the clones that might have caused these hits.