  With the increasing affordability and accessibility of genome sequencing
data, de novo genome assembly is an important first step to a wide variety of
downstream studies and analyses. Therefore, bioinformatics tools that enable
the generation of high-quality genome assemblies in a computationally efficient
manner are essential. Recent developments in long-read sequencing technologies
have greatly benefited genome assembly work, including scaffolding, by
providing long-range evidence that can aid in resolving the challenging
repetitive regions of complex genomes. ntLink is a flexible and
resource-efficient genome scaffolding tool that utilizes long-read sequencing
data to improve upon draft genome assemblies built from any sequencing
technologies, including the same long reads. Instead of using read alignments
to identify candidate joins, ntLink utilizes minimizer-based mappings to infer
how input sequences should be ordered and oriented into scaffolds. Recent
improvements to ntLink have added important features such as overlap detection,
gap-filling and in-code scaffolding iterations. Here, we present three basic
protocols demonstrating how to use each of these new features to yield highly
contiguous genome assemblies, while still maintaining ntLink's proven
computational efficiency. Further, as we illustrate in the alternate protocols,
the lightweight minimizer-based mappings that enable ntLink scaffolding can
also be utilized for other downstream applications, such as misassembly
detection. With its modularity and multiple modes of execution, ntLink has
broad benefit to the genomics community, from genome scaffolding and beyond.
ntLink is an open-source project and is freely available from
https://github.com/bcgsc/ntLink.
