![]() ![]() In GenoREAD, this process is composed of two successive steps. Sequence verification is therefore a critical part of the workflow of many projects in the life sciences, yet it is probably the one that has received the least attention. Addgene provides a similar functionality to its users by sharing results of its own internal quality controls beside the sequence provided by the depositing scientist however, the website does not provide users with tools to easily compare the two sequences. Each read is aligned with the part’s reference sequence, and the verification status of the clone is clearly displayed for each physical distribution in the Registry. Since efforts to verify the collection of clones distributed to the iGEM students demonstrated the need for systematic quality control of submissions to the Registry of Standard Biological Parts ( 7), users of this community resource have been encouraged to upload sequencing trace files. The value of integrating sequencing data in database applications to manage large collections of biological parts is now well recognized. It is common practice in molecular biology to verify the sequence of a plasmid prior to publication or submission to a community resource like Addgene ( 6), the Registry of Standard Biological Parts ( 7) or the DNASU repository ( 8). The need to verify the sequence of clones and plasmids is not limited to gene synthesis it also applies to any plasmid containing inserts with known sequences, such as clones from ORFeome collections, irrespective of the way the plasmid was assembled. Sanger remains the most cost-effective sequencing technology for most gene synthesis projects focused on assembling sequences that do not exceed a few kilobases in length. While more expensive per base than newer sequencing technologies, Sanger is less expensive per run, making it more relevant to the job of clone-verification than it might be for a traditional genome-sized sequence verification project. In this fast-evolving landscape of sequencing technologies, Sanger sequencing still remains the most commonly used technology for sequence verification ( 4, 5). Since the verification of thousands of 1-kb building blocks is very different from the verification of a small number of 100-kb synthetic fragments, different sequencing technologies are used at different stages of synthetic genomics projects ( 3). Difference of throughput, price structure and access to sequencing resources should be considered in relation to the gene synthesis facility throughput, nature of the sequences it produces and other technical and economic constraints. The rapid development and commercial success of new high-throughput sequencing technologies calls for a careful analysis of the technology best suited to meet the sequence verification needs of gene synthesis operators. The limitations of the chemistries used at different steps of the process require scientists to verify the physical sequence of the clones they produce at the different stages of the assembly process. ![]() Gene synthesis ( 1, 2) is the process of manufacturing user-defined DNA sequences with base-level precision. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD can determine if a clone matches its reference sequence. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. Sequence validation is equally important for other kinds of curated clone collections. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision.
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