1. Automatic DNA and Genome Sequencing
Yuki Juan

2. Genetic Mapping

3. Automated DNA Sequencing
Principle of Sanger Sequencing
High-Throughput Sequencing
Reading Sequence Traces
Contig Assembly
Emerging Sequencing Methods

6. The First Cycle in PCR

7. The Second Cycle in PCR

8. The Third Cycle in PCR

9. The Principle of Dideoxy (Sanger) Sequencing
Basic chain terination method developed in 1974 by Frederick Sanger

10. Strategy of the Chain-termination Method for Sequencing DNA

11. Strategy of the Chain-termination Method for Sequencing DNA

12. Fluorescence Detection

13. High-Throughput Sequencing
The new techniques and equipment include:
Four-color fluorescent dyes have replaced the radioactive label.
Automatical trace reading
Improvement in the chemistry of template purification and the sequencing reaction.
Capillary electrophresis

14. Automated Sequencing Method

15. Automated Sequencing Method

16. ABI PRISM® 3700 DNA Sequencer

17. ABI PRISM® 3700 DNA Sequencer
Price: $65,50
A fully automated, multi-capillary electrophoresis instrument designed
Automatically analyze multiple runs of 96 samples

18. MegaBACE 1000 DNA Sequencer

19. MegaBACE 1000 DNA Sequencer
An automated machine capable of high-throughput DNA analysis, processing 96 samples in just a few short hours.
Applications :
DNA sequencing
genotyping
fragment analysis.
6 arrays of 16 capillaries with an interior diameter of about 100 µm.
The system uses high-pressure nitrogen gas to inject the capillaries with Linear Polyacrylamide, a denaturing gel.

20. Reading Sequence Traces
Base-calling
Using automated software
Phred program developed at the University of Washington.

21. Phred Program use algorithms to convert trace files into base sequences and assign quality values to each base call in the sequence

22. The Phred Base-calling Algorithm

23. Automated Sequence Chromatograms
SNP: Single nucleotide polymorphism

24. Phred Quality Value Distributions
Dark blue: Bases in each sequence
Light blue: All bases
The predicted error rate increases for longer fragments

25. Contig Assembly
Contig: A contiguous (touching; adjoining) stretch of cloned DNA
The finishing step in sequencing a multi-clone stretch of DNA, and involves alignment, editing, and error correction.
Sequence editing software（from the University of Washington）
phrap assembler
consed graphic editor

26. Phrap assembler http://www. mrc-lmb. cam. ac

27. An aligned reads window in Consed

28. Alignment algorithms
The Needleman-Wunsch method (1970) was the first computationally feasible algorithm for sequence alignment.

30. Alignments based on these algorithms may vary due to differences in the weighting of their default parameters.
weighting of the effects of indels relative to single base mismatches
weighting attached to quality scores of bases from contributing sequenc
weighting attached to frequency of mismatches

31. Emerging Sequencing Methods
Sequencing by Hybridization (SBH)
Mass Spectrophotometric Sequences
Direct Visualization of Single DNA Molecules by Atomic Force Microscopy (AFM)
Single Molecule Sequencing Techniques
Single Nucleotide Cutting

32. Sequencing by Hybridization (SBH)
Uses the complementarity of the two strands of DNA molecules to determine if a match to an oligonucleotides is present in the DNA.
Possible for short sequences

33. Mass Spectrophotometric Sequences
fragmented oligonucleotides can be identified by time of flight through a vacuum chamber
Useful for fragmented DNA molecules under 50 bases long
Likely possible to determine full sequence of molecule divided into all possible oligonucleotides
Methods fast, and should become cheap

34. Direct Visualization of Single DNA Molecules by AFM
Can observe bumps in ssDNA, but not resolve bases
Possibly hybridize molecule to Oligonucleotides with bulky modified side groups

35. Single Molecule Sequencing Technique
Extremely fast and relatively cheap
Can accommodate long DNA fragments
Nanopore sequencing

36. Single-molecule Nanopore Sequencing

37. Nanopore Sequencing
Protein pore channel in electrically polarized membrane
Single DNA molecule pulled through by electrophoreses
Nucleotides transiently block ion movement, resulting in drop in current resolutio
If slowed to about 1 base per millisecond, could sequence 1Kb per second, three orders of magnitude faster than capillary sequencers

38. Single Nucleotide Cutting
Can suspend long strand of DNA in a vacuum by molecular tweezers
Exonuclease molecule cuts off single nucleotides to be read by fluorescent signal or imprinting on grid

39. Genome Sequencing Hierarchical Sequencing Shotgun Sequencing
Sequence Verification

40. Hierarchical versus Shotgun Sequencing

41. Hierarchical versus Shotgun Sequencing
Both processes involve fragmenting the genome and aligning fragments due to overlapping sequences.
Both aim for 5-10x redundancy in sequence representation.
Main difference is that hierarchical sequencing attempts to align large cloned fragments (~100kb) into a tiling path.
shotgun sequencing omits this step. The entire genome is fragmented into small pieces which are then aligned using computer algorithms.
Hierarchical sequencing was the basis of the publicly funded Human Genome Project.
Shotgun sequencing was the basis of the privately funded Human Genome Project

42. Hierarchical Sequencing
Also known as top down,  map based, or clone by clone sequencing
Steps involved:
Shear DNA into manageable units ( kb) * This is accomplished by sonication * Amplification (PCR) * Clone into vector of choice (BAC'S usually)
Create DNA library * aim for 5-10x redundancy
Selection of a tiling path

43. Cloning Vectors Using in Genome Sequencing

44. Hierarchical Assembly of a Sequence-contig Scaffold

45. The Tiling Path Cab be assembled using a combination of three methods
Hybridization
Fingerprinting
End-sequencing

46. Hybridization Create probes for specific sequences
Often uses robots to replicate plate clones that show probe hybridization
The genome can be probed for many different sequences, leading to islands of overlapping clones that will be joined later in the process.
Chromosome walking - use the end sequence of a clone to create a probe for an adjacent clone.

47. Fingerprinting
Use restriction digest profile to determine sequence overlap
Done by complex computer algorithms

48. Alignment BAC clones by Hybridization and Fingerprinting

49. End-sequencing Sequence the end of BAC clones
Create a probe for that end sequence, and hope that it hybridizes near the middle of another clone

50. Assembly of The Draft Genome
3 steps:
Filtering
Removal of contaminating fragments
They may be bacterial in origin, or clones that show evidence of recombination.
Assembling the Layout
generating and ordering each BAC contig
Position of each contig can be confirmed by alignment with previously characterized Sequence Tagged Sites (STS)
Merging
Aligning BAC contigs that are known to be adjacent to each other

51. Shotgun Sequencing
Computer algorithms are used to assemble contigs from thousands of overlapping sequences

52. Tasks performed by Computational Algorithms
Screener
Overlapper
Unitigger
Scaffolder

53. Screener
Masks (marks & hide) sequences that contain repetitive DNA. e.g. Microsatellites, ALU repeats, ribosomal DNA
These sequences are not taken into consideration when determining overlap

54. Overlapper
Compares every unscreened read against every other unscreened read
Is essentially the same as performing a BLAST search
Searches for overlap of a predetermined length (40 bp for Human Genome Project)

55. Blast Output

56. A local Alignment

57. Unitigger
Unitig: a contig formed from a series of overlapping unambigously unique sequences

58. U-unitigs and Repeat Resolution

59. Scaffolder
Uses mate-pair information to link U-unitigs into scaffold contigs
Most of the remaining gaps at this point are due to repeat elements, and can be resolved by the following method:
Unitigs that were not classified as U-unitigs are placed in the gaps.
These are often referred to as overcollapsed unitigs
If their placement is supported by two or more mate-pairs, it is referred to as a ROCK.
If their placement is supported by one mate-pair, it is referred to as a STONE.
Small gaps can be filled in by chromosome walking

60. Assembly of a Mapped Scaffold

61. Proportion of Fly and Human Genomes in Large Scaffolds

62. Sequence Verification
Completeness
Accuracy
Validity of assembly

63. Alignment of Two Draft Human Genome Assemblies