DNA sequencing: methods

1
DNA sequencing methods

• I. Brief history of sequencing
• II. Sanger dideoxy method for sequencing
• III. Sequencing large pieces of DNA
• VI. The 1,000 dollar genome

On WebCT -- The 1000 genome -- review of new
sequencing techniques by George Church
2
Why sequence DNA?

• All genes available for an organism to use -- a
  very important tool for biologists
• Not just sequence of genes, but also positioning
  of genes and sequences of regulatory regions
• New recombinant DNA constructs must be sequenced
  to verify construction or positions of mutations
• Etc.

3
History of DNA sequencing
4
History of DNA sequencing
MC chapter 12
5
Methods of sequencing

1. Sanger dideoxy (primer extension/chain-termination
   ) method most popular protocol for sequencing,
   very adaptable, scalable to large sequencing
   projects
2. Maxam-Gilbert chemical cleavage method DNA is
   labelled and then chemically cleaved in a
   sequence-dependent manner. This method is not
   easily scaled and is rather tedious
3. Pyrosequencing measuring chain extension by
   pyrophosphate monitoring

6

• for dideoxy sequencing you need
• Single stranded DNA template
• A primer for DNA synthesis
• DNA polymerase
• Deoxynucleoside triphosphates and
  dideoxynucleotide triphosphates

7
Primers for DNA sequencing

• Oligonucleotide primers can be synthesized by
  phosphoramidite chemistry--usually designed
  manually and then purchased
• Sequence of the oligo must be complimentary to
  DNA flanking sequenced region
• Oligos are usually 15-30 nucleotides in length

8
DNA templates for sequencing

• Single stranded DNA isolated from recombinant M13
  bacteriophage containing DNA of interest
• Double-stranded DNA that has been denatured
• Non-denatured double stranded DNA (cycle
  sequencing)

9
One way for obtaining single-stranded DNA from a
double stranded source--magnets
10
Reagents for sequencing DNA polymerases

• Should be highly processive, and incorporate
  ddNTPs efficiently
• Should lack exonuclease activity
• Thermostability required for cycle sequencing

11
Sanger dideoxy sequencing--basic method
Single stranded DNA
5
3
3
5
a) Anneal the primer
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Sanger dideoxy sequencing basic method
5
Direction of DNA polymerase travel
b) Extend the primer with DNA polymerase in the
presence of all four dNTPs, with a limited amount
of a dideoxy NTP (ddNTP)
3
13
DNA polymerase incorporates ddNTP in a
template-dependent manner, but it works best if
the DNA pol lacks 3 to 5 exonuclease
(proofreading) activity
14
Sanger dideoxy sequencing basic method
5
3
T
T
T
T
3
5
ddATP in the reaction anywhere theres a T in
the template strand, occasionally a ddA will be
added to the growing strand
ddA
ddA
ddA
ddA
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How to visualize DNA fragments?

• Radioactivity
• Radiolabeled primers (kinase with 32P)
• Radiolabelled dNTPs (gamma 35S or 32P)

• Fluorescence
• ddNTPs chemically synthesized to contain fluors
• Each ddNTP fluoresces at a different wavelength
  allowing identification

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Analysis of sequencing products

• Polyacrylamide gel electrophoresis--good
  resolution of fragments differing by a single
  dNTP
• Slab gels as previously described
• Capillary gels require only a tiny amount of
  sample to be loaded, run much faster than slab
  gels, best for high throughput sequencing

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DNA sequencing gels old school
Different ddNTP used in separate reactions
Analyze sequencing products by gel
electrophoresis, autoradiography
Radioactively labelled primer or dNTP in
sequencing reaction
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cycle sequencing denaturation occurs during
temperature cycles
94CDNA denatures 45C primer
anneals 60-72C thermostable DNA pol extends
primer Repeat 25-35 times Advantages dont
need a lot of template DNA Disadvantages DNA
pol may incorporate ddNTPs poorly
20
Animation of cycle sequencing see http//www.dnai
.org/ Click on manipulation techniques s
orting and sequencing
21
An automated sequencer
The output
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Current trends in sequencing
It is rare for labs to do their own
sequencing --costly, perishable
reagents --time consuming --success rate
varies Instead most labs send out for
sequencing --You prepare the DNA (usually
plasmid, M13, or PCR product), supply the primer,
company or university sequencing center does the
rest --The sequence is recorded by an automated
sequencer as an electropherogram
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BREAK UP THE GENOME, PUT IT BACK TOGETHER
Assemble sequences by matching overlaps
160 kbp
BAC sequence
1 kbp
BAC overlaps give genome sequence
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Sequencing large pieces of DNAthe shotgun
method

• Break DNA into small pieces (typically sizes of
  around 1000 base pairs is preferable)
• Clone pieces of DNA into M13
• Sequence enough M13 clones to ensure complete
  coverage (eg. sequencing a 3 million base pair
  genome would require 5x to 10x 3 million base
  pairs to have a reliable representation of the
  genome)
• Assemble genome through overlap analysis using
  computer algorithms, also polish sequences
  using mapping information from individual clones,
  characterized genes, and genetic markers
• This process is assisted by robotics

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Sequencing done by TIGR (Maryland) and The Sanger
Institute (Cambridge, UK)
Here we report an analysis of the genome
sequence of P. falciparum clone 3D7, including
descriptions of chromosome structure, gene
content, functional classification of proteins,
metabolism and transport, and other features of
parasite biology.
26
Sequencing strategy A whole chromosome shotgun
sequencing strategy was used to determine the
genome sequence of P. falciparum clone 3D7. This
approach was taken because a whole genome shotgun
strategy was not feasible or cost-effective with
the technology that was available at the
beginning of the project. Also, high-quality
large insert libraries of (A - T)-rich P.
falciparum DNA have never been constructed in
Escherichia coli, which ruled out a
clone-by-clone sequencing strategy. The
chromosomes were separated on pulsed field gels,
and chromosomal DNA was extracted
27
The shotgun sequences were assembled into
contiguous DNA sequences (contigs), in some cases
with low coverage shotgun sequences of yeast
artificial chromosome (YAC) clones to assist in
the ordering of contigs for closure. Sequence
tagged sites (STSs)10, microsatellite
markers11,12 and HAPPY mapping7 were also used to
place and orient contigs during the gap closure
process. The high (A /T) content of the genome
made gap closure extremely difficult79.
Chromosomes 15, 9 and 12 were closed, whereas
chromosomes 68, 10, 11, 13 and 14 contained 337
gaps (most less than 2.5 kb) per chromosome at
the beginning of genome annotation. Efforts to
close the remaining gaps are continuing.
28
Methods Sequencing, gap closure and
annotation The techniques used at each of the
three participating centres for sequencing,
closure and annotation are described in the
accompanying Letters79. To ensure that each
centres annotation procedures produced roughly
equivalent results, the Wellcome Trust Sanger
Institute (Sanger) and the Institute for
Genomic Research (TIGR) annotated the
same100-kb segment of chromosome 14. The number
of genes predicted in this sequence by the two
centres was 22 and 23 the discrepancy being due
to the merging of two single genes by one centre.
Of the 74 exons predicted by the two centres, 50
(68) were identical, 9 (2) overlapped, 6 (8)
overlapped and shared one boundary, and the
remainder were predicted by one centre but not
the other. Thus 88 of the exons predicted by the
two centres in the 100-kb fragment were identical
or overlapped.
29
The 1000 dollar genome Venter Foundation
(2003) The first group to produce a technology
capable of a 1000 human genome will win 500,000
X - Prize Foundation no, 5 - 20 million
National Institutes of Health (2004) 70
million grant program to reach the 1000 genome
30
Previous sequencing techniques one DNA molecule
at a time Needed many DNA molecules at a time --
arrays One of these pyrosequencing Cut a
genome to DNA fragments 300 - 500 bases
long Immobilize single strands on a very small
plastic bead (one piece of DNA per bead) Amplify
the DNA on each bead to cover each bead to boost
the signal Separate each bead on a plate with up
to 1.6 million wells
31
Sequence by DNA polymerase -dependent chain
extension, one base at a time in the presence of
a reporter (luciferase) Luciferase is an enzyme
that will emit a photon of light in response to
the pyrophosphate (PPi) released upon nucleotide
addition by DNA polymerase Flashes of light and
their intensity are recorded
32
Extension with individual dNTPs gives a readout
A
B
The readout is recorded by a detector that
measures position of light flashes and intensity
of light flashes
A
B
33
25 million bases in about 4 hours
From www.454.com
APS Adenosine phosphosulfate
34
Height of peak indicates the number of dNTPs added
This sequence TTTGGGGTTGCAGTT
35
DNA sequencing methods

• I. Brief history of sequencing
• II. Sanger dideoxy method for sequencing
• III. Sequencing large pieces of DNA
• VI. The 1,000 dollar genome

On WebCT -- The 1000 genome -- review of new
sequencing techniques by George Church
36
Introduction to bioinformatics

1. Making biological sense of DNA sequences
2. Online databases a brief survey
3. Database in depth NCBI
4. What is BLAST?
5. Using BLAST for sequence analysis
6. Biology workbench, etc.

www.ncbi.nlm.nih.gov www.tigr.org http//workbench
.sdsc.edu
37
Theres plenty of DNA to make sense of
http//www.genomesonline.org/
(2006)
38

• Making sense of genome sequences
• Genes
• Protein-coding
• Where are the open reading frames?
• What are the ORFs most similar to? (What is the
  function/structure/evolution history?)
• RNA
• Non-genes
• Regulation promoters and factor-binding sites
• Transactions replication, repair, and
  segregation, DNA packaging (nucleosomes)

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Sequence output
Raw data
Computer calls GNNTNNTGTGNCGGATACAATTCCCCTCTAGAAA
TAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCACCAC CAC
CACCACCCCATGGGTATGAATAAGCAAAAGGTTTGTCCTGCTTGTGAATC
TGCGGAACTTATTTATGATCCAGAAAG GGGGGAAATAGTCTGTGCCAAG
TGCGGTTATGTAATAGAAGAGAACATAATTGATATGGGTCCTAAGTGGCG
TGCTTTTG ATGCTTCTCAAAGGGAACGCAGGTCTAGAACTGGTGCACCA
GAAAGTATTCTTCTTCATGACAAGGGGCTTTCAACTGCA ATTGGAATTG
ACAGATCGCTTTCCGGATTAATGAGAGAGAAGATGTACCGTTTGAGGAAG
TGGCANTCCANATTANGAGT TAGTGATGCAGCANANAGGAACCTAGCTT
TTGCCCTAAGTGAGTTGGATAGAATTNCTGCTCAGTTAAAACTTCCNNGA
C ATGTAGAGGAAGAAGCTGCAANGCTGNACANAGANGCAGNGNGANAGG
GACTTATTNGANGCAGATCTATTGAGAGCGTT ATGGCGGCANGTGTTTA
CCCTGCTTGTAGGTTATTAAAAGNTCCCGGGACTCTGGATGAGATTGCTG
ATATTGCTAGAGC
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atgttgtatttgtctgaagaaaataaatccgtatccactccttgccctcc
tgataagattatctttgatgcagagaggggggagtacatttgctctgaaa
ctggagaagttttagaagataaaattatagatcaagggccagagtggagg
gccttcacgccagaggagaaagaaaagagaagcagagttggagggccttt
aaacaatactattcacgataggggtttatccactcttatagactggaaag
ataaggatgctatgggaagaactttagaccctaagagaagacttgaggca
ttgagatggagaaagtggcaaattaga
What does this sequence do? Could it encode a
protein?
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Looking for ORFs (Open Reading Frames)using DNA
Strider
42
ORF map

1. Where are the potential starts (ATG) and stops
   (TAA, TAG, TGA)?
2. Which reading frame is correct?

ATG
stop codon
Reading frame 1 appears to encode a protein
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Cautions in ORF identification

• Not all genes initiate with ATG, particularly in
  certain microbes (archaea)
• What is the shortest possible length of a real
  ORF? 50 amino acids? 25 amino acids? Cut-off is
  somewhat arbitrary.
• In eukaryotes, ORFs can be difficult to identify
  because of introns
• Are there other sequences surrounding the ORF
  that indicate it might be functional?
• promoter sequences for RNA polymerase binding
• Shine-Dalgarno sequences for ribosome binding?

44
What is the function of the sequenced gene?
Classical methods -- mutate gene, characterize
phenotype for clues to function (genetics) --
purify protein product, characterize in vitro
(biochemistry)
Comparison to previously characterized genes --
genes sequences that have high sequence
similarity usually have similar functions -- if
your gene has been previously characterized
(using classical methods) by someone else, you
want to know right away! (avoid duplication of
labor)
45
NCBI
NCBI home page --Go to www.ncbi.nlm.nih.gov for
the following pages
Pubmed search tool for literature--search by
author, subject, title words, etc. All databases
a retrieval system for searching several linked
databases BLAST Basic Local Alignment Sequence
Tool OMIM Online Mendelian Inheritance in
Man Books many online textbooks available Tax
Browser A taxonomic organization of organisms
and their genomes Structure Clearinghouse for
solved molecular structures
46
What does BLAST do?

1. Searches chosen sequence database and identifies
   sequences with similarity to test sequence
2. Ranks similar sequences by degree of homology (E
   value)
3. Illustrates alignment between test sequence and
   similar sequences

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Alignment of sequences The principle two
homologous sequences derived from the same
ancestral sequence will have at least some
identical (similar) amino acid residues Fraction
of identical amino acids is called percent
identity Similar amino acids some amino acids
have similar physical/chemical properties, and
more likely to substitute for each other--these
give specific similarity scores in alignments
Gaps in similar/homologous sequences are rare,
and are given penalty scores
48
Homology of proteins Homology similarity of
biological structure, physiology, development,
and evolution, based on genetic
inheritance Homologous proteins statistically
similar sequence, therefore similar functions
(often, but not always)
Alignment of TFB and TFIIB sequences
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High sequence similarity correlates with
functional similarity
enzymes
Non-enzymes
40-20 identity fold can be predicted by
similarity but precise function cannot be
predicted (the 40 rule)
50
Programs available for BLAST searches Protein
sequence (this is the best option) blastp--compare
s an amino acid query sequence against a protein
sequence database tblastn--compares a protein
query sequence against a nucleotide sequence
database translated in all reading frames DNA
sequence blastn--compares a nucleotide query
sequence against a nucleotide sequence
database blastx--compares a nucleotide query
sequence translated in all reading frames against
a protein sequence database tblastx--compares
the six-frame translations of a nucleotide query
sequence against the six-frame translations of a
nucleotide sequence database.
51
BLAST considers all possible combinations of
matches mismatches gaps in any given
alignment Gives the best (highest scoring)
alignment of sequences Three scores 1) percent
identity 2) similarity score 3)
E-value--probability that two sequences will have
the similarity they have by chance (lower number,
higher probability of evolutionary homology,
higher probability of similar function)
52
What is the E-value? The E value represents the
chance that the similarity is random and
therefore insignificant. Essentially, the E value
describes the random background noise that exists
for matches between sequences. For example, an E
value of 1 assigned to a hit can be interpreted
as meaning that in a database of the current size
one might expect to see 1 match with a similar
score simply by chance. You can change the
Expect value threshold on most main BLAST search
pages. When the Expect value is increased from
the default value of 10, a larger list with more
low-scoring hits can be reported.
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E values (continued) From the BLAST
tutorial Although hits with E values much
higher than 0.1 are unlikely to reflect true
sequence relatives, it is useful to examine hits
with lower significance (E values between 0.1 and
10) for short regions of similarity. In the
absence of longer similarities, these short
regions may allow the tentative assignment of
biochemical activities to the ORF in question.
The significance of any such regions must be
assessed on a case by case basis.
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Relationship between E-value and function
Single domain proteins
Multi-domain proteins
E value greater than 10-10, similar structure but
possibly different functions
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What does this sequence do? Cue up BLAST..
Raw data
Computer calls GNNTNNTGTGNCGGATACAATTCCCCTCTAGAAA
TAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCACCAC CAC
CACCACCCCATGGGTATGAATAAGCAAAAGGTTTGTCCTGCTTGTGAATC
TGCGGAACTTATTTATGATCCAGAAAG GGGGGAAATAGTCTGTGCCAAG
TGCGGTTATGTAATAGAAGAGAACATAATTGATATGGGTCCTAAGTGGCG
TGCTTTTG ATGCTTCTCAAAGGGAACGCAGGTCTAGAACTGGTGCACCA
GAAAGTATTCTTCTTCATGACAAGGGGCTTTCAACTGCA ATTGGAATTG
ACAGATCGCTTTCCGGATTAATGAGAGAGAAGATGTACCGTTTGAGGAAG
TGGCANTCCANATTANGAGT TAGTGATGCAGCANANAGGAACCTAGCTT
TTGCCCTAAGTGAGTTGGATAGAATTNCTGCTCAGTTAAAACTTCCNNGA
C ATGTAGAGGAAGAAGCTGCAANGCTGNACANAGANGCAGNGNGANAGG
GACTTATTNGANGCAGATCTATTGAGAGCGTT ATGGCGGCANGTGTTTA
CCCTGCTTGTAGGTTATTAAAAGNTCCCGGGACTCTGGATGAGATTGCTG
ATATTGCTAGAGC
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Find the open reading frame(s) Translate it
MKCPYCKSRDLVYDRQHGEVFCKKCGSILATNLVDSELSRKTKTNDIPRY
TKRIGEFTREKIYRLRKWQKKISSERNLVLAMSELRRLSGMLKLPKYVEE
EAAYLYREAAKRGLTRRIPIETTVAACIYATCRLFKVPRTLNEIASYSKT
EKKEIMKAFRVIVRNLNLTPKMLLARPTDYVDKFADELELSERVRRRTVD
ILRRANEEGITSGKNPLSLVAAALYIASLLEGERRSQKEIARVTGVSEMT
VRNRYKELA
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• BLAST against (go to genomes page)
• -- Microbial genomes
• -- environmental sequences (genomes)
• Results
• Distribution of hits query sequence and
  positions in sequence that gave alignments
• Sequences producing significant alignments
• Accession number (this takes you to the sequence
  that yielded the hit gene or contig)
• Name of sequence (sometimes identifies the gene)
• Similarity score
• E-value
• Alignments arranged by E value, with links to
  gene reports

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1) Homology? the function is only inferred (NOT
known)
Two problems with BLAST
2) Large percentages of coding proteins cannot be
assigned function based on homology
59
For a current list of databases and
bioinformatics tools see Nucleic Acids Research
annual bioinformatics issue (comes out every
January). List of all the databases described,
by category http//www.oxfordjournals.org/nar/da
tabase/cap/
Guide to NCBI see Webct
60
Bioinformaticsmaking sense of biological
sequence

• New DNA sequences are analyzed for ORFs (Open
  Reading Frames protein)
• Any DNA or protein sequence can then be compared
  to all other sequences in databases, and similar
  sequences identified
• There is much more -- a great diversity of
  programs and databases are available

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Massively parallel measurements of gene
expression microarrays

• Defining the transcriptome
• The northern blot revisited
• Detecting expression of many genes arrays
• A typical array experiment
• What to do with all this data?
• Brown and Botstein (1999) Exploring the new
  world of the genome with DNA microarrays Nature
  Genetics 21, p. 33-37.

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(we have this)
genome
(we want these)
DNA
transcriptome
RNA
proteome
protein
63
The value of DNA microarrays for studying gene
expression

1. Study all transcripts at same time
2. Transcript abundance usually correlates with
   level of gene expression--much gene control is at
   level of transcription
3. Changes in transcription patterns often occur as
   a response to changing environment--this can be
   detected with a microarray

64
Detection of mRNA transcripts

• Northern Blot -- immobilize mRNA on membrane,
  detect specific sequence by hybridization with
  one labeled probe--requires a separate blotting
  for each probe
• DNA microarray -- immobilize many probes
  (thousands) in an ordered array, hybridize (base
  pair) with labelled mRNA or cDNA

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Generating an array of probes

• Identify open reading frames (orfs)
• PCR each orf (several for each orf), attach
  (spot) each PCR product to a solid support in a
  specific order (pioneered by Pat Browns lab,
  Stanford)
• Chemically synthesize orf-specific
  oligonucleotide probes directly on microchip
  (Affymetrix)

66
http//derisilab.ucsf.edu/microarray/ (Derisi Lab
at UCSF)
The chip defines the genes you are measuring
The RNA comes from the cells and conditions you
are interested in
The hybridization represents the measurement
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A print head for generating arrays of
probes Print head travels from DNA probe source
(microtiter plate) to solid support (treated
glass slide) Small amount of DNA probe is put on
a specific spot at a specific location Each spot
(DNA probe sequence) has a specific address
Print head
Printing needles
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A yeast array experiment
vegetative
sporulating
Isolate mRNA
Prepare fluorescently labeled cDNA with two
different-colored fluors
read-out
hybridize
72
Example microarray data
Green mRNA more abundant in vegetative cells
Yellow equivalent mRNA abundance in vegetative
and sporulating cells
Red mRNA more abundant in sporulating cells
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What to do with all that data?

• Overarching patterns may become apparent
• Organize data by hierarchical clustering,
  profiling to find patterns
• Display data graphically to allow
  assimilation/comprehension

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(Cell synchronization method)
All yeast cell cycle-regulated genes
(phase in which gene is expressed)
High mRNA levels
low mRNA levels
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MIAME The Minimum Information About a Microarray
Experiment
(6 helps correct for variations in the quantity
of starting RNA, and for variable labelling and
detection efficiencies)
76
(we have this)
genome
(we want these)
DNA
transcriptome
RNA
proteome
protein
77
Analysis of the proteome proteomics

• Which proteins are present and when?
• What are the proteins doing?
• What interacts with what?
• Protein-DNA interactions (chromatin
  immunoprecipitation)
• Protein-protein interactions
• Functions of proteins?
• Phizicky et al. (2003) Protein analysis on a
  proteomic scale Nature 422, p. 208-215

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Which proteins are expressed?

• Classical method
• Detect presence of a specific protein
• Using antibodies or specific assay
• Measure changes in protein levels with changing
  environment, in different tissues
• Very labor intensive, expensive to scale up to
  proteome

79
Massively parallel detection and identification
of proteins

• 2D gel electrophoresis
• Separate proteins in a given organism or tissue
  type by migration in gel electrophoresis
• Identify protein (cut out of gel, sequence or
  mass-spec)
• Pattern of spots like a barcode for hi-throughput
  studies
• Mass spectrometry
• Separate individual proteins from cell by charge
  and mass, individual proteins can be identified
  (but need genome sequence information for this)
• Microarrays isolate things that bind proteins

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2D gel electrophoresis

• 1) Separate proteins on the basis of isoelectric
  point

10
4
This technique is usually done on a long, narrow
gel
81
2D gel electrophoresis
Lay gel containing isoelectrically focused
protein on SDS page gel, separate on the basis of
size
E.coli protein profile From swissprot database,
www.expasy.ch
82
Mass spectrometry for identifying proteins in a
mixture
Liquid chromatography and tandem mass spectrometry
Software for processing data
From J.R. Yates 1998 Mass spectrometry and the
age of the proteome J Mass Spec. 33, p 1-19
83
Defining protein function

• Classical methods
• Define activity of protein, develop an assay for
  activity
• Biochemistry use assay to purify protein from
  cell, characterize structure/function of protein
  in vitro
• Genetics obtain mutants with change in activity,
  characterize phenotype of mutant, obtain
  suppressors to identify genes that interact with
  protein of interest
• Time intensive, expensive

84
Protein activity at the proteome level

• Protein-DNA interactions identifying binding
  sites for DNA-binding proteins regulation of
  gene expression
• Massively parallel screens for activity--protein
  arrays

85
chromatin immunoprecipitation (ChIP)
1) Grow cells, add formaldehyde to cross-link
everything to everything (including DNA to
protein)
2) Lyse cells, break up DNA by shearing
3) Retrieve protein of interest (and the DNA it
is bound to) using specific antibody to that
protein (immunoprecipitation)
4) Determine presence of DNA by quantitative PCR
V. Orlando (2000) TIBS 25, p. 99
86
Massively parallel Ch-IP
PCR, label with fluorescent dyes
87
Protein arrays for function
Proteins immobilized, usually by virtue of a tag
sequence (6 x his tag, biotin, etc.)
Probe all proteins at once for a specific activity
88
Example of a protein microarray
Proteins fused to GST with 6 x histidine tags,
immobilized on Ni matrix Anti-GST tells how
much protein is immobilized on surface Specific
assays identify proteins with specific
activities--calmodulin binding, phosphoinositide
binding
89
(we have this)
genome
(we want these)
DNA
transcriptome
RNA
proteome
protein