Abstract
BackgroundWhereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation.ResultsWe have developed an Automated Clone Evaluation (ACE) system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set.ConclusionACE was designed to facilitate high throughput clone sequence verification projects. The software has been used successfully to evaluate more than 55,000 clones at the Harvard Institute of Proteomics. The software dramatically reduced the amount of time and labor required to evaluate clone sequences and decreased the number of missed sequence discrepancies, which commonly occur during manual evaluation. In addition, ACE helped to reduce the number of sequencing reads needed to achieve adequate coverage for making decisions on clones.
Highlights
Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process
We present the Automated Clone Evaluation (ACE) system, which is an automated software application that simultaneously manages the process of sequence validation for thousands of plasmid clones
In order to test the performance of ACE, we compared the processing times and outcomes for an experienced operator using ACE with a researcher experienced at manual analysis using commercial software to analyze one plate of clones for 93 unique genes ranging in length from 130
Summary
Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. There is a substantial need to develop high throughput (HT) tools that will accelerate the study of the thousands of proteins not yet examined, a field referred to as functional proteomics. Protein function studies start by producing proteins using cloned copies of the genes that encode them. Recognition of this has led to the production of large collections of cloned genes configured in a protein expression-ready format (ORF collections). Despite the well-recognized importance of sequence verifying cloned genes, it has been performed on less than a handful of protein-expression clone collections currently in existence [1,2,3,4,5,6]
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