'PACLIMS': a component LIM system for high-throughput functional genomic analysis

Abstract

Background: Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the approximately 11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required.

Results: The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured.

Conclusion: Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors.

Figure 1

Schematic of data acquisition and transfer to PACLIMS from multiple universities.

Figure 2

A schematic overview of the flow in information and materials managed by PACLIMS. Module 1: entry point for PACLIMS, accessed by scanning or entering a plate or plant rack's barcode; the user is directed to the appropriate web form. Modules 2 and 3: displays web forms for data entry on a new, or "parent" plate, including fields for information on mutants in each of the 24 wells. Module 4: provides access to a web form for entry of growth rate data that is collected from the parent plate and allows the user to create "copies" of the parent plate for assaying other phenotypes. Module 5: guides and documents the transfer of paper disks from four 24-well "parent" plates into the four quadrants of three 96-well plates, used for permanent storage and shipping, the latter process being recorded with Module 6. Module 7: records the revival of cultures from permanent storage, specifically the reversion of the 96-well format into four 24-well plates for pathogenicity assays. Module 8: directs the inoculation process for each 24-well plate of spores, beginning with entering a barcode for a rack of plants to be inoculated, and culminating in entering pathological data for each mutant isolate. Module 9: records phenotypic data via four different web forms, each of which records specific phenotypes; module 1 controls the particular data entry form that is accessed.

Figure 3

(A) Front page for barcode scanning into PACLIMS. (B-D) Depending upon which barcode has been scanned, the user will be transferred by Module 1 to the next step in the experimental process.

Figure 4

Data entry page for pathogenicity screen results.