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

Area of Science:

• Genomics
• Bioinformatics
• Molecular Biology

Background:

• Accurate reference sequences of complex genomic regions are challenging to obtain.
• Existing clone-based methods like SHIMS have been improved but still require significant manual effort for repetitive regions.

Purpose of the Study:

• To describe SHIMS 3.0, an enhanced protocol for generating high-accuracy reference sequences.
• To leverage ultra-long nanopore reads to resolve repetitive structures in previously difficult-to-assemble clones.

Main Methods:

• Integration of ultra-long nanopore reads with existing Illumina-based SHIMS 2.0 assemblies.
• Application of the protocol to sequence structurally complex palindromic regions on primate X chromosomes.

Main Results:

• SHIMS 3.0 minimizes manual finishing of draft assemblies, enabling accurate sequencing of challenging targets.
• The protocol successfully produced reference sequences for complex palindromes in chimpanzee and rhesus macaque X chromosomes.
• Finished assemblies are achievable in approximately 2 weeks per clone at a cost of $80 USD.

Conclusions:

• SHIMS 3.0 provides an accessible and efficient method for obtaining reference sequences of structurally complex genomic regions.
• This advancement overcomes limitations of whole-genome shotgun data and reduces the impractical manual effort previously required.