Hybrid Long Read Assembly (optional)

Last updated on 2025-02-19 | Edit this page

Estimated time: 12 minutes

Overview

Questions

  • What is hybrid assembly, and how does it combine different sequencing technologies?
  • How can you perform hybrid assembly using both types of long-read data?
  • What are the key steps in hybrid assembly, including polishing and scaffolding?
  • How do you evaluate the quality of a hybrid assembly using bioinformatics tools?

Objectives

  • Understand the concept of hybrid assembly and its advantages in genome sequencing.
  • Learn how to perform hybrid assembly using long-read sequencing data from PacBio and ONT platforms.
  • Explore the key steps involved in hybrid assembly, including polishing and scaffolding.
  • Evaluate the quality of a hybrid assembly using bioinformatics tools such as QUAST and Compleasm.

Flye for Hybrid Assembly

For hybrid assembly using flye, first, run the pipeline with all your reads in the –pacbio-raw mode (you can specify multiple files, no need to merge all you reads into one). Also add –iterations 0 to stop the pipeline before polishing. Once the assembly finishes, run polishing using either PacBio or ONT reads only. Use the same assembly options, but add –resume-from polishing. Here is an example of a script that should do the job:

BASH 

ml --force purge
ml biocontainers
ml flye
# reads
PBREADS="At_pacbio-hifi-filtered.fastq"
ONTREADS="At_ont-reads-filtered.fastq"
# round 1
flye \
    --pacbio-raw $PBREADS $ONTREADS \
    --iterations 0 \
    --out-dir hybrid_flye_out \
    --genome-size 135m \
    --threads ${SLURM_CPUS_ON_NODE}
# round 2
flye \
   --pacbio-raw $PBREADS \
   --resume-from polishing \
   --out-dir hybrid_flye_out  \
   --genome-size 135m \
   --threads ${SLURM_CPUS_ON_NODE}

Evaluating Assembly Quality

For quick evaluation, we will run quast and compleasm on the hybrid assembly output.

BASH 

ml --force purge
ml biocontainers
ml quast
ml compleasm
fasta="hybrid_flye_out/assembly.fasta"
quast.py \
  --fast \
  --threads ${SLURM_CPUS_ON_NODE} \
  -o quast_basic \
    ${fasta}
compleasm \
compleasm run \
   -a ${fasta} \
   -o compleasm_out \
   -l brassicales_odb10  \
   -t ${SLURM_CPUS_ON_NODE}

Scaffolding with Bionano

To scaffold the assembly using Bionano data, we will use the bionano solve. We can run the following script to scaffold the assembly:

BASH 

ml --force purge
export PATH=$PATH:/apps/biocontainers/exported-wrappers/bionano/3.8.0
fasta="hybrid_flye_out/assembly.fasta"
run_hybridscaffold.sh \
  -c /opt/Solve3.7_10192021_74_1/HybridScaffold/1.0/hybridScaffold_DLE1_config.xml\
  -b workshop_assembly/col-0_bionano/Evry.OpticalMap.Col-0.cmap \
  -n ${fasta} \
  -u CTTAAG \
  -z results_bionano_hybrid_scaffolding.zip \
  -w log.txt \
  -B 2 \
  -N 2 \
  -g \
  -f \
  -r /opt/Solve3.7_10192021_74_1/RefAligner/1.0/sse/RefAligner \
  -p /opt/Solve3.7_10192021_74_1/Pipeline/1.0 \
  -o bionano_hybrid_scaffolding

Once this completes, you can generate the final scaffold-level assembly by merging placed and unplaced contigs in the bionano_hybrid_scaffolding/hybrid_scaffolds directory.

BASH 

cd bionano_hybrid_scaffolding/hybrid_scaffolds
cat *HYBRID_SCAFFOLD.fasta *_HYBRID_SCAFFOLD_NOT_SCAFFOLDED.fasta \
   > ../../assembly_scaffolds.fasta
cd ../..

You can evaluate the final assembly using quast and compleasm as before.

BASH 

fasta="assembly_scaffolds.fasta"
quast.py \
  --fast \
  --threads ${SLURM_CPUS_ON_NODE} \
  -o quast_scaffolds \
    ${fasta}
compleasm run \
   -a ${fasta} \
   -o compleasm_scaffolds_out \
   -l brassicales_odb10  \
   -t ${SLURM_CPUS_ON_NODE}

Hybrid Assembly Summary

In this section you have learned how to perform a hybrid assembly using Flye, polish the assembly, scaffold it using Bionano, and evaluate the final assembly quality. This workflow combines the advantages of ONT and PacBio sequencing, improves structural accuracy with Bionano scaffolding, and ensures a high-quality genome assembly. The steps involved are:

  1. Run Hybrid Assembly with Flye
    • Use Flye in --pacbio-raw mode to assemble both PacBio and ONT reads.
    • Set --iterations 0 to stop before polishing.
  2. Polishing the Assembly
    • Polish the assembly using either PacBio or ONT reads by resuming Flye with --resume-from polishing.
  3. Evaluate Assembly Quality
    • Run QUAST for basic assembly metrics (contig count, N50, genome size, misassemblies).
    • Use Compleasm to assess genome completeness based on conserved single-copy genes.
  4. Scaffold Assembly with Bionano Optical Mapping
    • Use Bionano Solve to integrate optical maps and scaffold the assembly.
    • Run run_hybridscaffold.sh with the reference .cmap optical map file.
  5. Generate Final Scaffold-Level Assembly
    • Merge placed and unplaced contigs from the Bionano scaffolding output to create the final genome assembly.
  6. Final Evaluation of Scaffolds
    • Re-run QUAST and Compleasm to validate improvements and ensure genome completeness after scaffolding.

Key Points

  • 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.