Yuki Juan2003.5.5

Automatic DNA and Genome Sequencing

Genetic Mapping

Automated DNA Sequencing

Principle of Sanger SequencingHigh-Throughput SequencingReading Sequence TracesContig AssemblyEmerging Sequencing Methods

http://www.mun.ca/biology/scarr/4241chaptertwo/Biology4241chaptertwo/

Chapter2GenomeSequencingandAnnotation.htm#sanger

The First Cycle in PCR

The Second Cycle in PCR

The Third Cycle in PCR

Basic chain terination method developed in 1974 by Frederick Sanger

The Principle of Dideoxy (Sanger) Sequencing

Strategy of the Chain-termination Method for Sequencing DNA

Strategy of the Chain-termination Method for Sequencing DNA

Fluorescence Detection

High-Throughput Sequencing

The new techniques and equipment include:

Four-color fluorescent dyes have replaced the radioactive label.Automatical trace readingImprovement in the chemistry of template purification and the sequencing reaction.Capillary electrophresis

Automated Sequencing Method

Automated Sequencing Method

ABI PRISM® 3700 DNA Sequencer

ABI PRISM® 3700 DNA Sequencer

Price: $65,50A fully automated, multi-capillary electrophoresis instrument designedAutomatically analyze multiple runs of 96 samples

MegaBACE 1000 DNA Sequencer

MegaBACE 1000 DNA Sequencer

An automated machine capable of high-throughput DNA analysis, processing 96 samples in just a few short hours.Applications :

DNA sequencinggenotyping 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.

Base-callingUsing automated softwarePhred program developed at the University of Washington.

Reading Sequence Traces

Phred Programuse algorithms to convert trace files into base sequences and assign quality values to

each base call in the sequence

The Phred Base-calling Algorithm

SNP: Single nucleotide polymorphism

Automated Sequence Chromatograms

Phred Quality Value Distributions

Dark blue: Bases 100-400 in each sequenceLight blue: All basesThe predicted error rate increases for longer fragments

Contig: A contiguous (touching; adjoining) stretch of cloned DNAThe 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 assemblerconsed graphic editor

Contig Assembly

Phrap assemblerhttp://www.mrc-lmb.cam.ac.uk/pubseq/manual/gap4_unix_90.ht

ml

An aligned reads window in Consed

Alignment algorithms

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

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 mismatchesweighting attached to quality scores of bases from contributing sequencweighting attached to frequency of mismatches

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

Emerging Sequencing Methods

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

Sequencing by Hybridization (SBH)

fragmented oligonucleotides can be identified by time of flight through a vacuum chamberUseful for fragmented DNA molecules under 50 bases longLikely possible to determine full sequence of molecule divided into all possible oligonucleotidesMethods fast, and should become cheap

Mass Spectrophotometric Sequences

Can observe bumps in ssDNA, but not resolve basesPossibly hybridize molecule to Oligonucleotides with bulky modified side groups

Direct Visualization of Single DNA Molecules by AFM

Extremely fast and relatively cheapCan accommodate long DNA fragmentsNanopore sequencing

Single Molecule Sequencing Technique

Single-molecule Nanopore Sequencing

Protein pore channel in electrically polarized membraneSingle DNA molecule pulled through by electrophoresesNucleotides transiently block ion movement, resulting in drop in current resolutioIf slowed to about 1 base per millisecond, could sequence 1Kb per second, three orders of magnitude faster than capillary sequencers

Nanopore Sequencing

Can suspend long strand of DNA in a vacuum by molecular tweezersExonuclease molecule cuts off single nucleotides to be read by fluorescent signal or imprinting on grid

Single Nucleotide Cutting

Hierarchical SequencingShotgun SequencingSequence Verification

Genome Sequencing

Hierarchical versus Shotgun Sequencing

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

Also known as top down,  map based, or clone by clone sequencing Steps involved:

Shear DNA into manageable units (50 - 200 kb)

* This is accomplished by sonication* Amplification (PCR)* Clone into vector of choice (BAC'S usually)Create DNA library

* aim for 5-10x redundancySelection of a tiling path

Hierarchical Sequencing

Cloning Vectors Using in Genome Sequencing

Hierarchical Assembly of a Sequence-contig Scaffold

The Tiling Path

Cab be assembled using a combination of three methods

HybridizationFingerprintingEnd-sequencing

Create probes for specific sequencesOften uses robots to replicate plate clones that show probe hybridizationThe 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.

Hybridization

Use restriction digest profile to determine sequence overlapDone by complex computer algorithms

Fingerprinting

Alignment BAC clones by Hybridization and Fingerprinting

End-sequencing

Sequence the end of BAC clonesCreate a probe for that end sequence, and hope that it hybridizes near the middle of another clone

3 steps: Filtering

Removal of contaminating fragmentsThey may be bacterial in origin, or clones that show evidence of recombination.

Assembling the Layout generating and ordering each BAC contigPosition 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

Assembly of The Draft Genome

Computer algorithms are used to assemble contigs from thousands of overlapping sequences

Shotgun Sequencing

ScreenerOverlapperUnitiggerScaffolder

Tasks performed by Computational Algorithms

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

Screener

Compares every unscreened read against every other unscreened readIs essentially the same as performing a BLAST searchSearches for overlap of a predetermined length (40 bp for Human Genome Project)

Overlapper

Blast Output

A local Alignment

Unitigger

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

U-unitigs and Repeat Resolution

Scaffolder

Uses mate-pair information to link U-unitigs into scaffold contigsMost 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 unitigsIf 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

Assembly of a Mapped Scaffold

Proportion of Fly and Human Genomes in Large Scaffolds

CompletenessAccuracyValidity of assembly

Sequence Verification

Alignment of Two Draft Human Genome Assemblies