Note

When we start this project, the accuarcy of Nanopore sequencing is <90%, but for now, the continuous improvement of Nanopore sequencing accuracy (>95%) make the long-read-only strategy possible, so we simplified the hybrid strategy framwork and developed the long-read-only strategy. Please see NanoPhase for detailed usage.

Brief introduction

• HCBHA stands for the Hierachical Clustering Based Hybrid Assembly, which could be applied for the complete and high-quality/high-contiguity genome reconstruction from highly complex ecosystems by integrating the iterative strategy (hybrid assembly, bining and reads mapping). The genome reconstruction methods require both the short reads and long reads from the same sample. Please note that the iterative strategy is well described in our previous work High-Quality Bacterial Genomes of a Partial-Nitritation/Anammox System by an Iterative Hybrid Assembly Method

• The HCBHA workflow is a haplotype-resolved genome reconstruction approach. The key idea is phasing short and long reads into candidate bacterial/archaeal haplotypes and then hybrid assembling these genomes individually. We designed a seven-step workflow to achieve this goal: 1) short and long reads preparation; 2) long-read-only de novo assembly, aiming to generate high-contiguity error-prone contigs; 3) initial binning, aiming to obtain candidate genome bins; 4) initial bins polish, aiming to improve contigs accuracy; 5) re-binning, aiming to improve bins accuracy; 6) re-assembly, aiming to further improve contigs contiguity and 7) final binning, aiming to obtain the high-accurate MAGs.

• For instructions, usage and explanations of the HCBHA approach, please refer to the HCBHA wiki!

• Aside from the evaluation of the proposed iterative HCBHA approach using the Mock dasets, we also demostrated the performance of the approach using a highly complex metagenomic sample, the Activated Sludge sample from Shatin wastewater treatment plant, Hong Kong.

• If you are using the HCBHA workflow for the genome reconstruction, please cite the corresponding reference papers, particullary the paper described the metaFlye, Unicycler

  • metaFlye: scalable long-read metagenome assembly using repeat graphs
  • Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads
    
  • MetaWRAP—a flexible pipeline for genome-resolved metagenomic data analysis
  • Minimap2: pairwise alignment for nucleotide sequences
  • Fast gapped-read alignment with Bowtie 2
  • seqtk (https://github.com/lh3/seqtk)
  • SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation

Citing our paper

If you found this iterative haplotype-resolved framework useful in your research, a citation would be appreciated!

• Charting the complexity of an activated sludge microbiome through a hybrid sequencing strategy
• High-Quality Bacterial Genomes of a Partial-Nitritation/Anammox System by an Iterative Hybrid Assembly Method