Genome Assembly: Hands-on Training: Key Points

Introduction to Genome assembly

Genome assembly reconstructs complete genome sequences from fragmented DNA reads.
De novo assembly builds genomes without a reference, while reference-guided assembly uses existing genomes.
Sequencing technologies like Illumina, PacBio HiFi, and Oxford Nanopore offer different read lengths and error rates.
Challenges include repetitive elements, heterozygosity, and error correction.
Tools many programs are available for data QC, assembly, post-processing, and evaluation - choice depends on data type and research goals.

Genome assembly strategy depends on read type, genome complexity, and computational resources, with PacBio HiFi, ONT, and hybrid approaches offering different advantages in accuracy, cost, and contiguity.
Assembly evaluation is critical for assessing completeness and accuracy, using tools like BUSCO for gene completeness, QUAST for structural integrity, and Merqury for k-mer-based validation.
Scaffolding methods like Bionano OGM and Hi-C improve genome organization, resolving large structural variations and ordering contigs into chromosome-level assemblies.
A well-assembled genome is essential for downstream applications such as annotation, comparative genomics, and structural variation analysis, with missing or misassembled regions potentially leading to incorrect biological conclusions.

Data Quality Control: Assessing and filtering raw sequencing data is essential for accurate genome assembly.
NanoPlot: Visualizes read length distributions, quality scores, and other metrics to evaluate sequencing data quality.
Filtlong: Filters long-read sequencing data based on length and quality to retain high-quality reads.
GenomeScope: Profiles genomes using k-mer frequency distributions to estimate genome size, heterozygosity, and repeat content.

HiFiasm is a specialized assembler for PacBio HiFi reads, providing high-quality, haplotype-resolved genome assemblies.
It leverages the high accuracy of HiFi reads to generate phased assembly graphs, preserving haplotype information.
HiFiasm is optimized for resolving complex regions and distinguishing haplotypes in diploid or polyploid organisms.
The assembler generates primary contigs and haplotype-resolved contigs, offering valuable information for downstream analyses.
By adjusting purging levels and using parental kmer profiles, users can improve haplotype resolution and assembly quality.

ONT provides long-read sequencing data with high error rates.
Flye is a long-read assembler optimized for handling ONT data and producing highly contiguous assemblies.
The Flye assembly workflow involves read preprocessing, repeat graph construction, graph resolution, polishing, and post-processing.
Flye output includes the final assembly sequence, assembly graph, and summary information for evaluation.
Polishing the assembly can improve base-level accuracy and overall assembly quality.
Flye provides built-in polishing capabilities, and other tools like Racon, Nanopolish, and Medaka can be used for further refinement.

Hybrid assembly with Flye combines ONT and PacBio reads to leverage long-read continuity and high-accuracy sequencing, with separate polishing steps to refine base-level errors.
Assembly quality assessment using QUAST and Compleasm provides critical insights into contiguity, completeness, and potential misassemblies, ensuring reliability before scaffolding.
Bionano Optical Genome Mapping (OGM) improves hybrid assemblies by scaffolding contigs, resolving misassemblies, and enhancing genome continuity, leading to chromosome-scale assemblies.
Final scaffolding validation and quality assessment ensure the integrity of the genome assembly, with QUAST and Compleasm used to confirm improvements after Bionano integration.

Bionano optical genome mapping (OGM) provides long-range structural information for scaffolding genome assemblies.
Bionano Solve hybrid scaffolding integrates optical maps with sequence assemblies to improve contiguity and accuracy.
The Bionano Solve pipeline involves in silico map generation, conflict resolution, hybrid scaffolding, and final alignment.
The output of Bionano Solve includes scaffolded genome assemblies in AGP and FASTA formats, alignment results, and conflict resolution information.
Quality assessment of hybrid scaffolds involves evaluating alignment statistics, conflict resolution, scaffold N50 values, and completeness of the assembly.