  Golden Gate cloning has revolutionized synthetic biology. Its concept of
modular, highly characterized libraries of parts that can be combined into
higher order assemblies allows engineering principles to be applied to
biological systems. The basic parts, typically stored in level 0 plasmids, are
sequence validated by the method of choice and can be combined into higher
order assemblies on demand. Higher order assemblies are typically
transcriptional units, and multiple transcriptional units can be assembled into
multi-gene constructs. Higher order Golden Gate assembly based on defined and
validated parts usually does not introduce sequence changes. Therefore, simple
validation of the assemblies, e.g. by colony PCR or restriction digest pattern
analysis, is sufficient. However, in many experimental setups, researchers do
not use defined parts, but rather part libraries, resulting in assemblies of
high combinatorial complexity where sequencing again becomes mandatory. Here we
present a detailed protocol for the use of a highly multiplexed dual barcode
amplicon sequencing using the Nanopore sequencing platform for in-house
sequence validation. The workflow, called DuBA.flow, is a start-to-finish
procedure that provides all necessary steps from a single colony to the final
easy-to-interpret sequencing report.
