1 Falcon https://github.com/PacificBiosciences/pb-assembly

2 Canu assembler http://canu.readthedocs.io/en/latest/index.html

3 Miniasm assembler https://github.com/lh3/miniasm

4 PBJelly scaffolding tool https://sourceforge.net/projects/pb-jelly/

5 ARCS scaffolding tool https://github.com/bcgsc/arcs

6 Redundans reduction and scaffolding tool https://github.com/Gabaldonlab/redundans

7 Arrow error correction https://github.com/PacificBiosciences/ GenomicConsensus

8 PILON error correction https://github.com/broadinstitute/pilon/wiki

9 BUSCO single copy gene markers http://busco.ezlab.org/

10 Bandage graph assembly viewer https://rrwick.github.io/Bandage/

11 Gepard dotter http://cube.univie.ac.at/gepard

12 MUMmer aligner and plotter http://mummer.sourceforge.net/

Address of the bookmark: https://github.com/wtmatlock/flanker

Availability and implementation: The software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/iva

https://pubmed.ncbi.nlm.nih.gov/25725497/

Address of the bookmark: https://github.com/sanger-pathogens/iva

• Homology-based misassembly correction
• Homology-based assembly scaffolding and patching
• Scaffold merging

Address of the bookmark: https://github.com/malonge/RagTag

1. Merge re-sequenced FastQ files (cat)
2. Read QC (FastQC)
3. Adapter trimming (fastp)
4. Removal of host reads (Kraken 2; optional)
5. Variant calling
   1. Read alignment (Bowtie 2)
   2. Sort and index alignments (SAMtools)
   3. Primer sequence removal (iVar; amplicon data only)
   4. Duplicate read marking (picard; optional)
   5. Alignment-level QC (picard, SAMtools)
   6. Genome-wide and amplicon coverage QC plots (mosdepth)
   7. Choice of multiple variant calling and consensus sequence generation routes (iVar variants and consensus; default for amplicon data || BCFTools, BEDTools; default for metagenomics data)
      • Variant annotation (SnpEff, SnpSift)
      • Consensus assessment report (QUAST)
      • Lineage analysis (Pangolin)
      • Clade assignment, mutation calling and sequence quality checks (Nextclade)
      • Individual variant screenshots with annotation tracks (ASCIIGenome)
   8. Intersect variants across callers (BCFTools)
6. De novo assembly
   1. Primer trimming (Cutadapt; amplicon data only)
   2. Choice of multiple assembly tools (SPAdes || Unicycler || minia)
      • Blast to reference genome (blastn)
      • Contiguate assembly (ABACAS)
      • Assembly report (PlasmidID)
      • Assembly assessment report (QUAST)
7. Present QC and visualisation for raw read, alignment, assembly and variant calling results (MultiQC)

You can try this here, or try it later on your own data.

Get data

We will use the same Illumina data as we used above:

• illumina_R1.fastq.gz: the Illumina forward reads
• illumina_R2.fastq.gz: the Illumina reverse reads

Assemble

Run Spades:

spades.py -1 illumina_R1.fastq.gz -2 illumina_R2.fastq.gz --careful --cov-cutoff auto -o spades_assembly_all_illumina

• -1 is input file of forward reads
• -2 is input file of reverse reads
• --careful minimizes mismatches and short indels
• --cov-cutoff auto computes the coverage threshold (rather than the default setting, “off”)
• -o is the output directory

Results

Move into the output directory and look at the contigs:

infoseq contigs.fasta

Address of the bookmark: https://www.ncbi.nlm.nih.gov/nuccore/ON563414

MitoHiFi was first developed to assemble the mitogenomes for a wide range of species in the Darwin Tree of Life Project (DToL)

https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-023-05385-y 

Address of the bookmark: https://github.com/marcelauliano/MitoHiFi

Key Findings of the Study:

1. Alu Insertion and Tail Loss:
   The researchers discovered an Alu-mediated genetic change in a common ancestor of modern apes and humans. This change disrupted the normal function of a gene involved in tail development, leading to the suppression of tail formation.

2. Gene Disruption Mechanism:
   The Alu insertion was found within a regulatory region of the TBXT gene (also known as T or Brachyury), which is crucial for tail development in vertebrates. This insertion likely altered the gene's expression patterns, leading to tail reduction over evolutionary time.

3. Functional Evidence from Model Organisms:
   To test their hypothesis, the researchers introduced similar genetic modifications in mice. The modified mice exhibited shortened or absent tails, supporting the idea that the identified mutation played a role in tail loss in hominoids.

4. Evolutionary Implications:
   The findings suggest that small, random genomic changes—such as transposable element insertions—can have profound effects on body morphology. This study provides evidence that mobile DNA elements (like Alu) can drive major evolutionary transitions.

5. Relevance to Human Evolution:
   Understanding the genetic basis of tail loss helps in reconstructing the evolutionary history of hominins (the lineage that includes humans and our extinct relatives). It also sheds light on how genetic variations contribute to anatomical diversity among primates.

Significance of the Study:

This research highlights the role of transposable elements in shaping evolutionary traits and provides a concrete genetic explanation for a defining characteristic of humans and great apes. It also demonstrates how mutations in regulatory regions of developmental genes can lead to significant anatomical changes.