ntLink: A Toolkit for De Novo Genome Assembly Scaffolding and Mapping Using Long Reads

Abstract

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. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ntLink scaffolding using overlap detection Basic Protocol 2: ntLink scaffolding with gap‐filling Basic Protocol 3: Running in‐code iterations of ntLink scaffolding Alternate Protocol 1: Generating long‐read to contig mappings with ntLink Alternate Protocol 2: Using ntLink mappings for genome assembly correction with Tigmint‐long Support Protocol: Installing ntLink

Post-embryonic tail development through molting of the freshwater shrimpNeocaridina denticulata

“…The five assemblies were combined into a single assembly using quickmerge version 0.3 (44). Subsequently, the assembly was scaffolded three times using ntlink version 1.3.9 and Flye polishing version 2.9.1 (45, 46). Haplotype duplication was removed and the mitochondrial genome was eliminated using purge_haplotigs version 1.1.2 (47).…”

High-quality genome of the zoophytophagous stink bug,Nesidiocoris tenuis, informs their food habit adaptation

“…Scaffolding was performed with the Hi-C reads by using SALSA v2.3 (Ghurye et al, 2019). We next performed raw long-read-based scaffolding by using ntLink (Coombe et al, 2023) and then finally checked manually for remaining gaps. We used GENESPACE (Wang et al, 2012) for the synteny comparison of scaffolded genomes and visualization of the results.…”

“…Genome contamination from symbiotic microorganisms was removed by MEGAN and DIAMOND (Bagci et al, 2021). We lastly performed 1-kbp or less raw long-read assembly by using ntLink with the "-gap_fill" option (Coombe et al, 2023).…”

Multi-genome synteny detection using minimizer graph mappings

“…Today, an increasing number of bioinformatics tools are utilizing minimizer sketches 30 , which represent underlying sequences using a particular subset of k -mers (substrings of length k ) for various applications, including mapping 25 , estimating sequence divergence 31 , and assembly scaffolding 32,33 . Sketching greatly reduces the computational cost of comparing and analyzing sequences, making it an attractive approach for scalable tool development in support of large-scale genomics research.…”