Although Unicycler is a well-performed assembler when you have deep-sequenced short reads and higher error rate long reads, with the continuous improvement of Nanopore sequencing accuracy and throughput, we prefer a long-read-first framework, please consider to use NanoPhase (with --hybird parameter)
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IHA (Iterative Hybrid Assembly) is a Unicycler-based workflow for high-quality and high-contiguity genome reconstruction from
the enrichment system. The proposed Iterative method requires that high-quality and high-contiguity genomes could be retrieved from the initial hybrid assembly using Unicycler. If MAGs are highly fragmented, other assemblers might be considered. Notably, compared with short-read assembly and binning process, the hyrbid assembly process might take longer time (2-3x). It depends on the complexity of your metagenomic dataset, if too much time consumed, please consider subsampling your short and/or long read dataset. However, the job for hybrid assembly process will be finished for a shorter time as the metagenomic dataset is simplifying. Normally, A workstaion with 40 threads and 256 Gbp RAM is good enough to handle the process. -
As Unicycler is designed for bacterial isolates, the Unicycler author expressly cautions against the use of this assembler for the analysis of data other than bacterial isolates. So we first established the feasibility of using Unicycler to retrieve genomes by different mock datasets (ZymoBIOMICS Microbial Community Standards (EVEN), Nicholls et. al., 2019), Human Microbiome Project (HMP), Kuleshov et. al., 2014 and GIS20, Bertrand et al., 2019). The evaluation results presented the robustness of this assembly tool. However, if you applied Unicycler to assemble high-complexity metagenomes, e.g., soil or activated sludge samples, the performance might be not so positive due to the algorithms and assumptions of Unicycler and SPAdes. To resolve this question, we have also developed the hybrid assembly workflow for highly complex environmental samples and integrated with this iterative hybrid assembly/binning method (
will come soon). -
Here we provided some basic
bash scripts, so it would be easier for the beginer to follow. -
If you are using the IHA workflow for the genome reconstruction, please cite the corresponding reference papers, particullary the paper described the Unicycler!
- 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
- Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads
- If you found IHA workflow useful in your research, a citation would be appreciated!
High-Quality Bacterial Genomes of a Partial-Nitritation/Anammox System by an Iterative Hybrid Assembly Method