1. Introduction to Sequencing
BI420 – Introduction to Bioinformatics
Introduction to Sequencing

2. The nuclear genome (chromosomes)

3. The genome sequence
the primary template on which to outline functional features of our genetic code (genes, regulatory elements, secondary structure, tertiary structure, etc.)

4. Completed genomes Humans, mouse ~3,000 Mb D. melanogaster ~120 Mb
C. elegans
~100 Mb
E. coli
~5 Mb

5. Main genome sequencing strategies
Whole-genome shotgun sequencing
Celera Genomics, Inc.
Clone-based shotgun sequencing
Human Genome Project

6. Hierarchical genome sequencing
BAC (bacterial artificial
chromosome)
library construction
clone mapping
shotgun subclone library construction
sequencing
sequence reconstruction (sequence assembly)
Lander et al. Nature 2001

7. BACs
The genome is broken down into chunks kb long, and these are individually integrated into BACs (bacterial artificial chromosomes).
The BACs are then grown in bacterial cells to produce a large number of duplicate copies of the kb chunk.
The original location of each BAC in the human genome is “fingerprinted” based on the lengths of sequences generated when restriction enzymes are applied.

8. Clone mapping – “sequence ready” map
Restriction enzymes (ex. A, B, C, D, E) are applied to the chunk, generating a set of fragments of particular lengths. These provide a “fingerprint” for where the chunk came from in the genome.

9. Hierarchical genome sequencing
BAC library construction
clone mapping
shotgun subclone library construction
sequencing/read processing
sequence reconstruction (sequence assembly)
Lander et al. Nature 2001

10. Shotgun subclone library construction
cloning vector
BAC primary clone
subclone insert
sequencing vector
The BAC sequence is fragmented into short reads, and these are sequenced.

11. Hierarchical genome sequencing
BAC library construction
clone mapping
shotgun subclone library construction
sequencing/read processing
sequence reconstruction (sequence assembly)
Lander et al. Nature 2001

12. Traditional gel-based sequencing
Polymerize off single-strand in the presence of some radioactive dideoxy nucleotides, which cap a DNA sequence and will leave a film signal due to radioactivity. Then run in a gel, which separates segments by length. Repeat 4 times with each of dideoxy A, C, G and T

13. Sequencing using fluorescence
Use dideoxy nucleotides that fluoresce under UV light with a different color for each of A, C, G, T. Instead of gel do electrophoresis in a capillary tube.

14. Robotic automation
Lander et al. Nature 2001

15. Base calling PHRED base = A Q = 40
Software such as PHRED is used to interpret the chromatogram and generate base calls.

16. Vector clipping
Because the sequence was generated in a BAC, there may be some overhangs of bacterial sequence (pink). These should be removed.

17. Hierarchical genome sequencing
BAC library construction
clone mapping
shotgun subclone library construction
sequencing/read processing
sequence reconstruction (sequence assembly)
Lander et al. Nature 2001

18. Sequence assembly PHRAP
Software such as PHRAP is used to find sequences which overlap one another

19. Repetitive DNA may confuse assembly

20. region of low sequence coverage and/or quality
Sequence completion (finishing)
region of low sequence coverage and/or quality
gap
CONSED, AUTOFINISH
Software such as CONSED produces an assembled genome, taking into account base quality.

21. Main genome sequencing strategies
Whole-genome shotgun sequencing
Celera Genomics, Inc.
Clone-based shotgun sequencing
Human Genome Project

22. Whole-genome shotgun sequencing characteristics
PROS
WGS less labor-intensive than clone-based sequencing
Faster
Very effective in re-sequencing projects where the scaffold of the genome is known, e.g. human genome population studies
CONS
WGS has greater uncertainty in mapping of read positions
Difficult to use WGS to de novo assemble genomes that have not previously been sequenced or where structure is not known (seawater sampling, soil sampling, or other metagenomic studies)

23. Current usage
Whole genome shotgun is used for re-sequencing studies, i.e. those where at least one individual of the species has already been fully sequenced.
This is because indviduals in a species have very similar genomes, making it easy to assemble any new individual off the existing scaffold.

24. DNA Sequencing: Instructor Demo
Platform: UNIX (bioclass.bc.edu)
Instructions:
Data: bioclass:~marth/CLONE.tar.gz or