BME 130 – Genomes

Lecture 5

Genome assembly IThe good old days

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

Genomics in the newsGenomic 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): e1000495. doi:10.1371/journal.pbio.1000495

Figure 4.10 Genomes 3 (© Garland Science 2007)

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

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

Sequence assembly

de novo

reference-guided

overlap layout consensus

s1

s2

s3

s4

s5

s6

s1 s2 s3 s4 s5 s6s1

s2

s5

s3

s4

s6

s1

s2

s5

s3

s4

s6

s1s2

s5 s3 s4s6

Reference sequence

de novo sequence assembly

overlap

s1

s2

s3

s4

s5

s6

s1 s2 s3 s4 s5 s6

Most CPU and memory demanding

stage

Phusion: group reads sharing >= 11 k-mers of 17 bases

Phrap: “banded” alignment of reads around k-mer matches; tolerate alignment mismatches of low-quality bases

Celera: k-mer seed and extend alignment of reads

Arachne: 24-mer seed and extend alignment of reads

newbler: flowgram similarities (?)

Generate alignments s1

s2

s5

s3

s4

s6

de novo sequence assembly

Wide range of strategies for the layout stage, many using mate-pair

information

s1

s2

s3

s4

s5

s6

s1 s2 s3 s4 s5 s6

Find connected

components

s1 s2

s3

s4

s5

s6

consensus

s1

s2

s5

s3

s6

de novo Sequence assembly

s4

PHRAPConsensus base is base with

highest quality score Quality score for position is based

on all reads quality scores

PCAP/CAP3Sum up quality scores for each

base take base with highest sumQuality score for position:

highest sum – all other sums

s1

s2

s5 s3 s4

s6

Reference sequence

Reference-guidedsequence assembly

Advantages(much) faster

(much) less memory

DisadvantagesIndels/rearragements

Lack of closely related referenceBias towards reference similarity

Pop M et al., “Comparative Genome Assembly”Brief Bioinform. 2004 Sep;5(3):237-48.

Figure 4.11a Genomes 3 (© Garland Science 2007)

Why is this called a sequence gap and not a physical gap?

Closing a physical gap means finding a physical clone to

sequence that will span the gap

Figure 4.11b Genomes 3 (© Garland Science 2007)

Genomic DNA is template for this PCR

Figure 4.12 Genomes 3 (© Garland Science 2007)

Chromosome walking(is slow)

Figure 4.13 Genomes 3 (© Garland Science 2007)

PCR from clone libraryInsert 1 connects to who?

Figure 4.14 Genomes 3 (© Garland Science 2007)

Figure 4.15 Genomes 3 (© Garland Science 2007)

Figure 4.15a Genomes 3 (© Garland Science 2007)

Figure 4.15b Genomes 3 (© Garland Science 2007)

Figure 4.15c Genomes 3 (© Garland Science 2007)

Figure 4.15d Genomes 3 (© Garland Science 2007)

Figure 4.16 Genomes 3 (© Garland Science 2007)

Assembly can by validated by mate-pair information

Figure 4.16a Genomes 3 (© Garland Science 2007)

Figure 4.16b Genomes 3 (© Garland Science 2007)

Figure 4.17a Genomes 3 (© Garland Science 2007)

Figure 4.17b Genomes 3 (© Garland Science 2007)

Figure 4.18 Genomes 3 (© Garland Science 2007)