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/ biology Genome Sequence

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

Repeat with 4 different ddNTPs ddNTPc

Denaturation

dNTPc

Identifying the sequence

A C T G G C T A

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 >> Less demanding during assembly p(Recombination of plasmids) Contamination More demanding during assembly Better Orientation Assembly of Scaffold Worst Orientation Assembly of Scaffold 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 sequenced Finding 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

R1

Optical Mapping

Original Genome Sequence Strain 1 Strain n R2 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 A C C T T G 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 N N Pol N N N N Pol N Pol N N N Pol N N N N N N N N Pol N N N Pol N N N N Polony Pol N N N N Pol N N N

Sequencing Polonies

A CC

dATP dCTP

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 (10 6 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