1. BME 130 – Genomes
Lecture 5
Genome assembly I
The good old days

2. Administrivia
Homework 1 – on the website today, due Friday; homework policy
Student-led paper discussion; choose groups and pick paper
Guest lecture Friday – Bob Kuhn will demo the UCSC genome browser

3. Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses
Genomics in the news
Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses
Citation: Gilbert C, Feschotte C (2010) Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses. PLoS Biol 8(9): e doi: /journal.pbio

4. Figure 4.10 Genomes 3 (© Garland Science 2007)

5. Figure 4.10 part 1 of 2 Genomes 3 (© Garland Science 2007)

6. Figure 4.10 part 2 of 2 Genomes 3 (© Garland Science 2007)

7. Sequence assembly de novo reference- guided overlap layout consensus
Reference sequence s1 s5 s3 s4 s2 s6

8. Most CPU and memory demanding stage
de novo sequence assembly
Most CPU and memory demanding stage
overlap s1 s2 s3 s4 s5 s6
Phrap: “banded” alignment of reads around k-mer matches; tolerate alignment mismatches of low-quality bases
Phusion: group reads sharing >= 11 k-mers of 17 bases
Celera: k-mer seed and extend alignment of reads
Arachne: 24-mer seed and extend alignment of reads
newbler: flowgram similarities (?)

9. de novo sequence assembly
Generate alignments s1 s2 s3 s4 s5 s6 s5 s1 s5
Find connected
components s1 s2 s2 s3 s3 s4 s6 s4 s6
Wide range of strategies for the layout stage, many using mate-pair information

10. de novo Sequence assembly
consensus
PHRAP
Consensus base is base with highest quality score
Quality score for position is based on all reads quality scores s1 s5 s2 s3
PCAP/CAP3
Sum up quality scores for each base take base with highest sum
Quality score for position:
highest sum – all other sums s4 s6

11. Reference-guided sequence assembly Advantages (much) faster
Reference sequence
Advantages
(much) faster
(much) less memory
Disadvantages
Indels/rearragements
Lack of closely related reference
Bias towards reference similarity
Pop M et al., “Comparative Genome Assembly”
Brief Bioinform Sep;5(3):

12. Why is this called a sequence gap and not a physical gap?
Figure 4.11a Genomes 3 (© Garland Science 2007)

13. Closing a physical gap means finding a physical clone to sequence that will span the gap

14. Genomic DNA is template for this PCR
Figure 4.11b Genomes 3 (© Garland Science 2007)

15. Chromosome walking (is slow)
Figure Genomes 3 (© Garland Science 2007)

16. PCR from clone library Insert 1 connects to who?
Figure Genomes 3 (© Garland Science 2007)

17. Figure 4.14 Genomes 3 (© Garland Science 2007)

18. Figure 4.15 Genomes 3 (© Garland Science 2007)

19. Figure 4.15a Genomes 3 (© Garland Science 2007)

20. Figure 4.15b Genomes 3 (© Garland Science 2007)

21. Figure 4.15c Genomes 3 (© Garland Science 2007)

22. Figure 4.15d Genomes 3 (© Garland Science 2007)

23. Assembly can by validated by mate-pair information
Figure Genomes 3 (© Garland Science 2007)

24. Figure 4.16a Genomes 3 (© Garland Science 2007)

25. Figure 4.16b Genomes 3 (© Garland Science 2007)

26. Figure 4.17a Genomes 3 (© Garland Science 2007)

27. Figure 4.17b Genomes 3 (© Garland Science 2007)

28. Figure 4.18 Genomes 3 (© Garland Science 2007)