What is Genome Assembly?

Genome assembly involves piecing together short DNA sequence reads generated by sequencing platforms (e.g., Illumina, PacBio, Oxford Nanopore) into longer, contiguous sequences called contigs. This can be performed as:

• De Novo Assembly: Without a reference genome.
• Reference-Guided Assembly: Using a reference genome to guide the assembly process.

Step 1: Preparing Your Data

Before starting the assembly, ensure that your raw sequencing data is high quality.

1. Input Data

   • Short Reads: Illumina sequencing generates short, accurate reads ideal for scaffolding.
   • Long Reads: PacBio and Nanopore sequencing provide long reads for resolving repetitive regions.

2. Quality Control (QC)
   Use tools like FastQC or MultiQC to assess the quality of your reads:

   fastqc reads.fastq multiqc .

   Look for issues like low-quality bases, adapter contamination, or overrepresented sequences.

3. Read Trimming and Filtering
   Trim low-quality bases and adapters using Trimmomatic or Cutadapt:

   trimmomatic PE reads_R1.fastq reads_R2.fastq trimmed_R1.fastq trimmed_R2.fastq \ ILLUMINACLIP:adapters.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36

Step 2: Choosing an Assembly Strategy

Select an assembly strategy based on your data type:

• Short-Read Assemblers:

  • SPAdes: Popular for microbial genomes.
  • Velvet: Fast for smaller genomes.

• Long-Read Assemblers:

  • Canu: Ideal for long-read datasets.
  • Flye: Versatile for small and large genomes.

• Hybrid Assemblers:

  • MaSuRCA: Combines short and long reads.
  • Unicycler: Optimized for bacterial genomes.

Step 3: Running the Assembly

3.1. SPAdes (Short-Read Assembly)

SPAdes is an excellent choice for small genomes, such as bacteria.

The output includes assembled contigs (contigs.fasta) and scaffolds (scaffolds.fasta).

3.2. Canu (Long-Read Assembly)

Canu is designed for high-error long reads from PacBio or Nanopore.

The output will be in canu_output/genome.contigs.fasta.

3.3. Hybrid Assembly with Unicycler

Unicycler combines short and long reads for improved assemblies.

Step 4: Assessing Assembly Quality

After assembly, evaluate its quality using the following tools:

1. QUAST
   QUAST generates assembly statistics, such as N50, genome size, and GC content:

   quast contigs.fasta -o quast_output

2. BUSCO
   BUSCO checks genome completeness by identifying conserved genes:

   busco -i contigs.fasta -o busco_output -l fungi_odb10 -m genome

3. Assembly Graph Visualization
   Visualize assembly graphs with Bandage:

   Bandage load assembly_graph.gfa

Step 5: Post-Assembly Steps

1. Polishing
   Improve assembly accuracy using tools like Pilon (for short reads) or Racon (for long reads).

   racon long_reads.fasta mapped_reads.sam contigs.fasta > polished_contigs.fasta

2. Scaffolding
   Link contigs into scaffolds using tools like SSPACE or Opera-LG if required.

3. Annotation
   Annotate the assembled genome using Prokka for prokaryotes or Maker for eukaryotes.

   prokka --outdir annotation_output --prefix genome contigs.fasta

Step 6: Sharing and Archiving

1. Submit to Public Repositories
   Share your assembly in databases like NCBI GenBank, ENA, or DDBJ.

2. Metadata Preparation
   Include detailed metadata for your submission, such as organism name, sequencing platform, and coverage.

Best Practices

• Always perform quality checks at each stage to ensure data integrity.
• Use multiple tools to cross-validate results when working with complex genomes.
• Document parameters and software versions for reproducibility.

Conclusion

Genome assembly is a powerful process that transforms raw sequencing data into a coherent representation of an organism’s genome. By following this step-by-step guide, you can successfully assemble genomes and uncover valuable biological insights. Whether you’re assembling a microbial genome or tackling the complexities of a eukaryotic genome, these tools and strategies will set you on the path to success.

We have developed panHiTE, a comprehensive and accurate pipeline for TE detection in large-scale population genomes. It has been successfully applied to hundreds of plant population genomes, demonstrating its effectiveness and scalability.

For detailed instructions, please refer to the panHiTE tutorial.

Address of the bookmark: https://github.com/CSU-KangHu/HiTE

Stable version with pip:

pip3 install --user chromosight

Stable version with conda:

conda install -c bioconda -c conda-forge chromosight

or, if you want to get the latest development version:

pip3 install --user -e git+https://github.com/koszullab/chromosight.git@master#egg=chromosight

Address of the bookmark: https://github.com/koszullab/Chromosight

Address of the bookmark: https://bioinformaticsworkbook.org/dataAnalysis/GenomeAssembly/GeneticMaps/creating-genetic-maps.html

GoJS offers many advanced features for user interactivity such as drag-and-drop, copy-and-paste, in-place text editing, tooltips, context menus, automatic layouts, templates, data binding and models, transactional state and undo management, palettes, overviews, event handlers, commands, and an extensible tool system for custom operations.

GoJS is pure JavaScript, so users get interactivity without requiring round-trips to servers and without plugins. GoJS normally runs completely in the browser, rendering to an HTML5 Canvas element or SVG without any server-side requirements. GoJS does not depend on any JavaScript libraries or frameworks, so it should work with any HTML or JavaScript framework or with no framework at all.          

More at http://gojs.net/latest/index.html

Address of the bookmark: http://gojs.net/latest/index.html

https://en.wikipedia.org/wiki/Sankey_diagram

Address of the bookmark: https://christophergandrud.github.io/networkD3/#sankey

R script that makes a plotly interactive and/or static (png/pdf) dot plot.

Shiny app available for testing

Address of the bookmark: https://github.com/tpoorten/dotPlotly

More recently, we've been enthused about using k-mer based similarity measures and computing and searching k-mer-based sketch search databases for all the things.

But I haven't spent too much talking about using k-mers for taxonomy, although that has become an ahem area of interest recently, if you read into our papers a bit.

In this blog post I'm going to fix this by doing a little bit of a literature review and waxing enthusiastic about other people's work. Then in a future blog post I'll talk about how we're building off of this work in fun! and interesting? ways!

Address of the bookmark: http://ivory.idyll.org/blog/2017-something-about-kmers.html

The position is open for immediate appointment and available for two years and then extendable for additional one year. The applicant will be appointed as Research Associate based on qualifications as detailed below:

Research Associate:

-Master’s degree with bioinformatics with at least 2 years of research experience in Next Generation sequencing data analysis as evidence from Fellowship/ Associateship / Training / other engagements.

-Familiarity with bioinformatics tools, database development, programming skills

-Minimum 1 publication in any peer reviewed journal

Salary will be as per ICAR rules and guidelines. Application will be shortlisted based on CV, reference letters from mentors and telephonic interview. Candidates will be called for a personal interview at Bangalore before appointment. No travel expense will be provided for attending interview at Bangalore.

Interested candidates may send a Letter of Interest and CV by email to: keshav@ibioinformatics.org before September 29, 2015.

http://sfg.stanford.edu/denovo.html

http://sfg.stanford.edu/sequencing.html

http://sfg.stanford.edu/BLAST.html

http://sfg.stanford.edu/denovo.html 

Address of the bookmark: http://sfg.stanford.edu/guide.html