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BASICS ON MOLECULAR BIOLOGY

Cell – DNA – RNA – protein
Sequencing methods
arising questions for handling the data, making sense of it
next two week lectures: sequence alignment and genome
assembly

Cells
•
•

•

2

Fundamental working units of every living system
...

Prokaryotes and Eukaryotes are descended from primitive cells and the results of
3
...


Prokaryotes and Eukaryotes
•

According to the most recent
evidence, there are three
main branches to the tree of
life

•

Prokaryotes include Archaea
(“ancient ones”) and bacteria

•

Eukaryotes are kingdom
Eukarya and includes plants,
animals, fungi and certain
algae
Lecture: Phylogenetic trees,
this topic in more detail

3

All Cells have common Cycles

• Born, eat, replicate, and die
4

Common features of organisms
•

Chemical energy is stored in ATP

•

Genetic information is encoded by DNA

•

Information is transcribed into RNA

•
•

There is a common triplet genetic code
–
some variations are known, however
Translation into proteins involves ribosomes

•

Shared metabolic pathways

•

Similar proteins among diverse groups of organisms

5

All Life depends on 3 critical molecules
• DNAs (Deoxyribonucleic acid)
– Hold information on how cell works

• RNAs (Ribonucleic acid)
– Act to transfer short pieces of information to different parts of cell
– Provide templates to synthesize into protein

• Proteins
– Form enzymes that send signals to other cells and regulate gene
activity
– Form body’s major components
6

DNA structure
•

DNA has a double helix structure
which is composed of
– sugar molecule
– phosphate group
– and a base (A,C,G,T)

•

By convention, we read DNA
strings in direction of
transcription: from 5’ end to 3’
end
5’ ATTTAGGCC 3’
3’ TAAATCCGG 5’

7

DNA is contained in chromosomes

In eukaryotes, DNA is packed into linear chromosomes

In prokaryotes, DNA is usually contained in a single, circular
chromosome

8
http://en
...
org/wiki/Image:Chromatin_Structures
...
wikipedia
...
It is usually only a single strand
...


tRNA linear and 3D view:
10

http://www
...
ucsf
...
gif

DNA, RNA, and the Flow of Information
Replication

Transcription

”The central dogma”

Translation
Is this true?

11

Denis Noble: The principles of Systems Biology illustrated using the virtual heart
http://velblod
...
net/2007/pascal/eccs07_dresden/noble_denis/eccs07_noble_psb_01
...
wikimedia
...
png

Amino acids

13

How DNA/RNA codes for protein?
•

•
•

14

DNA alphabet contains four
letters but must specify protein,
or polypeptide sequence of 20
letters
...


•

Proteins do all essential work for the cell
– build cellular structures
– digest nutrients
– execute metabolic functions
– mediate information flow within a cell and among cellular communities
...


15

Genes
•

“A gene is a union of genomic sequences encoding a coherent set of
potentially overlapping functional products”

•

A DNA segment whose information is expressed either as an RNA
molecule or protein
(translation)

(folding)
MSG …

(transcription)
5’

… a t g a g t g g a …

3’

3’

… t a c t c a c c t …

5’

16

http://fold
...

•

•
•

Prokaryotes are typically haploid:
they have a single (circular)
chromosome
DNA is usually inherited vertically
(parent to daughter)
Inheritance is clonal
– Descendants are faithful copies
of an ancestral DNA
– Variation is introduced via
mutations, transposable
elements, and horizontal transfer
of DNA
Chromosome map of S
...
mgc
...
cn/ShiBASE/circular_Sd197
...
Proc
...
9x109 bases)

•

Reads have to be assembled!

•

28

Problems
•
•

•
•

29

Sanger sequencing error rate per base varies from 1% to 3%1
Repeats in DNA
– For example, ~300 base longs Alu sequence repeated is over million times in
human genome
– Repeats occur in different scales
What happens if repeat length is longer than read length?
Shortest superstring problem
– Find the shortest string that ”explains” the reads
– Given a set of strings (reads), find a shortest string that contains all of them

Sequence assembly and combination locks

•

30

What is common with sequence assembly and opening keypad locks?

Whole-genome shotgun sequence
•

Whole-genome shotgun sequence assembly starts with a large sample of
genomic DNA
1
...

3
...


31

Sample is randomly partitioned into inserts of length > 500 bases
Inserts are multiplied by cloning them into a vector which is used to infect
bacteria
DNA is collected from bacteria and sequenced
Reads are assembled

Assembly of reads with Overlap-LayoutConsensus algorithm
•
•
•

•

32

Overlap
– Finding potentially overlapping reads
Layout
– Finding the order of reads along DNA
Consensus (Multiple alignment)
– Deriving the DNA sequence from the layout
Next, the method is described at a very abstract level, skipping a lot of details

Finding overlaps
•

First, pairwise overlap alignment of
reads is resolved

•

Reads can be from either DNA strand:
The reverse complement r* of each
read r has to be considered

acggagtcc
agtccgcgctt
r1
5’

… a t g a g t g g a …

3’

3’

… t a c t c a c c t …

5’

r2

33

r1: tgagt, r1*: actca
r2: tccac, r2*: gtgga

Example sequence to assemble
5’ – CAGCGCGCTGCGTGACGAGTCTGACAAAGACGGTATGCGCATCG
TGATTGAAGTGAAACGCGATGCGGTCGGTCGGTGAAGTTGTGCT - 3’

• 20 reads:
#
1
2
3
4
5
6
7
8
9
10
34

Read
CATCGTCA
CGGTGAAG
TATGCGCA
GACGAGTC
CTGACAAA
ATGCGCAT
ATGCGGTC
CTGCGTGA
GCGTGACG
GTCGGTGA

Read*
TCACGATG
CTTCACCG
TGCGCATA
GACTCGTC
TTTGTCAG
ATGCGCAT
GACCGCAT
TCACGCAG
CGTCACGC
TCACCGAC

#
11
12
13
14
15
16
17
18
19
20

Read
GGTCGGTG
ATCGTGAT
GCGCTGCG
GCATCGTG
AGCGCGCT
GAAGTTGT
AGTGAAAC
ACGCGATG
GCGCATCG
AAGTGAAA

Read*
CACCGACC
ATCACGAT
CGCAGCGC
CACGATGC
AGCGCGCT
ACAACTTC
GTTTCACT
CATCGCGT
CGATGCGC
TTTCACTT

Finding overlaps
•

Overlap between two reads can
be found with a dynamic
programming algorithm

Overlap(1, 6) = 3
6 ATGCGCAT

– Errors can be taken into account

•

•

12 ATCGTGAT

Dynamic programming will be
discussed more during the next
two weeks
Overlap scores stored into the
overlap matrix
– Entries (i, j) below the diagonal
denote overlap of read ri and rj*

35

1 CATCGTCA

Overlap(1, 12) = 7

1

6

12

3

7

Finding layout & consensus
•

Method extends the assembly
greedily by choosing the best
overlaps

•

Both orientations are considered

•

Sequence is extended as far as
possible

consensus sequence
36

Ambiguous bases

7*
GACCGCAT
6=6* ATGCGCAT
14
GCATCGTG
1
CATCGTGA
12
ATCGTGAT
19
GCGCATCG
13* CGCAGCGC
--------------------CGCATCGTGAT

Finding layout & consensus
•

We move on to next best
overlaps and extend the
sequence from there

•

The method stops when there are
no more overlaps to consider

•

A number of contigs is produced

•

Contig stands for contiguous
sequence, resulting from merging
reads

37

2
CGGTGAAG
10
GTCGGTGA
11
GGTCGGTG
7
ATGCGGTC
--------------------ATGCGGTCGGTGAAG

Whole-genome shotgun sequencing:
summary
Original genome sequence

…

…

Reads
Non-overlapping
read

Overlapping reads
=> Contig

•

Ordering of the reads is initially unknown

•

Overlaps resolved by aligning the reads

•

In a 3x109 bp genome with 500 bp reads and 5x coverage, there are ~107 reads and
~107(107-1)/2 = ~5x1013 pairwise sequence comparisons

38

Repeats in DNA and genome assembly
Two instances of the same repeat

39

Repeats in DNA cause problems in
sequence assembly
•

•

•

Recap: if repeat length exceeds read length, we might not get the correct
assembly
This is a problem especially in eukaryotes
– ~3
...

2
...

–
...
wikipedia
...
, A WholeGenome Assembly of Drosophila,
Science 24, 2000
Genome size 120 Mbp

Sequencing of the Human Genome
•

The (draft) human genome was
published in 2001

•

Two efforts:
– Human Genome Project (public
consortium)
– Celera (private company)

•

HGP: BAC-by-BAC approach

•

Celera: whole-genome shotgun
sequencing

HGP: Nature 15 February 2001
Vol 409 Number 6822

Celera: Science 16 February 2001
Vol 291, Issue 5507

45

Sequencing of the Human Genome
• The (draft) human genome
was published in 2001
• Two efforts:
– Human Genome Project
(public consortium)
– Celera (private company)

• HGP: BAC-by-BAC approach
• Celera: whole-genome
shotgun sequencing

HGP: Nature 15 February 2001
Vol 409 Number 6822

Celera: Science 16 February 2001
Vol 291, Issue 5507

46

Next-gen sequencing: 454
•
•

Sanger sequencing is the prominent first-generation sequencing method
Many new sequencing methods are emerging

•

Genome Sequencer FLX (454 Life Science / Roche)
– >100 Mb / 7
...
5% accuracy / base in a single run
– >99
...

A mixture of DNA fragments with agarose beads
containing complementary oligonucleotides to the
adapters at the fragment ends are mixed in an
approximately 1:1 ratio
...

The resulting beads are decorated with
approximately 1 million copies of the original
single-stranded fragment, which provides
sufficient signal strength during the
pyrosequencing reaction that follows to detect
and record nucleotide incorporation events
...


Next-gen sequencing: Illumina Solexa
•

49

Illumina / Solexa Genome Analyzer
– Read length 35 - 50 bp
– 1-2 Gb / 3-6 day run
– > 98
...
99% accuracy / consensus with 3x coverage

The Illumina sequencing-by-synthesis
approach
...
The cluster strands are extended
by one nucleotide
...
Once imaging is
completed, chemicals that effect cleavage of
the fluorescent labels and the 3 -OH blocking
groups are added to the flow cell, which
prepares the cluster strands for another round
of fluorescent nucleotide incorporation
...
94% accuracy / base
– >99
...
In a manner similar to Roche/454 emulsion PCR amplification, DNA
fragments for SOLiD sequencing are amplified on the surfaces of 1- m magnetic
beads to provide sufficient signal during the sequencing reactions, and are then
deposited onto a flow cell slide
...
Each ligation step
is followed by fluorescence detection, after which a regeneration step removes
bases from the ligated 8mer (including the fluorescent group) and concomitantly
prepares the extended primer for another round of ligation
...
Because each fluorescent group on a ligated 8mer identifies a
two-base combination, the resulting sequence reads can be screened for basecalling errors versus true polymorphisms versus single base deletions by aligning
the individual reads to a known high-quality reference sequence

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