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

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NN

N

N

N

N

N

N

N

N

N

N

N

N

N

N

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