High%20Throughput%20Genomic%20DNA%20Sequencing%20and%20Bioinformatics

1
High Throughput Genomic DNA Sequencing and
Bioinformatics
2
The Human Genome Project

• The Human genome is now officially sequenced.
• That was a big job.
• How did they do it?
• Is there anything that a knowledge of
  bioinformatics tells us that we should watch out
  for in the human genome sequence?

3
What is DNA Sequencing?

• A DNA sequence is the order of the bases on one
  strand.
• By convention, we order the DNA sequence from 5
  to 3, from left to right.
• Often, only one strand of the DNA sequence is
  written, but usually both strands have been
  sequenced as a check.

4
DNA Sequencing was Awarded the Nobel Prize

• Walter Gilbert and Fred Sanger were awarded the
  Nobel Prize in Chemistry for the development of
  two different methods of DNA sequencing.
• http//www.nobel.se/chemistry/laureates/1980/
• (Oh yes, and Paul Berg for Recombinant DNA- a big
  year!)

5
Two Methods of DNA Sequencing

• Maxam - Gilbert Method, in which a DNA sequence
  is end-labeled with P-32 phosphate and
  chemically cleaved to leave a signature pattern
  of bands.
• Sanger Method, in which a DNA sequence is
  annealed to an oligonucleotide primer, which is
  then extended by DNA polymerase using a mixture
  of dNTP and ddNTP (chain terminating) substrates.
  This is the main method used now.

6
Sanger Method is a Form of DNA Synthesis

• DNA to be sequenced acts as a template for the
  enzymatic synthesis of new DNA strand starting at
  a defined primer.
• Polymerases used are Pol I type polymerases.
• Incorporation of a dideoxynucleotide blocks
  further synthesis of the new DNA strand.

7
Remember the Rules of In Vivo DNA Replication
8
Remember the Rules of In Vivo DNA Replication
9
How the Reaction Works

• If the DNA is double stranded, the reaction is
  started by heating until the two strands of DNA
  separate.
• Lower the temperature and the primer sticks to
  its intended location by H bonds.
• DNA polymerase starts elongating the primer.
• If allowed to go to completion, a new strand of
  DNA would be the result.

10
How the Reaction Works

• If we start with a billion identical pieces of
  template DNA, we'll get a billion new copies of
  one of its strands.
• We run the reactions, however, in the presence of
  a dideoxyribonucleotide.
• This is just like regular DNA, except it has no
  3' hydroxyl group - once it's added to the end of
  a DNA strand, there's no way to continue
  elongating it.

11
(No Transcript)
12
(No Transcript)
13
Original Sanger Sequencing

• A mixture of dNTPs and a single ddNTP is used in
  the reaction tubes.
• We can start with 4 different reaction tubes,
  each with all four dNTPS (dATP, dGTP, dTTP, dCTP)
  and ONLY one of either ddA, ddC, ddG and ddT
  (only 1).
• The key is MOST of the nucleotides are regular
  ones, and just a fraction of them are
  dideoxynucleotides.

14
An Example of a T tube

• MOST of the time when a 'T' is required to make
  the new strand, the enzyme will get a good one
  and it continues to elongate.
• MOST of the time after adding a T, the enzyme
  will go ahead and add more nucleotides.
• However, about 1 of the time, the enzyme will
  get a dideoxy-T, and that strand can never again
  be elongated.
• It eventually breaks away from the enzyme,
  leaving a dead end DNA that cant be further
  extended.

15
Original Sanger Sequencing

• Sooner or later ALL of the copies will get
  terminated by a T.
• But each time the enzyme makes a new strand, the
  place it gets stopped will be random.
• In millions of starts, there will be strands
  stopping at every possible T along the way.

16
Specific Primers Start the Sequence

• ALL of the strands we make started at one exact
  position.
• ALL of them end with a T. There are billions of
  them ... many millions at each possible T
  position.
• To find out where all the T's are in our newly
  synthesized strand, all we have to do is find out
  the sizes of all the terminated products!

17
(No Transcript)
18
Non-Radioactive DNA Labels

• Add a chemical tag to each ddNTP that can emit a
  fluorescent color when excited by a laser.
• We can add a different dye to each ddNTP and each
  is excited by a different laser wave length.
• Run the reactions in only one tube, not 4 tubes!
• This is easier and faster. A big contribution to
  high throughput sequencing.

19
Automated DNA Sequencing

• We don't even have to 'read' the sequence from
  the gel - the computer does that for us!
• This is a plot of the colors detected in one
  'lane' of a gel (one sample), scanned from
  smallest fragments to largest.
• The computer even interprets the colors by
  printing the nucleotide sequence across the top
  of the plot.
• This is just a fragment of the entire file, which
  would span around 700 or so nucleotides of
  accurate sequence.

20
Automated DNA Sequence Readouts
21
(No Transcript)
22
The Biology of DNA Sequencing

• Virtually all DNA sequencing, (both automated and
  manual) relies on the Sanger method
• DNA replication with dideoxy chain termination
• separation of the resulting molecules by
  polyacrylamide gel electrophoresis.
• The DNA fragment to be sequenced must first be
  cloned into a vector (plasmid or lambda).
• Then the cloned DNA must be copied in a test tube
  (in vitro ) by a DNA polymerase enzyme to obtain
  a sufficient quantity to be sequenced.

23
(No Transcript)
24
Sample DNA Sequence from ABI sequencer
25

• Automated sequencing machines,
• particularly those made by PE Applied
  Biosystems, use 4 colors of dye, so they can read
  all 4 bases at once.

26
Challenges of DNA Sequencing

• One technician with an automated DNA sequencer
  can produce over 20 KB of raw sequence data per
  day.
• The real challenge of DNA sequencing is in the
  analysis of the data

27
J. Craig Venter

• Proposed a whole-genome shotgun sequencing method
  to NIH in 1991. Proposal rejected.
• Sets up The Institute for Genomic Research (TIGR)
  in 1992 (private and non-profit)
• TIGR publishes the first complete genome sequence
  in 1995 (Haemophilis influenzae)
• Forms Celera Genomics in 1998 to sequence human
  genome in three years (private, for-profit)
• The Sequence of the Human Genome is published in
  Science. February 2001
• Venter departs Celera. 2002

28
Human Genome Project Sequencing Strategy

• Clone-based physical mapping
• Digest genome and make Bacterial Artificial
  Chromosomes (BACs, 150,000 bp each)
• Digest BACs to create fingerprints
• Organize BACs to form contigs
• Select BAC clones for sequencing
• Shear BACs and shotgun clone
• Sequence clones and assemble overlaps

29
(No Transcript)
30
(No Transcript)
31
Celera Sequencing Strategy

• Whole-genome shotgun sequencing of five
  individuals with 5 fold coverage
• Computer assembles overlapping sequences to form
  contigs
• Contigs are assembled into scaffolds
• Scaffolds are mapped to the genome by two or more
  Sequence Tagged Site (STS) markers

32
(No Transcript)
33
Technology Breakthroughs

• Development of Expressed Sequence Tag (EST)
  method to discover and map human genes
• Development of Bacterial Artificial Chromosomes
  (BACs) to clone large DNA fragments
• Development of an automated high-throughput
  capillary DNA sequencer in 1998 (Applied
  Biosystems ABI PRISM 3700 DNA Analyzer)
• Development of powerful computers and software to
  analyze sequence data

34
Genome Questions

• Has every base in our genome been sequenced?
• What is the total number of genes and where are
  they located?
• How many genes have an unknown function?
• What percent of our DNA encodes genes and what is
  the remainder?
• Do we share DNA sequences with other organisms?
• How much sequence variation is there between
  individuals?

35
Genome SequencingHTG, GSS,(WGS)
Whole BAC insert (or genome)
shredding
sequencing
cloning isolating
GSS division or trace archive
assembly
Draft Sequence (HTG division)
36
GSS Division Genome Survey Sequences

• Genomic equivalent of ESTs
• BAC and other first pass surveys
• BAC end sequences
• Whole Genome Shotgun (some)
• RAPIDS and other anonymous loci

SP6 end
T7 end
37
Working Draft Sequence
38
Technology Limitations

• Sequences can only be determined in approximately
  400-800 base pair sections known as reads.
• This is due to both the biochemistry of the DNA
  polymerase enzyme and the resolution of
  polyacrylamide gel electrophoresis.
• Most genes contain many thousands of bp and many
  modern sequencing projects are intended to
  produce complete sequences of large genomic
  regions (millions of bp)

39
Assembly of Contigs

• As a result, all sequencing projects must involve
  the division of the target DNA into a set of
  overlapping 500 bp fragments.
• Then these fragments are assembled into complete
  sequences (contigs)
• Contig contiguous sequenced region
• Assembly of overlapping fragments is a
  computational problem

40
(No Transcript)
41
Contig Assembly Problems

• 1) The 500 bp reads of sequence data have errors
  of both incorrectly determined bases and
  insertions/deletions.
• 2) The error rate is highest at the beginning and
  ends of the reads - precisely the regions that
  must be overlapped.
• 3) Some sequence from cloning vectors is often
  included at the ends of sequence reads.

42
Sequence Assembly Algorithms

• Different than similarity searching
• Look for ungapped overlaps at end of fragments
• (method of Wilbur and Lipman, (SIAM J. Appl.
  Math. 44 557-567, 1984)
• High degree of identity over a short region
• Want to exclude chance matches, but not be thrown
  off by sequencing errors
• Vector removal uses similar approach, but less
  stringent
• should recognize small regions of identity and
  tolerate more mismatches

43
Celera Innovation Clone End Tracking

• Create 3 libraries with 2, 10, and 50 KB inserts
• Use information from clone ends distance and
  orientation
• Can span some gaps between contigs and determine
  the size of gaps

44
Overlap at ends, not internal
45
Software determines strategy

• Based on their faith in the speed and
  reliability of sequence analysis/assembly
  software, researchers have generally taken one of
  three different approaches to planning sequencing
  projects
• Ordered sub-cloning
• Primer walking
• Shotgun sequencing

46
Ordered cloning

• People who don't trust software generally put a
  lot of time into dividing large pieces of DNA
  into small ordered overlapping fragments
• This strategy requires much more initial cloning
  work in the laboratory.
• but it minimizes the number of actual sequencing
  reads required to complete a project.
• It is easy to assemble the reads since it is
  known how they should fit together to form the
  final contig.

47
Primer Walking

• Make a new primer from the end of each new
  sequence read
• It requires very fast and accurate analysis of
  sequence reads since each step uses information
  from the previous read
• Skips sub-cloning step entirely since all
  sequencing reactions can be done on one large
  clone
• Expensive to make a lot of PCR primers
• but the price of primer synthesis keeps dropping
  there is an economy of scale
• Assembly problems are minimized since both the
  order and the amount of overlap of reads are known

48
Shotgun Sequencing

• Shotgun sequencing takes maximum advantage of the
  speed and low cost of automated sequencing
• relies totally on software to assembly a jumble
  of essentially random sequence reads into a
  coherent and accurate contig
• TIGR demonstrated proof of concept on the
  genomes of Haemophilus influenzae, Methanococcus
  jannaschii, and Mycoplasma genitalium
• Celera Genomics demonstrated the ability to
  shotgun sequence the entire human genome (?)

49
Human Genome Assembly

• The HGP vs. Celera race to sequence the entire
  human genome was a classic battle of different
  strategies
• The HGP used an ordered cloning approach
• Breaking the genome into mapped BAC clones, then
  shotgun sequencing the BACs
• Celera used a modified shotgun method
• Random clones of various sizes (size selected
  libraries)
• Plus relative mapping of clone ends (they must be
  located in the assembly at the correct distance
  and orientations
• Created custom software to handle the assembly
• Celera did make use of the scaffold built by
  the HGP

50
Other Large Sequencing Projects

• Phylogenetic identification/analysis
• medical studies of bacteria
• environmental samples
• EST sequencing - differential expression
• cDNA studies
• alternate splicing
• full length transcripts
• Genotyping
• score known alleles
• identify new mutations

51
Automation

• The "pipeline" approach
• Vector removal
• Assembly of identical and/or overlapping
  fragments
• Identify genes
• Lookup on genome if fully sequenced organism
• Or genome contigs for partially sequences
  organsims
• BLAST search of GeneBank for similar genes
• Lookup in specialized database of "predicted
  genes"
• ie. ENSEMBL
• Project specific analysis
• differentials between sets
• Phylogenetics

52
DATABASE!!

• What these projects all share is a need to keep
  track of a lot of data
• Hundreds to thousands of sequences
• Many fields of information about each one
• Organism, library, plate ID for each clone
• the sequence itself
• cluster/contig membership
• best BLAST hit (accession , e-value, alignment)
• genome position
• Can't keep track just using folders and text
  files on your hard drive
• Design the database to include all possible
  fields
• (its a lot harder to add info later)

53
Computer tools for sequencing

• A wide variety of different software tools have
  been created to aid DNA sequencing projects.
• Each genome project lab has built its own custom
  software
• UNIX
• Based on a particular workflow design
• PHRED, PHRAP, and Consed
• Many packages for the individual investigator -
  included in most comprehensive molecular
  biology products MacVector, LaserGene, DNA,
  etc.
• I will focus on the assembly tools in GCG.

54
The GCG Fragment Assembly System

• GCG has a complete set of programs that allow
  data entry, and assembly of overlapping
  nucleotide sequence fragments into one contig
• SEQED a single sequence editor
• GELSTART creates fragment assembly projects
• GELENTER adds sequences (reads) to an assembly
  project, input of new sequences from keyboard,
  digitizer, or import of existing text files
• GELMERGE assembles individual sequences into
  contigs, can automatically remove vector
  sequences
• GELASSEMBLE multiple sequence editor for viewing
  and editing contigs, allows manual alignment of
  fragments insertion/deletion of gaps and changing
  of individual bases
• GELDISASSEMBLE breaks up contigs into individual
  sequences within a project
• GELVIEW displays contigs as a schematic display
  of overlapping fragments

55
(No Transcript)
56
SeqLab has a Chromatogram viewer
57
Other Chromatogram Viewers

• Applied Biosystems has a free viewer/editor
  program for sequence chromatograms
• It is called EditView and it is a Macintosh only
  program (does not work in System 9.1 and newer)
• http//cancer-seqbase.uchicago.edu/documents/EditV
  iew.hqx
• There are a couple of viewers for Windows
  machines
• ABIView is free from David H. Klatte
• http//bioinformatics.weizmann.ac.il/software/abiv
  iew/abiinfo.html
• Chromas is 50 shareware from Conor McCarthy,
  Technelysium Pty Ltd in Australiahttp//www.techne
  lysium.com.au/chromas.html

58
The Genome Sequencing Era
59
Complex Genomes Jan. 2003

• Chordates
• Human
• Mouse
• Rat
• Pufferfish
• Sea squirt (Ciona)
• Arthropods
• D. melanogaster
• D. simulans
• A. gambiae

• Higher plants
• Arabidopsis
• Rice
• Fungi
• Aspergillus terreus

60
Coming soon

• In progress
• purple sea urchin
• zebrafish
• NHGRIs Priority Organisms
• Chicken
• Cow
• Dog
• Chimpanzee
• Honeybee
• Tetrahymena
• Oxytrichia
• Several fungi
• Over 100 bacterial genomes

61
Some Books on the Human Genome Project

• The Common Thread A Story of Science, Politics,
  Ethics and the Human Genome by John Sulston,
  Georgina Ferry
• The Gene Masters How a New Breed of Scientific
  Entrepeneurs Raced for the Biggest Prize in
  Biology by Ingrid Wickelgren
• The Genome War How Craig Venter Tried to Capture
  the Code of Life and Save the World by James
  Shreeve

62
Controversy and Issues

• Does human DNA sequence information belong to
  everyone?
• Should publication require the release of all
  data?
• Did Celera use public information to complete the
  human sequence?
• Should a gene or life form be patented?
• Should personal genetic information be protected
  from public release?