Xiaole Shirley Liu (lead instructor)
Joshua Starmer
Martin Hemberg
Ting Wang
Feng Yue

Ming Tang
Yang Liu
Jack Kang
Scarlett Ge
Jiazhen Rong
Phillip Nicol
Maartin De Vries

We thank many colleagues in the community, who helped Dr. Liu in prepare the STAT115/215 BIO/BST282 course over the years. 

Address of the bookmark: https://liulab-dfci.github.io/bioinfo-combio/

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.

ancestor -- Any organism, population, or species from which some other organism, population, or species is descended by reproduction.

apomorphy -- specialized (=derived) characters of an organism.

basal group -- The earliest diverging group within a clade; for instance, to hypothesize that sponges are basal animals is to suggest that the lineage(s) leading to sponges diverged from the lineage that gave rise to all other animals.

biological classification -- The orderly arrangement of organisms in hierarchical system that ideally reflects evolutionary history.

cDNA -- Complementary DNA; DNA that is synthesized, by reverse transcriptase, from a Messenger RNA template ( Messenger RNA contains the coded information for protein synthesis).

character -- Heritable trait possessed by an organism.

character state -- characters are usually described in terms of their states, for example: "hair present" vs. "hair absent," where "hair" is the character, and "present" and "absent" are its states.

clade -- A monophyletic taxon; a group of organisms which includes the most recent common ancestor of all of its members and all of the descendants of that most recent common ancestor. From the Greek word "klados", meaning branch or twig.

cladogenesis -- The development of a new clade; the splitting of a single lineage into two distinct lineages; speciation.

cladogram -- A diagram, resulting from a cladistic analysis, which depicts a hypothetical branching sequence of lineages leading to the taxa under consideration. The points of branching within a cladogram are called nodes. All taxa occur at the endpoints of the cladogram.

convergence -- Similarities which have arisen independently in two or more organisms that are not closely related. Contrast with homology. 

crown group -- All the taxa descended from a major cladogenesis event, recognized by possessing the clade's synapomorphy. See: stem group.

derived -- Describes a character state that is present in one or more subclades, but not all, of a clade under consideration. A derived character state is inferred to be a modified version of the primitive condition of that character, and to have arisen later in the evolution of the clade. For example, "presence of hair" is a primitive character state for all mammals, whereas the "hairlessness" of whales is a derived state for one subclade within the Mammalia.

diversity -- Term used to describe numbers of taxa, or variation in morphology. 

evolution -- Darwin's definition: descent with modification. The term has been variously used and abused since Darwin to include everything from the origin of man to the origin of life.

evolutionary tree -- A diagram which depicts the hypothetical phylogeny of the taxa under consideration. The points at which lineages split represent ancestor taxa to the descendant taxa appearing at the terminal points of the cladogram.

expressed sequence tag (EST) -- A partial coding sequence isolated at random from a cDNA library, used for identification and mapping of coding sequences, for discovery of new genes and (by reference to sequence data banks) for discovery of identities with other genes.

extinction -- When all the members of a clade or taxon die, the group is said to be extinct.

genetic marker -- A DNA sequence that can be recognized and thus used to characterize the larger DNA sequence and the chromosome in which it occurs. 

homolog -- A feature that appears similar in two or more taxa with a common ancestor that also possessed that feature.

homology -- Two structures are considered homologous when they are inherited from a common ancestor which possessed the structure. This may be difficult to determine when the structure has been modified through descent.

hypothesis -- A concept or idea that can be falsified by various scientific methods.

ingroup -- In a cladistic analysis, the set of taxa which are hypothesized to be more closely related to each other than any are to the outgroup.

lineage -- Any continuous line of descent; any series of organisms connected by reproduction by parent of offspring.

monophyletic -- Term applied to a group of organisms which includes the most recent common ancestor of all of its members and all of the descendants of that most recent common ancestor. A monophyletic group is called a clade.

outgroup -- In a cladistic analysis, any taxon used to help resolve the polarity of characters, and which is hypothesized to be less closely related to each of the taxa under consideration than any are to each other.

paraphyletic -- Term applied to a group of organisms which includes the most recent common ancestor of all of its members, but not all of the descendants of that most recent common ancestor.

parsimony -- Refers to a rule used to choose among possible cladograms, which states that the cladogram implying the least number of changes in character states is the best.

phylogenetics -- Field of biology that deals with the relationships between organisms. It includes the discovery of these relationships, and the study of the causes behind this pattern.

phylogeny -- The evolutionary relationships among organisms; the patterns of lineage branching produced by the true evolutionary history of the organisms being considered.

plesiomorphy -- A primitive character state for the taxa under consideration.

polarity of characters -- The states of characters used in a cladistic analysis, either original or derived. Original characters are those acquired by an ancestor deeper in the phylogeny than the most recent common ancestor of the taxa under consideration. Derived characters are those acquired by the most recent common ancestor of the taxa under consideration.

polyphyletic -- Term applied to a group of organisms which does not include the most recent common ancestor of those organisms; the ancestor does not possess the character shared by members of the group.

primitive -- Describes a character state that is present in the common ancestor of a clade. A primitive character state is inferred to be the original condition of that character within the clade under consideration. For example, "presence of hair" is a primitive character state for all mammals, whereas the "hairlessness" of whales is a derived state for one subclade within the Mammalia.

radiation -- Event of rapid cladogenesis, believed to occur under conditions where a new feature permits a lineage to move into a new niche or new habitat, and is then called an adaptive radiation.

rank -- In traditional taxonomy, taxa are ranked according to their level of inclusiveness. Thus a genus contains one or more species, a family includes one or more genera, and so on.

relatedness -- Two clades are more closely related when they share a more recent common ancestor between them than they do with any other clade.

repetitive DNA -- Sequences of DNA that are found to be repeated, sometimes thousands of times over.  

reticulation -- Joining of separate lineages on a phylogenetic tree, generally through hybridization or through lateral gene transfer. Fairly common in certain land plant clades; reticulation is thought to be rare among metazoans.

selection -- Process which favors one feature of organisms in a population over another feature found in the population. This occurs through differential reproduction -- those with the favored feature produce more offspring than those with the other feature, such that they become a greater percentage of the population in the next generation.

sister group -- The two clades resulting from the splitting of a single lineage.

stem group -- All the taxa in a clade preceding a major cladogenesis event. They are often difficult to recognize because they may not possess synapomorpies found in the crown group.

sympleisiomorphy – A ancestral character shared by the taxa under consideration

synapomorphy -- A character which is derived, and because it is shared by the taxa under consideration, is used to infer common ancestry (shared derived state).

synteny -- Portions of chromosomes in which gene order is conserved. 

systematics -- Field of biology that deals with the diversity of life. Systematics is usually divided into the two areas of phylogenetics and taxonomy.

taxon -- Any named group of organisms, not necessarily a clade

taxonomy -- The science of naming and classifying organisms. 

Tools

• Parsnp - Core-genome alignment and analysis
• Gingr - Interactive visualization of alignments, trees and variants
• HarvestTools - Archiving and postprocessing

Address of the bookmark: https://harvest.readthedocs.io/en/latest/

Other tools

https://github.com/shenwei356/taxonkit

• ETE, version: 3.1.1
• BioPython, version: 1.73
• taxadb, version: 0.10.1
• TaxonKit, version: 0.5.0

Address of the bookmark: https://pypi.org/project/ete3/3.1.1/

Address of the bookmark: http://www.phytools.org/Cordoba2017/ex/2/Intro-to-phylogenies.html