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Unicycler assembly of SARS-CoV-2 genome with preprocessing to remove human genome reads

Contributors

Questions

Objectives

last_modification Last modification: Jul 26, 2021

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SARS-CoV-2

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The severe acute respiratory syndrome coronavirus, known as SARS-CoV-2, is a betacoronavirus which belongs to the subfamily Coronavidinae, family Coronavidae.


SARS-CoV-2 genome structure

Structure of the SARS CoV 2 genome, a 5' utr, a polyprotein pp1ab/pp1b, and several structural and accessory proteins before the 3' utr. The pp1ab polyprotein is shown exapnded into a series of non-structural proteins labelled nsp1 to nsp16.


SARS-CoV-2 structure

A graphic of the virus as a sphere with spikes (S1 and S2) coming out from the membrane, envelope proteins embedded within the membrane, and then a nucelocapsid inside. The 3d protein structures are shown in two styles of the spike proteins with the receptor binding domain highlighted. .


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Hybrid assembly

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.reduce70[ 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. ]

Cartoon of hybrid assembly. Step 1 shows short reads from Illumina and long reads from Nanopore. In step 2 these are assembled separately and there are ambiguities in sequence assembly. In step 3, hybrid assembly shows the assembly done with both sets of data and it helps resolve ambiguities with higher coverage.


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Data sources

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Illumina reads

A picture of an illumina sequencer. It is a large white and grey box with a computer screen.

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Oxford Nanopore reads

A picture of someone holding a nanopore device, approximately the size of an oversize usb stick with a chip visible.

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Data sources: Illumina sequencing

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Cartoon of illumina sequence, DNA is fragmented and adapters added. This binds to nanowells with oligonucleotides. Then the DNA bends and attaches to another binder. A primer attaches to the adapter and polymerase adds flourescently tagged dNTPs. Imaging happens while these are added. Then it is split, the DNA strand is denaturalised and now it there are two strands, bound to different adapters in the well. This process is repeated many times.


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Data sources: Nanopore sequencing

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Single nucleic acid molecules pass through a nanopore and changes in electrical field are measured. The magnitude of the current density depends on which nucleotide occupies the nanopore. This produces a graph which is then rad into individual bases.


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Quality control

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Quality control, read trimming and filtering are essential preprocessing steps required to garantee accurate results from RNA-seq datasets. Due to their very different nature, Illumina and Nanopore reads should be processed by using different tools.

Schematic of a workflow, RNA-seq dataset is input which consists of illumina and nanopore reads. Those go through quality assessment with fastp and nanoplot, trimming, and filtering, before producing processed reads.

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Subtraction of reads mapping to the human reference genome

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Since the SARS-CoV-2 samples were obtained from human tissues, it is necessary to retain only the reads that don’t map to the human genome, i.e those of potential viral origin.


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Subtraction of reads mapping to the human reference genome

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.image-75[A set of reads and a human genome are put together, mapping done with bowtie2 or minimap2 to identify reads which map to the human genome. (Then these are removed.). ]

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As with quality control, differential characteristics of Illumina and Nanopore reads require different tools for mapping the reads to the human genome:

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Genome assembly

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Now everything is ready to perform genome assembly!

A picture of a jigsaw puzzle with a DNA image, and several missing pieces scattered around.

Genome assembly is a complex computational process whose objetive is to reconstruct a genome from the reads obtained by sequencing technologies.


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De novo genome assembly

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De novo assembly is a method for constructing genomes from a large number of DNA fragments, with no a priori knowledge of the correct sequence or order of those fragments.

Two common types of de novo assemblers are greedy algorithm assemblers and graph method assemblers.


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De novo genome assembly algorithms

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Greedy algorithm assemblers

It finds overlaps between reads, then builds a consensus sequence from the aligned overlapping reads.

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.image-75[Rainbow coloured reads are aligned locally to make small high quality overlaps. These are then built up into a larger consensus with the entire rainbow.]

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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.

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.image-75[A graph with many nodes connected by lines in a large tangle.]

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Graph methods assemblers: de Brujin graphs

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.reduce70[ 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.

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.image-75[Reads are provided to the algorithm, they are in the colours of the rainbow. Next overlaps are identified and the rainbow resolves itself. A subset of that is highlighted and points to reads connected by overlaps with many arrows going between the bluegreen fragments that are highlighted. This goes to the hamiltonian path identified with a re-run arrow between, indicating some mount of backtracking needed to find the best path. Finally the hamiltonian produces a consensus sequence with the correct final ordering.]


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Graph methods assemblers: de Brujin graphs

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The de Brujin graph assembly tutorial provides a detailed explanation about this topic.


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Assembly genome with Unicycler

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.image-50[unicycler logo. ]

Unicycler is a software tool designed specifically for hybrid assembly of small genomes.


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Assembly genome with Unicycler

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It employs a multi-step process that utilizes a set of software tools.

schematic of the unicycler pipeline, illumina short reads are assembled with spades into an assembly graph in one branch. Another branch brings nanopore reads through miniasm and racon to assemble and polish into long read contigs. Bridge application and contig merging combin the assembly graph and long reads, and this is sent to bowtie2 and pilon for polishing, producing the final assembly.


Key Points

Thank you!

This material is the result of a collaborative work. Thanks to the Galaxy Training Network and all the contributors! Galaxy Training Network This material is licensed under the Creative Commons Attribution 4.0 International License.