Transcript Bioinformatics tools as JAWB (Just another Western Blot)
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