SHIMS 3.0: Highly efficient single-haplotype iterative mapping and sequencing using ultra-long nanopore reads

The reference sequence of structurally complex regions can only be obtained through a highly accurate clone-based approach that we call Single-Haplotype Iterative Mapping and Sequencing (SHIMS). In recent years, improvements to SHIMS have reduced the cost and time required by two orders of magnitude...

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Bibliographic Details
Published in:PloS one 2022-06, Vol.17 (6), p.e0269692-e0269692
Main Authors: Bellott, Daniel W, Cho, Ting-Jan, Jackson, Emily K, Skaletsky, Helen, Hughes, Jennifer F, Page, David C
Format: Article
Language:English
Subjects:
Accuracy
Analysis
Animals
Assemblies
Biology and Life Sciences
Chromosomes
Cloning
DNA sequencing
Engineering and Technology
Experiments
Gene mapping
Genomes
Haplotypes
High-Throughput Nucleotide Sequencing - methods
Imidoesters
Lab Protocol
Macaca mulatta
Mapping
Nanopores
Nucleotide sequencing
Palindromes
Repetitive structures
Research and Analysis Methods
Sequence Analysis, DNA - methods
Sex chromosomes
X chromosomes
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Summary:The reference sequence of structurally complex regions can only be obtained through a highly accurate clone-based approach that we call Single-Haplotype Iterative Mapping and Sequencing (SHIMS). In recent years, improvements to SHIMS have reduced the cost and time required by two orders of magnitude, but internally repetitive clones still require extensive manual effort to transform draft assemblies into reference-quality finished sequences. Here we describe SHIMS 3.0, using ultra-long nanopore reads to augment the Illumina data from SHIMS 2.0 assemblies and resolve internally repetitive structures. This greatly minimizes the need for manual finishing of Illumina-based draft assemblies, allowing a small team with no prior finishing experience to sequence challenging targets with high accuracy. This protocol proceeds from clone-picking to finished assemblies in 2 weeks for about $80 (USD) per clone. We recently used this protocol to produce reference sequence of structurally complex palindromes on chimpanzee and rhesus macaque X chromosomes. Our protocol provides access to structurally complex regions that would otherwise be inaccessible from whole-genome shotgun data or require an impractical amount of manual effort to generate an accurate assembly.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0269692