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

AbstractThe 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 introduce SHIMS 3.0, using ultra-long nanopore reads to resolve internally repetitive structures and minimize the need for manual finishing of Illumina-based draft assemblies. This protocol proceeds from clone-picking to finished assemblies in 2 weeks for about 80 dollars per clone. We have used SHIMS 3.0 to finish the structurally complex TSPY array on the human Y chromosome, which could not be resolved by previous sequencing methods. 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.

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Selective single molecule sequencing and assembly of a human Y chromosome of African origin

10.1101/342667 ◽

2018 ◽

Cited By ~ 1

Author(s):

Lukas F.K. Kuderna ◽

Esther Lizano ◽

Eva Julià ◽

Jessica Gomez-Garrido ◽

Aitor Serres-Armero ◽

...

Keyword(s):

Y Chromosome ◽

Single Molecule ◽

Genomic Analysis ◽

European Ancestry ◽

African Origin ◽

Repeat Content ◽

Y Chromosomes ◽

Reference Quality ◽

Novel Strategy ◽

Human Y Chromosome

Mammalian Y chromosomes are often neglected from genomic analysis. Due to their inherent assembly difficulties, high repeat content, and large ampliconic regions1, only a handful of species have their Y chromosome properly characterized. To date, just a single human reference quality Y chromosome, of European ancestry, is available due to a lack of accessible methodology2–5. To facilitate the assembly of such complicated genomic territory, we developed a novel strategy to sequence native, unamplified flow sorted DNA on a MinION nanopore sequencing device. Our approach yields a highly continuous and complete assembly of the first human Y chromosome of African origin. It constitutes a significant improvement over comparable previous methods, increasing continuity by more than 800%6, thus allowing a chromosome scale analysis of human Y chromosomes. Sequencing native DNA also allows to take advantage of the nanopore signal data to detect epigenetic modifications in situ7. This approach is in theory generalizable to any species simplifying the assembly of extremely large and repetitive genomes.

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Cost-effective, high-throughput, single-haplotype iterative mapping and sequencing for complex genomic structures

10.1101/157206 ◽

2017 ◽

Author(s):

Daniel W. Bellott ◽

Ting-Jan Cho ◽

Jennifer F. Hughes ◽

Helen Skaletsky ◽

David C. Page

Keyword(s):

High Throughput ◽

Cost Effective ◽

Reference Sequence ◽

Sequencing Technologies ◽

A Genome ◽

Time Required ◽

Genomic Regions ◽

The Cost ◽

Genome Center ◽

Library Preparation Protocol

AbstractReference sequence of structurally complex regions can only be obtained through highly accurate clone-based approaches. We and others have successfully employed Single-Haplotype Iterative Mapping and Sequencing (SHIMS 1.0) to assemble structurally complex regions across the sex chromosomes of several vertebrate species and in targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 was expensive and time consuming, requiring the resources that only a genome center could command. Here we introduce SHIMS 2.0, an improved SHIMS protocol to allow even a small laboratory to generate high-quality reference sequence from complex genomic regions. Using a streamlined and parallelized library preparation protocol, and taking advantage of high-throughput, inexpensive, short-read sequencing technologies, a small group can sequence and assemble hundreds of clones in a week. Relative to SHIMS 1.0, SHIMS 2.0 reduces the cost and time required by two orders of magnitude, while preserving high sequencing accuracy.

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Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes

Nature ◽

10.1038/news020722-10 ◽

2002 ◽

Keyword(s):

Whole Genome Shotgun ◽

Fugu Rubripes ◽

Whole Genome ◽

Shotgun Assembly

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Use of cold tolerant eucalyptus species as a partial replacement for southern mixed hardwoods

TAPPI Journal ◽

10.32964/tj11.7.29 ◽

2012 ◽

Vol 11 (7) ◽

pp. 29-35 ◽

Cited By ~ 5

Author(s):

PETER W. HART ◽

DALE E. NUTTER

Keyword(s):

Potential Method ◽

The United States ◽

Partial Replacement ◽

Growth Time ◽

Eucalyptus Species ◽

Cold Tolerant ◽

Hardwood Pulp ◽

Time Required ◽

The Cost ◽

Operational Data

During the last several years, the increasing cost and decreasing availability of mixed southern hardwoods have resulted in financial and production difficulties for southern U.S. mills that use a significant percentage of hardwood kraft pulp. Traditionally, in the United States, hardwoods are not plantation grown because of the growth time required to produce a quality tree suitable for pulping. One potential method of mitigating the cost and supply issues associated with the use of native hardwoods is to grow eucalyptus in plantations for the sole purpose of producing hardwood pulp. However, most of the eucalyptus species used in pulping elsewhere in the world are not capable of surviving in the southern U.S. climate. This study examines the potential of seven different cold-tolerant eucalyptus species to be used as replacements for, or supplements to, mixed southern hardwoods. The laboratory pulping and bleaching aspects of these seven species are discussed, along with pertinent mill operational data. Selected mill trial data also are reviewed.

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