CLONEQC: lightweight sequence verification for synthetic biology

Abstract

Synthetic biology projects aim to produce physical DNA that matches a designed target sequence. Chemically synthesized oligomers are generally used as the starting point for building larger and larger sequences. Due to the error rate of chemical synthesis, these oligomers can have many differences from the target sequence. As oligomers are joined together to make larger and larger synthetic intermediates, it becomes essential to perform quality control to eliminate intermediates with errors and retain only those DNA molecules that are error free with respect to the target. This step is often performed by transforming bacteria with synthetic DNA and sequencing colonies until a clone with a perfect sequence is identified. Here we present CloneQC, a lightweight software pipeline available as a free web server and as source code that performs quality control on sequenced clones. Input to the server is a list of desired sequences and forward and reverse reads for each clone. The server generates summary statistics (error rates and success rates target-by-target) and a detailed report of perfect clones. This software will be useful to laboratories conducting in-house DNA synthesis and is available at http://cloneqc.thruhere.net/ and as Berkeley Software Distribution (BSD) licensed source.

Figure 1.

Flowchart of the CloneQC sequence validation pipeline. See ‘Materials and Methods’ section for details.

Figure 2.

Summary results for CloneQC run as a web application. ‘STATISTICS’ provides summary statistics for each synthetic target matched by at least one clone. Following are summary tables for each target sequence (only the first two shown), giving the identities of clones that contain perfect physical DNA for the target (Passing Clones), have discrepancies between reads but may have perfect physical DNA (Check Clones) or have errors that are fixable by reamplification (Fixable Clones).