https://www.nist.gov/news-events/news/2016/09/nist-releases-new-family-standardized-genomes

Address of the bookmark: https://jimb.stanford.edu/giab/

Address of the bookmark: https://github.com/CMU-SAFARI/SneakySnake

All genomes in the dataset were sequenced to at least 30x coverage using Illumina technology. The sequencing reads were mapped and genotyped using a customized procedure that was optimized for population genetic analysis. The researchers eliminated bias of alleles toward matching the human genome reference sequence, and determined genotypes on a single-sample basis to avoid preferential calling of genotypes from populations that had more individuals represented.

Address of the bookmark: https://www.simonsfoundation.org/simons-genome-diversity-project/

Please cite:

Kim, D., Paggi, J.M., Park, C. et al. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37, 907–915 (2019). https://doi.org/10.1038/s41587-019-0201-4

Address of the bookmark: http://daehwankimlab.github.io/hisat2/download/#h-sapiens

Address of the bookmark: https://github.com/cschin/Peregrine

https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html

Address of the bookmark: https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html

We have sequenced the CHM13hTERT human cell line with a number of technologies. Human genomic DNA was extracted from the cultured cell line. As the DNA is native, modified bases will be preserved. The data includes 30x PacBio HiFi, 120x coverage of Oxford Nanopore, 70x PacBio CLR, 50x 10X Genomics, as well as BioNano DLS and Arima Genomics HiC. Most raw data is available from this site, with the exception of the PacBio data which was generated by the University of Washington/PacBio and is available from NCBI SRA.

A UCSC browser is available for v2.0 (as well as legacy v1.0 and v1.1 versions). An interactive dotplot visualization of all genomic repeats is also available from resgen.io. Known issues identified in the assembly are tracked at CHM13 issues.

MORE at https://github.com/marbl/CHM13

Address of the bookmark: https://www.science.org/doi/10.1126/science.abj6987

Address of the bookmark: https://github.com/legumeinfo/gcv

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.