Unicycler assembly of SARS-CoV-2 genome with preprocessing to remove human genome reads
Requirements
Before diving into this slide deck, we recommend you to have a look at:
- Sequence analysis
- Quality Control: slides slides - tutorial hands-on
SARS-CoV-2
The severe acute respiratory syndrome coronavirus, known as SARS-CoV-2, is a betacoronavirus which belongs to the subfamily Coronavidinae, family Coronavidae.
Genome characteristics:
- Positive-sense single-stranded RNA (+ssRNA) virus of 30 kb.
- Encode 9860 aminoacids.
- Includes 14 functional ORFs.
- Codify 4 structural proteins and 23 non-structural proteins.
Hybrid assembly
Hybrid assembly consists in using a combination of long and short reads to produce genome sequence.
Long reads are used to resolve ambiguities that exist in genomes previously assembled using the short reads. In addition, low rate-error short reads are used to correct errors that exist in the error-prone long reads.
Subtraction of reads mapping to the human reference genome
As with quality control, differential characteristics of Illumina and Nanopore reads require different tools for mapping the reads to the human genome:
Bowtie2: It is optimized for the read lengths and error modes yielded by typical Illumina sequencers.
Minimap2: It is particularly efficient for mapping Nanopore long reads.
De novo genome assembly algorithms
Greedy algorithm assemblers
It finds overlaps between reads, then builds a consensus sequence from the aligned overlapping reads.
- Relative efficiency
- Do not work well for large read sets because only takes into account local information
- Do not perform well with repeat regions
Graph method assemblers
Basically it represent reads as a set of nodes, and overlaps between these reads as directed edges which connect these nodes to form a complete graph.
- Computationally more expensive
- Aim for global optima
- Perform well on large read sets, specially when they contain repeat regions.
Graph methods assemblers: de Brujin graphs
De Bruijn graphs is the graph model used by most genome assemblers.
During the assembly process reads are broken into smaller fragments of a specified size, the k-mers, whichs are then used as nodes in the graph assembly. Nodes that overlap are then connected by an edge, which represents the reads. An ideal genome assembly corresponds to the path that visits every node exactly once.
Graph methods assemblers: de Brujin graphs
The de Brujin graph assembly tutorial provides a detailed explanation about this topic.
keypoints Key points
Certain types of NGS samples can be heavily contaminated with sequences from other genomes.
Reads from known/expected contaminating sources can be identified by mapping to the respective genomes.
The different characteristics of Illumina and Nanopore sequencing technologies require processing by different tools.
Hybrid genome assembly allows to obtain high quality genome sequences.
Thank You!
This material is the result of a collaborative work. Thanks to the Galaxy Training Network and all the contributors!
| Author(s) |
 
Cristóbal Gallardo
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| Reviewers |
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Tutorial Content is licensed under Creative Commons Attribution 4.0 International License.