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MichaelSchatz_Genome_Sequencing_Assembly.md

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Assembly theory

Assembling a Genome

Why are genomes hard to assemble?

  1. Biological

    (Very) High ploidy, heterozygosity, repeat content

  2. Sequencing

    (Very) large genomes, imperfect sequencing

  3. Computational

    (Very) Large genomes, complex structure

  4. Accuracy

    (Very) Hard to assess correctness

Detangle graph with long reads, mates, and other links

De Bruijn and Overlap graph

Ingredients for a good assembly

  • Coverage

    Typical sequencing coverage

  • Read Length

    Coverage and Read Length

  • Error

  • Repeats composition

Whole Genome Alignment & visualization

Goal of WGA

For two genomes, A and B, find a mapping from each position in A to its corresponding position in B

Not so fast...

Genome A may have insertions, deletions, translocations, inversions, duplications or SNPs with respect to B (sometimes all of the above)

WGA visualization

Genome assemblers

ALLPATHS-LG: recommended for Illumina-only projects

Celera Assembler: recommended for PacBio projects

Assembly Complexity

Long Read Sequencing Technology

  • Moleculo Sequencing

    Clever library preparation technique to turn a short read sequencer into a quazi-long read sequencer

  • PacBio SMRT Sequencing

    Imaging of fluorescently phospholinked labeled nucleotides as they are incorporated by a polymerase anchored to a Zero-Mode Waveguide (ZMW)

  • Oxford Nanopore MinION

Consensus Accuracy and Coverage

Application: Her2 amplified breast cancer

SK-BR-3: Most commonly used Her2-amplified breast cancer cell line

❓ Can we resolve the complex structural varia4ons, especially around Her2?

Improving SMRTcell Performance

Structural variant discovery with long reads

PacBio and Illumina coverage values are highly correlated but Illumina shows greater variance because of poorly mapping reads

Confirmed both known gene fusions in this region

Joint coverage and breakpoint analysis to discover underlying events

Cancer lesion Reconstruction

Oncogene Analysis