Genome Sequencing and Assembly Rui Alves
Dec 28, 2015
Genome Sequencing and Assembly
Rui Alves
Outline of the Talk
• Genomes• Methods for Genome Sequencing
– (Y)BAC-to-(Y)BAC (HSG)– Shotgun Sequencing– Primer walking– Optical mapping– Direct Reads– Polonies– High density reactors
What are genomes?
What are genomes?
• A genome is the whole native DNA content within a cell for any given organism
• Genomes code for all proteins an organism needs to survive
What are genomes?
• Sequencing and annotating a genome reveals the proteins, tRNAs, rRNAs that each organisms possesses to adapt to their environment
Why sequence genomes?
A, B, C, …, Z Dictionary Write/ Read books
ACTG Genes/
Proteins
Understand/
manipulate biology
Genome Sequence
AACTGGTCCAT
Methods for Genome Sequencing
• Methods for Genome Sequencing– (Y)BAC-to-(Y)BAC (HSG)– Shotgun Sequencing– Primer walking– Optical mapping– Direct Reads– Polonies– High density reactors
(Y)BAC-to-(Y)BAC Sequencing
Restriction Enzymes
(Yeast) Bacterial Artificial Chromosome
Replication
(Y)BAC-to-(Y)BAC Sequencing
Same
Restriction
Enzyme
(Y)BAC-to-(Y)BAC Sequencing
Sequence fed
into computer
program
How sequencing works: The Sanger Method
Denaturation
ddNTPc
dNTPc
Repeat with 4 different ddNTPs
Identifying the sequenceA C T G
ATCG
Computer assembly
ACT…GTC CTA …ATC … …GGGG
Methods for Genome Sequencing
• Methods for Genome Sequencing– (Y)BAC-to-(Y)BAC (HSG)– Shotgun Sequencing– Primer walking– Optical mapping– Direct Reads– Polonies– High density reactors
Shotgun Sequencing
Restriction Enzymes
Computer assembly
ACT…GTC CTA …ATC … …GGGG
Comparison
(Y)BAC WGS
p(Recombination of (Y)BACs) >>
Contamination
p(Recombination of plasmids)
Contamination
Less demanding during assembly
Better Orientation of Scaffold Assembly
More demanding during assembly
Worst Orientation of Scaffold Assembly
Better Coverage for Repeats Worst Coverage for Repeats
Methods for Genome Sequencing
• Methods for Genome Sequencing– (Y)BAC-to-(Y)BAC (HSG)– Shotgun Sequencing– Primer walking– Optical mapping– Direct Reads– Polonies– High density reactors
Primer Walking
• Sequencing an initial fragment.
• Use the end of this sequence to design a primer
• Primer will capture next fragment to be sequenced
• Repeat until the end of the chromosome is reached
Primer Walking
ACT…GTC CTA …ATC … …GGGG
New challenges in genome sequencing
• Many organisms have been sequencedFinding intraspecific variations (e.g. SNPs)
Study evolution of strains
• Faster & Cheaper sequencing methods are needed
• These should also be able to deal with single molecules
Methods for Genome Sequencing
• Methods for Genome Sequencing– (Y)BAC-to-(Y)BAC (HSG)– Shotgun Sequencing– Primer walking– Optical mapping– Direct Reads– Polonies– High density reactors
Optical Mapping
Optical Mapping
Original Genome Sequence
Strain 1
Strain n
R1R2
Ri
Determining the new sequence
• Combining the know original genome sequence with Restriction Enzymes specificity predicts mutation
• Small scale re-sequencing of appropriate regions
Methods for Genome Sequencing
• Methods for Genome Sequencing– (Y)BAC-to-(Y)BAC (HSG)– Shotgun Sequencing– Primer walking– Optical mapping– Direct Reads– Polonies– High density reactors
Direct reads
• Bases have different emission spectra
• Fix straight DNA into slide
• run slide through spectophotometer
• Identify each base
Direct Reads
A C T G
A C T G
Methods for Genome Sequencing
• Methods for Genome Sequencing– (Y)BAC-to-(Y)BAC (HSG)– Shotgun Sequencing– Primer walking– Optical mapping– Direct Reads– Polonies– High density reactors
Polonies
• Polonies are colonies of PCR amplicons derived from a single molecule of nucleic acid
Polonies
Pol
Pol
PolPol
Pol
PolPol
Pol
Pol
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NPolony
Sequencing Polonies
dATP
A
dCTP
CC
Methods for Genome Sequencing
• Methods for Genome Sequencing– (Y)BAC-to-(Y)BAC (HSG)– Shotgun Sequencing– Primer walking– Optical mapping– Direct Reads– Polonies– High density reactors
High density reactors
Fragments bound to beads, 1 per bead
PCR of single DNA molecules
(106 clones)
Beads with imobilized enzymes for pyrophosphate sequencing
How are genomes assembled?
• Fragment Chromosome into manageable pieces
• Take fragments
• Feed them into computer programs
• Assembled them onto scaffold
• Fill in the gaps