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.
0 Comments
Leave a Reply. |