2. Genome analyses

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Progress in sequencing technology

• • Dideoxy method by fluorescence (Sanger method)
    • Polymerase Chain Reaction
      • See: [Movie] The Polymerase Chain Reaction https://www.youtube.com/watch?v=2KoLnIwoZKU by DNA Learning Center
    • Sanger Sequencing (dideoxy method)
      • See: [Movie] DNA Sanger Sequencing https://www.youtube.com/watch?v=6ldtdWjDwes by DNA Learning Center

Next Generation Sequencing (NGS)

• • Illumina – Massively parallel high throughput by The “sequencing-by-synthesis” technology.
    • See: [Movie] Illumina Sequencing – https://www.youtube.com/watch?v=fCd6B5HRaZ8by Illumina Inc
    • See: What is the Illumina method of DNA sequencing?
  •  PacBio – Single Molecule / Long read
    • See: [Movie] Introduction to SMRT Sequencing by PacBio
  •  Nanopore – portable / Single Molecule / Long read
    • See: [Movie] Sequencing DNA (or RNA) | Real-time, Ultra Long-Reads, Scalable Technology from Oxford Nanopore by Oxford Nanopore Technologies

Big Sequencing projects enabled by the emergence of NGS’s

From 1k to 10K genomes and now more.

• • • • Human deepsequencing / Human microbiome
        • 1000 human genomes project: A Deep Catalog of Human Genetic Variation – See [Movie] Introduction to 1000 Genomes Tutorial Gil McVean
        • UK10K: Rare Genetic Variants in Health and Disease – See [Movie]
          About the 100,000 Genomes Project
        • Human Microbiome Project – See [Movie] Human Microbiome Project by Harvard Biostatistics
      • Other 10k-ordered genomics / metagenomics
        • Genome 10K Project – See: [Movie] The Genome 10K Project
        • Global Genome Biodiversity Network (GGBN) – See: [Movie] The GGI and the GGBN
        • DivSeek – Sequencing project for crop diversity – See: [Movie] What is DivSeek?
        • Earth Microbiome Project – See: [Movie] Earth Microbiome Project: Rick Stevens at TEDxNaperville

Sequence archives in the INSDC

The International Nucleotide Sequence Database Collaboration (INSDC) is a long-standing foundational initiative that operates between DDBJ, EMBL-EBI and NCBI. INSDC covers the spectrum of data raw reads, though alignments and assemblies to functional annotation, enriched with contextual information relating to samples and experimental configurations. (The INSDC site)

Data type	DDBJ	EMBL-EBI	NCBI
Next generation reads	Sequence Read Archive	European Nucleotide Archive (ENA)	Sequence Read Archive
Capillary reads	Trace Archive		Trace Archive
Annotated sequences	DDBJ		GenBank
Samples	BioSample		BioSample
Studies	BioProject		BioProject

• • • DDBJ – DNA Data Bank of Japan
    • ENA – European Nucleotide Archive
    • GenBank – the NIH genetic sequence database

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Training

NCBI Search

NCBI’s comprehensive search system which can find anything related to sequences and articles.

• • 1. Search and open the Search NCBI.
    2. See what kind of databases’ complex.
    3. Find out an accession J00264 from Nucleotide DB for full length cDNA of interleukin-2 (hint: “interleukin-2” AND human AND cDNA … etc.)
    4. Check the description of each FEATURE.
    • Practice: Search papers in Pubmed as well.
    • See: [Movie] My NCBI by NCBI and take advantage of it.

GOLD

A database covering genome projects around the world

See: http://www.genomesonline.org

• • Studies – Metagenomic / Non-Metagenomic
  • Biosamples – Classification / Ecosystems (Host-associated, Engineered, Environmental)
  • Sequencing Projects: Complete Projects / Permanent Drafts / Incomplete Projects / Targeted Projects
  • Analysis Projects: Genome Analysis / Metagenome Analysis / etc.

• • Practice: How many genomes of E. coli O157 are determined? (hint: “Search” function)
  • Practice: How many E. coli genome projects exist?

NCBI Taxonomy

When you want to search biological taxonomy and sequence information

• • http://www.ncbi.nlm.nih.gov/Taxonomy
  • Practice: Search NCBI Taxonomy and GOLD for genome projects of species of your interest. If it doesn’t exist, find the closest organism instead.
  • See: [Movie] NCBI: Retrieve Sequences for an Organism by NCBI

Find and use genome sequence data

• • NCBI genomes – organizes information on genomes including sequences, maps, chromosomes, assemblies, and annotations
  • See: [Movie] Introducing the Genome Data Viewer, NCBI’s Genome Browser by NCBI
  • Ensembl – is a genome browser for vertebrate genomes that supports research in comparative genomics, evolution, sequence variation and transcriptional regulation.
    1. Ensembl Online Training 1 – 2017 by EBI
    2. Ensembl Online Training 2 – 2017 by EBI
    3. Ensembl online training 3 – 2017 by EBI
    4. Ensembl Online Training 4 – 2017 by EBI
    5. Ensembl Online Training 5 – 2017 by EBI
    6. Ensembl Online Training 6 – 2017 by EBI
    7. Ensembl online training 7 – 2017 by EBI

DDBJ’s NGS archive and analysis pipeline

• • NGS data archive: DDBJ SRA (DRA) http://trace.ddbj.nig.ac.jp/dra/
    • Practice: search data on material / methodology you are interested in. (hint: DRA search)

• • DFAST – DDBJ Fast Annotation and Submission Tool
    • Practice: See the Sample Result.

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