DOI: 10.5061/dryad.kkwh70sfr
title: Targeted sequencing and iterative assembly of near-complete genomes

description: Recent advances in long-read sequencing (LRS) and assembly algorithms have
 made it possible to create highly complete genome assemblies for humans,
 animals, plants, and other eukaryotes. However, there is a need for
 ongoing development to improve accessibility and affordability of the
 required data, increase the range of usable sample types, and reliably
 resolve the most challenging, repetitive genome regions.
 'Cornetto' is a new experimental paradigm in which the genome
 assembly process is adaptively integrated with programmable selective
 nanopore sequencing, with target regions being iteratively updated to
 focus LRS data production onto the unsolved regions of a nascent assembly.
 This improves assembly quality and streamlines the process, both for human
 individuals and diverse non-human vertebrates, including endemic
 Australian endangered species, tested here. Cornetto enables us to
 generate highly complete diploid human genome assemblies using only a
 single LRS platform, surpassing the quality of previous efforts at a
 fraction of the cost. Cornetto enables genome assembly from challenging
 sample types like human saliva, for the first time, further enhancing
 accessibility. Finally, we obtain complete and accurate assemblies for
 clinically-relevant repetitive loci at the extremes of the genome,
 demonstrating valid approaches for genetic diagnosis in
 facioscapulohumeral muscular dystrophy (FSHD) and MUC1-autosomal dominant
 tubulointerstitial kidney disease (MUC1-ADTKD) - inherited diseases for
 which diagnosis is complicated by an inability to sequence the genes
 involved. In summary, Cornetto will improve, accelerate, and democratise
 genome assembly, delivering impacts across a range of bioscience domains.
description: # Targeted sequencing and iterative assembly of near-complete genomes
 Dataset DOI:
 [10.5061/dryad.kkwh70sfr](https://doi.org/10.5061/dryad.kkwh70sfr) ##
 Description of the data and file structure These assemblies were generated
 using the cornetto adaptive sampling method described in our manuscript.
 The documentation and source code used for the process are available
 at [https://github.com/hasindu2008/cornetto](https://github.com/hasindu2008/cornetto). There are three files, namely cornetto-hg002-asm.tar.gz, cornetto-animal-asm.tar.gz and cornetto-hg002-asm-updates.tar.gz that are described below. ### File: cornetto-hg002-asm.tar.gz **Description:** This tarball contains the FASTA assemblies for the hg002 sample.The below files are found inside the cornetto-hg002-asm directory created when you download and extract the cornetto-hg002-asm.tar.gz | Assembly name | Assembly Description | Assembly File | | :---------------------------------- | :------------------------------------------------------------------------------------------------------- | :------------------------------------------------- | | hg002-Hifi-1 | Hg002 DNA; 1x HiFi SMRT cell; primary asm | hg002-Hifi-1/hg002-Hifi-1.fasta | | hg002-Hifi-2 | Hg002 DNA; 2x HiFi SMRT cell; primary asm | hg002-Hifi-2/hg002-Hifi-2.fasta | | hg002-Hifi-3 | Hg002 DNA; 3x HiFi SMRT cell; primary asm | hg002-Hifi-3/hg002-Hifi-3.fasta | | hg002-NonCornetto-1 | hg002 DNA; 1x HiFi SMRT cell + 3x ONT duplex FC (without cornetto); primary asm | hg002-NonCornetto-1/hg002-NonCornetto-1.fasta | | hg002-NonCornetto-2 | hg002 DNA; 1x HiFi SMRT cell + 9x ONT duplex FC (without cornetto); primary asm | hg002-NonCornetto-2/hg002-NonCornetto-2.fasta | | hg002-Cornetto-1.1 | Hg002 DNA; 1x HiFi SMRT cell + 1x ONT duplex FC (cornetto cycle 1); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.1.fasta | | hg002-Cornetto-1.2 | Hg002 DNA; 1x HiFi SMRT cell + 1x ONT duplex FC (cornetto cycle 2); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.2.fasta | | hg002-Cornetto-1.3 | Hg002 DNA; 1x HiFi SMRT cell + 1x ONT duplex FC (cornetto cycle 3); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.3.fasta | | hg002-Cornetto-1.4 | Hg002 DNA; 1x HiFi SMRT cell + 2x ONT duplex FC (cornetto cycle 4); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.4.fasta | | hg002-Cornetto-1.5 | Hg002 DNA; 1x HiFi SMRT cell + 2x ONT duplex FC (cornetto cycle 5); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.5.fasta | | hg002-Cornetto-1.6 | Hg002 DNA; 1x HiFi SMRT cell + 2x ONT duplex FC (cornetto cycle 6); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.6.fasta | | hg002-Cornetto-1.7 | Hg002 DNA; 1x HiFi SMRT cell + 3x ONT duplex FC (cornetto cycle 7); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.7.fasta | | hg002-Cornetto-1.8 | Hg002 DNA; 1x HiFi SMRT cell + 3x ONT duplex FC (cornetto cycle 8); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.8.fasta | | hg002-Cornetto-1.9 (final) | hg002 DNA; 1x HiFi SMRT cell + 3x ONT duplex FC (cornetto cycle 9); primary asm | hg002-Cornetto-1/hg002-Cornetto-1.9.fasta | | hg002-Cornetto-2.1 | Hg002 DNA; 1x HiFi SMRT cell + 1x ONT duplex FC (cornetto cycle 1); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.1.fasta | | hg002-Cornetto-2.2 | Hg002 DNA; 1x HiFi SMRT cell + 1x ONT duplex FC (cornetto cycle 2); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.2.fasta | | hg002-Cornetto-2.3 | Hg002 DNA; 1x HiFi SMRT cell + 1x ONT duplex FC (cornetto cycle 3); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.3.fasta | | hg002-Cornetto-2.4 | Hg002 DNA; 1x HiFi SMRT cell + 2x ONT duplex FC (cornetto cycle 4); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.4.fasta | | hg002-Cornetto-2.5 | Hg002 DNA; 1x HiFi SMRT cell + 2x ONT duplex FC (cornetto cycle 5); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.5.fasta | | hg002-Cornetto-2.6 | Hg002 DNA; 1x HiFi SMRT cell + 2x ONT duplex FC (cornetto cycle 6); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.6.fasta | | hg002-Cornetto-2.7 | Hg002 DNA; 1x HiFi SMRT cell + 3x ONT duplex FC (cornetto cycle 7); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.7.fasta | | hg002-Cornetto-2.8 | Hg002 DNA; 1x HiFi SMRT cell + 3x ONT duplex FC (cornetto cycle 8); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.8.fasta | | hg002-Cornetto-2.9 (final) | hg002 DNA; 1x HiFi SMRT cell + 3x ONT duplex FC (cornetto cycle 9); primary asm | hg002-Cornetto-2/hg002-Cornetto-2.9.fasta | | hg002-Base, primary | hg002 DNA; 1x ONT flow cell (LSK114, simplex reads, SUP); primary asm | hg002-ONT\_base/hg002-ONT\_base.fasta | | hg002-Base, h1 | hg002 DNA; 1x ONT flow cell (LSK114, simplex reads, SUP); haplotype 1 | hg002-ONT\_base/hg002-ONT\_base.hap1.fasta | | hg002-Base, h2 | hg002 DNA; 1x ONT flow cell (LSK114, simplex reads, SUP); haplotype 2 | hg002-ONT\_base/hg002-ONT\_base.hap2.fasta | | hg002-NonCornetto-3, primary | hg002 DNA; 3x ONT flow cell (LSK114, simplex reads, SUP); primary asm | hg002-NonCornetto-3/hg002-NonCornetto-3.fasta | | hg002-NonCornetto-3, h1 | hg002 DNA; 3x ONT flow cell (LSK114, simplex reads, SUP); haplotype 1 | hg002-NonCornetto-3/hg002-NonCornetto-3.hap1.fasta | | hg002-NonCornetto-3, h2 | hg002 DNA; 3x ONT flow cell (LSK114, simplex reads, SUP); haplotype 2 | hg002-NonCornetto-3/hg002-NonCornetto-3.hap2.fasta | | hg002-Cornetto-3.1, primary | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 1; LSK114, simplex reads, SUP); primary asm | hg002-Cornetto-3/hg002-Cornetto-3.1.fasta | | hg002-Cornetto-3.2, primary | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 2; LSK114, simplex reads, SUP); primary asm | hg002-Cornetto-3/hg002-Cornetto-3.2.fasta | | hg002-Cornetto-3.3 (final), primary | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP); primary asm | hg002-Cornetto-3/hg002-Cornetto-3.3.fasta | | hg002-Cornetto-4.1, h1 | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 1; LSK114, simplex reads, SUP); haplotype 1 | hg002-Cornetto-4/hg002-Cornetto-4.1.hap1.fasta | | hg002-Cornetto-4.1, h2 | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 1; LSK114, simplex reads, SUP); haplotype 2 | hg002-Cornetto-4/hg002-Cornetto-4.1.hap2.fasta | | hg002-Cornetto-4.2, h1 | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 2; LSK114, simplex reads, SUP); haplotype 1 | hg002-Cornetto-4/hg002-Cornetto-4.2.hap1.fasta | | hg002-Cornetto-4.2, h2 | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 2; LSK114, simplex reads, SUP); haplotype 2 | hg002-Cornetto-4/hg002-Cornetto-4.2.hap2.fasta | | hg002-Cornetto-4.3 (final), h1 | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP); haplotype 1 | hg002-Cornetto-4/hg002-Cornetto-4.3.hap1.fasta | | hg002-Cornetto-4.3 (final), h2 | hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP); haplotype 2 | hg002-Cornetto-4/hg002-Cornetto-4.3.hap2.fasta | **Note:** 'hg002-Cornetto-4.3 (final)' above is only applicable for the first version of the manuscript preprint. For the updated assemblies used in the updated version of the manuscript preprint (and the published paper), please see the last table. ### File: cornetto-animal-asm.tar.gz **Description:** This tarball contains FASTA assemblies for the animal samples (the base assembly and the assembly after the final cornetto cycle). The below files are found inside the cornetto-animal-asm directory created when you download and extract the cornetto-animal-asm.tar.gz | Assembly Name | Assembly Description | Assembly File | | :------------------------ | :---------------------------------------------------------------------------------------------------------------------- | :----------------------------------------- | | Petrel-Base, primary | Petrel DNA; 1x HiFi SMRT cell; primary asm | Petrel-Base/Petrel-Base.fasta | | Petrel-Cornetto, primary | Petrel DNA; 1x HiFi SMRT cell + 2x ONT duplex FC (7x cycles); primary asm | Petrel-Cornetto/Petrel-Cornetto.fasta | | Cichlid-Base, primary | Cichlid DNA; 1x HiFi SMRT cell; primary asm | Cichlid-Base/Cichlid-Base.fasta | | Cichlid-Cornetto, primary | Cichlid DNA; 1x HiFi SMRT cell + 2x ONT duplex FC (6x cycles); primary asm | Cichlid-Cornetto/Cichlid-Cornetto.fasta | | Turtle-Base, h1 | Turtle DNA; 1x ONT FC (standard; LSK114, simplex reads, SUP basecalling); haplotype 1 | Turtle-Base/Turtle-Base.hap1.fasta | | Turtle-Base, h2 | Turtle DNA; 1x ONT FC (standard; LSK114, simplex reads, SUP basecalling); haplotype 2 | Turtle-Base/Turtle-Base.hap2.fasta | | Turtle-Cornetto, h1 | Turtle DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; 3x cycles; LSK114, simplex reads, SUP basecalling); haplotype 1 | Turtle-Cornetto/Turtle-Cornetto.hap1.fasta | | Turtle-Cornetto, h2 | Turtle DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; 3x cycles; LSK114, simplex reads, SUP basecalling); haplotype 2 | Turtle-Cornetto/Turtle-Cornetto.hap2.fasta | | Parrot-Base, h1 | Parrot DNA; 2x ONT FC (standard; LSK114, simplex reads, SUP basecalling); haplotype 1 | Parrot-Base/Parrot-Base.hap1.fasta | | Parrot-Base, h2 | Parrot DNA; 2x ONT FC (standard; LSK114, simplex reads, SUP basecalling); haplotype 2 | Parrot-Base/Parrot-Base.hap2.fasta | | Parrot-Cornetto, h1 | Parrot DNA; 2x ONT FC (standard) + 1x ONT FC (cornetto; 4x cycles; LSK114, simplex reads, SUP basecalling); haplotype 1 | Parrot-Cornetto/Parrot-Cornetto.hap1.fasta | | Parrot-Cornetto, h2 | Parrot DNA; 2x ONT FC (standard) + 1x ONT FC (cornetto; 4x cycles; LSK114, simplex reads, SUP basecalling); haplotype 2 | Parrot-Cornetto/Parrot-Cornetto.hap2.fasta | ### File: cornetto-hg002-asm-updates.tar.gz **Description:** This tarball contains the updated 'hg002-Cornetto-4.3' assemblies created during the manuscript revision. This tarball also includes a copy of the 'T2TC-ONT' assemblies used for comparison. The below files are found inside the cornetto-hg002-asm-updates directory created when you download and extract the cornetto-hg002-asm-updates.tar.gz | Assembly name | Assembly Description | Assembly File | | :------------------------------- | :------------------------------------------------------------------------------------------------------------------------------------------ | :----------------------------------------------------------- | | hg002-Cornetto-4.3, ctg, h1 | partially phased asm; contigs; hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP); haplotype 1 | hg002-Cornetto-4-contigs/hg002-Cornetto-4.3.hap1.fasta | | hg002-Cornetto-4.3, ctg, h2 | partially phased asm; contigs; hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP); haplotype 2 | hg002-Cornetto-4-contigs/hg002-Cornetto-4.3.hap2.fasta | | hg002-Cornetto-4.3, scaf, h1 | partially phased asm; scaffolds; hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP); haplotype 1 | hg002-Cornetto-4-scaffolds/hg002-Cornetto-4.3.hap1.fasta | | hg002-Cornetto-4.3, scaf, h2 | partially phased asm; scaffolds; hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP); haplotype 2 | hg002-Cornetto-4-scaffolds/hg002-Cornetto-4.3.hap2.fasta | | hg002-Cornetto-4.3-HiC, ctg, h1 | fully phased asm; contigs; hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP) + HiC; haplotype 1 | hg002-Cornetto-4-HiC-contigs/hg002-Cornetto-4.3.hap1.fasta | | hg002-Cornetto-4.3-HiC, ctg, h2 | fully phased asm; contigs; hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP) + HiC; haplotype 2 | hg002-Cornetto-4-HiC-contigs/hg002-Cornetto-4.3.hap2.fasta | | hg002-Cornetto-4.3-HiC, scaf, h1 | fully phased asm; scaffolds; hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP) + HiC; haplotype 1 | hg002-Cornetto-4-HiC-scaffolds/hg002-Cornetto-4.3.hap1.fasta | | hg002-Cornetto-4.3-HiC, scaf, h2 | fully phased asm; scaffolds; hg002 DNA; 1x ONT FC (standard) + 1x ONT FC (cornetto; cycle 3; LSK114, simplex reads, SUP) + HiC; haplotype 2 | hg002-Cornetto-4-HiC-scaffolds/hg002-Cornetto-4.3.hap2.fasta | | T2TC-ONT, ctg, h1 | fully phased asm; contigs; Koren et al (Genome Research 2024); 50x duplex + 30x UL + PoreC; haplotype 1 | T2TC-ONT-contigs/asm.hap1.fasta | | T2TC-ONT, ctg, h2 | fully phased asm; contigs; Koren et al (Genome Research 2024); 50x duplex + 30x UL + PoreC; haplotype 2 | T2TC-ONT-contigs/asm.hap2.fasta | | T2TC-ONT, scaf, h1 | fully phased asm; scaffolds; Koren et al (Genome Research 2024); 50x duplex + 30x UL + PoreC; haplotype 1 | T2TC-ONT-scaffolds/asm.hap1.fasta | | T2TC-ONT, scaf, h2 | fully phased asm; scaffolds; Koren et al (Genome Research 2024); 50x duplex + 30x UL + PoreC; haplotype 2 | T2TC-ONT-scaffolds/asm.hap2.fasta | 
 
 ## Code/software Standard tar command in unix is adequate to extract the
 tar.gz file. Once extracted, the FASTA files can be opened using any text
 viewing/editing software or common genomics software such as samtools,
 seqtk or seqkit. ## Access information The raw FASTQ files that were used
 to generate the assemblies here are available to be downloaded from the
 ENA project PRJEB86853.