Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing...
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![Page 1: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/1.jpg)
Genome Sequencing and Assembly
Rui Alves
![Page 2: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/2.jpg)
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
![Page 3: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/3.jpg)
What are genomes?
![Page 4: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/4.jpg)
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
![Page 5: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/5.jpg)
What are genomes?
• Sequencing and annotating a genome reveals the proteins, tRNAs, rRNAs that each organisms possesses to adapt to their environment
![Page 6: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/6.jpg)
Why sequence genomes?
A, B, C, …, Z Dictionary Write/ Read books
ACTG Genes/
Proteins
Understand/
manipulate biology
Genome Sequence
AACTGGTCCAT
![Page 7: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/7.jpg)
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
![Page 8: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/8.jpg)
(Y)BAC-to-(Y)BAC Sequencing
Restriction Enzymes
(Yeast) Bacterial Artificial Chromosome
Replication
![Page 9: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/9.jpg)
(Y)BAC-to-(Y)BAC Sequencing
Same
Restriction
Enzyme
![Page 10: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/10.jpg)
(Y)BAC-to-(Y)BAC Sequencing
Sequence fed
into computer
program
![Page 11: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/11.jpg)
How sequencing works: The Sanger Method
Denaturation
ddNTPc
dNTPc
Repeat with 4 different ddNTPs
![Page 12: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/12.jpg)
Identifying the sequenceA C T G
ATCG
![Page 13: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/13.jpg)
Computer assembly
ACT…GTC CTA …ATC … …GGGG
![Page 14: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/14.jpg)
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
![Page 15: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/15.jpg)
Shotgun Sequencing
Restriction Enzymes
![Page 16: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/16.jpg)
Computer assembly
ACT…GTC CTA …ATC … …GGGG
![Page 17: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/17.jpg)
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
![Page 18: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/18.jpg)
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
![Page 19: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/19.jpg)
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
![Page 20: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/20.jpg)
Primer Walking
ACT…GTC CTA …ATC … …GGGG
![Page 21: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/21.jpg)
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
![Page 22: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/22.jpg)
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
![Page 23: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/23.jpg)
Optical Mapping
![Page 24: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/24.jpg)
Optical Mapping
Original Genome Sequence
Strain 1
Strain n
R1R2
Ri
![Page 25: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/25.jpg)
Determining the new sequence
• Combining the know original genome sequence with Restriction Enzymes specificity predicts mutation
• Small scale re-sequencing of appropriate regions
![Page 26: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/26.jpg)
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
![Page 27: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/27.jpg)
Direct reads
• Bases have different emission spectra
• Fix straight DNA into slide
• run slide through spectophotometer
• Identify each base
![Page 28: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/28.jpg)
Direct Reads
A C T G
A C T G
![Page 29: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/29.jpg)
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
![Page 30: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/30.jpg)
Polonies
• Polonies are colonies of PCR amplicons derived from a single molecule of nucleic acid
![Page 31: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/31.jpg)
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
![Page 32: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/32.jpg)
Sequencing Polonies
dATP
A
dCTP
CC
![Page 33: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/33.jpg)
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
![Page 34: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/34.jpg)
High density reactors
Fragments bound to beads, 1 per bead
PCR of single DNA molecules
(106 clones)
Beads with imobilized enzymes for pyrophosphate sequencing
![Page 35: Genome Sequencing and Assembly Rui Alves. Outline of the Talk Genomes Methods for Genome Sequencing –(Y)BAC-to-(Y)BAC (HSG) –Shotgun Sequencing –Primer.](https://reader030.fdocuments.in/reader030/viewer/2022033021/56649e595503460f94b52a3d/html5/thumbnails/35.jpg)
How are genomes assembled?
• Fragment Chromosome into manageable pieces
• Take fragments
• Feed them into computer programs
• Assembled them onto scaffold
• Fill in the gaps