Sequencing strategies Hierarchical genome shotgun HGS Human Genome Project map first sequence second clonebyclone cloning is performed twice BAC plasmid Sequencing strategies ID: 294330
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Slide1
Last lecture summarySlide2
Sequencing strategies
Hierarchical genome shotgun HGS – Human Genome Project
“map first, sequence second”clone-by-clone … cloning is performed twice (BAC, plasmid)Slide3
Sequencing strategies
Whole genome shotgun WGS – Celera
shotgun, no mapping
Coverage
- the average number of reads representing a given nucleotide in the reconstructed sequence. HGS: 8, WGS: 20Slide4
Human genome
3
billions bps, ~20 000 – 25 000 genesOnly 1.1 – 1.4 % of the genome sequence codes for proteins.
State of completion:
best estimate – 92.3% is complete
problematic unfinished regions: centromeres, telomeres (both contain highly repetitive sequences), some unclosed gapsIt is likely that the centromeres and telomeres will remain unsequenced until new technology is developed
Genome is stored in databases
Primary database – Genebank
(
http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucleotide
)
Additional
data and
annotation,
tools for visualizing and
searching
UCSCS (
http://genome.ucsc.edu
)
Ensembl (
http://www.ensembl.org
)Slide5
New stuffSlide6
Personal human genomes
Personal genomes had not been sequenced in the Human Genome Project to protect the identity of volunteers who provided DNA samples.
Following personal genomes were available by July 2011:
Japanese male (2010, PMID: 20972442
)
Korean male (2009, PMID: 19470904)Chinese male (2008, PMID: 18987735)Nigerian male (2008, PMID: 18987734)J. D. Watson (2008, PMID: 18421352)J. C. Venter (2007, PMID: 17803354
)
HGP sequence is haploid, however, the sequence maps
of
Venter and Watson
are diploid.Slide7
Next generation sequencing (NGS)
The completion of human genome was just a start of modern DNA sequencing era – “high-throughput next generation sequencing” (NGS).
New approaches, reduce time and cost.Holly Grail of sequencing – complete human genome below $ 1000.Slide8
1
st
and 2nd generation of sequencers
1
st
generation – ABI Prism 3700 (Sanger, fluorescence, 96 capillaries), used in HGP and in CeleraSanger method overcomes NGS by the read length (600 bps)2nd generation - birth of HT-NGS in 2005. 454 Life Sciences developed GS 20 sequencer. Combines PCR with
pyrosequencing
.
Pyrosequencing
–
sequencing-by-synthesis
Relies
on detection of pyrophosphate release on nucleotide incorporation rather than chain termination with
ddNTs
.
The release of pyrophosphate is detected by flash of light (
chemiluminiscence
).
Average read length: 400
bp
Roche GS-FLX 454 (successor of GS 20) used for J. Watson’s genome sequencing.Slide9
3
rd
generation2nd generation still uses PCR amplification which may introduce base sequence errors or favor certain sequences over others.
To overcome this, emerging 3
rd
generation of seqeuencers performs the single molecule sequencing (i.e. sequence is determined directly from one DNA molecule, no amplification or cloning).Compared to 2nd generation these instruments offer higher throughput, longer reads (~1000 bps), higher accuracy, small amount of starting material, lower costSlide10
Moore’s lawSlide11
source
: http://www.genome.gov/27541954
transition to 2
nd
generation
4,905 $
0.054$
5,000$Slide12
Illumina
HiSeq X Ten
14. 1. 2014 Illumina anounced the new HiSeq X Ten Sequencing System.Illumina claims they are enabling the $1,000
genome.
Uses Illumina SBS technology (sequencing-by-synthesis).
It sells for at least $10 million.Slide13
Human Longevity
4. 3. 2014 – Human Longevity was founded by Craig Venter
Its main aim: to slow down the process of ageingThe largest human DNA sequencing operation in the world, capable of processing 40,000 human genomes a
year.
DNA
data will be combined with other data on the health and body composition of the people whose DNA is sequenced, in the hope of gleaning insights into the molecular causes of aging and age-related illnesses like cancer and heart disease.Equipment: 2x Illumina
Hiseq
X TenSlide14
Which genomes were sequenced?
http
://www.ncbi.nlm.nih.gov/sites/genomeGOLD –
Genomes online database
(http://www.genomesonline.org/)information regarding complete and ongoing genome projectsSlide15
Important genomics projects
The analysis of personal genomes has demonstrated, how difficult is to draw medically or biologically relevant conclusions from individual sequences.
More genomes need to be sequenced to learn how genotype correlates with phenotype.
1000
Genomes
project (http://www.1000genomes.org/) started in 2009. Sequence the genomes of at least a 1000 people from around the world to create the detailed and medically useful picture of human genetic
variation.
2
nd
generation of sequencers is used in 1000 Genomes.
10 000 Genomes will start soon.Slide16
Important genomics projects
ENCODE project (ENCyclopedia Of DNA Elements,
http://www.genome.gov/ENCODE/)by NHGRI
identify all functional elements in the human genome sequence
Defined regions of the human genome corresponding to 30Mb (1%) have been selected.
These regions serve as the foundation on which to test and evaluate the effectiveness and efficiency of a diverse set of methods and technologies for finding various functional elements in human DNA.Slide17
Sequence AlignmentSlide18
What is sequence alignment ?
CTTTTCAAGGCTTA
GGCTTATTATTGC
CTTTTCAAGGCTTA
GGCTATTATTGC
CTTTTCAAGGCTTA
GGCT-ATTATTGC
Fragment overlapsSlide19
What is sequence alignment ?
CCCCATGGTGGCGGCAGGTGACAG
CATGGGGGAGGATGGGGACAGTCCGG TTACCCCATGGTGGCGGCTTGGGAAACTT
TGGCGGCTCGGGACAGTCGCGCATAAT
CCATGGTGGTGGCTGGGGATAGTA
TGAGGCAGTCGCGCATAATTCCG
TTACCCCATGGTGGCGGCTGGGGACAGTCGCGCATAATTCCG
CCCCATGGTGGCGGCAGGTGACAG
CATGGGGGAGGATGGGGACAGTCCGG
TTACCCCATGGTGGCGGCTTGGGAAACTT
TGGCGGCTCGGGACAGTCGCGCATAAT
CCATGGTGGTGGCTGGGGATAGTA
TGAGGCAGTCGCGCATAATTCCG
consensusSlide20
Sequence alphabet
side chain charge at physiological pH 7.4
Name
3 letters
1 letter
Positively charged side chains
Arginine
Arg
R
Histidine
His
H
Lysine
Lys
K
Negatively charged side chains
Aspartic Acid
Asp
D
Glutamic Acid
Glu
E
Polar uncharged side chains
Serine
Ser
S
Threonine
Thr
T
Asparagine
Asn
N
Glutamine
Gln
Q
Special
Cysteine
Cys
C
Selenocysteine
Sec
U
Glycine
Gly
G
Proline\
Pro
P
Hydrophobic side chains
Alanine
Ala
A
Leucine
Leu
L
Isoleucine
Ile
IMethionineMetMPhenylalaninePheFTryptophanTrpWTyrosineTyrYValineValV
Adenine
A
Thymine
T
Cytosine
G
Guanine
CSlide21
Sequence alignment
Procedure
of comparing sequencesPoint mutations – easyMore difficult example
However,
gaps
can be inserted to get something like this
ACGTCTGAT
A
CGCC
G
TAT
A
GTCTATCT
ACGTCTGAT
T
CGCC
C
TAT
C
GTCTATCT
AC
G
TC
T
GAT
A
CGCCG
TAT
AGTCTATCT
CT
G
AT
T
CGC
A
TCGTC
TAT
CT
ACGTCTGAT
A
CGCCGTAT
A
GTCTATCT
----CTGAT
T
CGC---AT
C
GTCTATCT
gapless alignment
gapped alignment
insertion × deletion
indel Slide22
Why align sequences – continuation
The draft human genome is available
Automated gene finding is possibleGene: AGTACGTATCGTATAGCGTAA
What does it do?
One approach: Is there a similar gene in another species?
Align sequences with known genesFind the gene with the “best” matchSlide23
Flavors of sequence alignment
pair-wise alignment
× multiple sequence alignmentSlide24
Flavors of sequence alignment
global alignment
× local alignment
global
local
align entire sequence
stretches of sequence with the highest density of matches
are aligned
,
generating islands
of matches or subalignments in the
aligned sequencesSlide25
Evolution
wikipedia.org
common ancestorsSlide26
Evolution of sequences
The sequences are the products of molecular evolution.
When sequences share a common ancestor, they tend to exhibit similarity in their sequences, structures and biological functions.
Similar function
Sequence similarity
Similar 3D structure
Protein1
Protein2
DNA1
DNA2
However, this statement is not a
rule. See
Gerlt JA, Babbitt PC. Can sequence determine function?
Genome
Bio
l.
2000
;1(5) PMID: 11178260
Similar sequences produce similar proteinsSlide27
Homology
During
the time period, the molecular sequences undergo random changes, some of which are selected during the process of evolution.Selected sequences accumulate mutations, they diverge over time
.
Two sequences are
homologous when they are descended from a common ancestor sequence.Traces of evolution may still remain in certain portions of the sequences to allow identification of the common ancestry.Residues performing key roles are preserved by natural selection, less crucial residues mutate more frequently
.Slide28
Orhology, paralogy I
Orthologs
– homologous proteins from different species that possess the same function (e.g. corresponding kinases in signal transduction pathway in humans and mice)
Paralogs
– homologous proteins that have different function in the same species (e.g. two kinases in different signal transduction pathways of humans)
However, these terms are controversially discussed:Jensen RA. Orthologs and paralogs - we need to get it right. Genome Biol. 2001;2(8), PMID: 11532207 and references thereinSlide29
Orthology,
paralogy II
Orthologs – genes separated by the
event
of
speciationSequences are direct descendants of a common ancestor.Most likely have similar domain structure, 3D structure and biological function.Paralogs
– genes
separated by the event of genetic
duplication
Gene duplication
: An extra copy of a gene. Gene duplication is a key mechanism in evolution. Once a gene is duplicated, the identical genes can undergo changes and diverge to create two different genes
.
http://www.globalchange.umich.edu/globalchange1/current/lectures/speciation/speciation.htmlSlide30
Gene duplication
Unequal cross-over
Entire chromosome is replicated twiceThis error will result in one of the daughter cells having an extra copy of the
chromosome.
If this cell fuses with another cell during reproduction, it may or may not result in a viable zygote.
RetrotranspositionSequences of DNA are copied to RNA and then back to DNA instead of being translated into proteins resulting in extra copies of DNA being present within cell.Slide31
Unequal cross-over
Homologous chromosomes are misaligned during meiosis.
The probability
of
misalignment is a function of the degree of sharing
the
repetitive elements.Slide32
Comparing sequences through alignment – patterns of conservation and variation can be identified.
The degree of sequence
conservation in the alignment reveals evolutionary relatedness of different sequencesThe variation between sequences reflects the changes that have occurred during evolution in the form of substitutions and/or indels.
Identifying the evolutionary relationships between sequences helps to characterize the function of unknown sequences.
Protein sequence comparison can identify homologous sequences from common ancestor 1 billions year ago (BYA). DNA sequences typically only 600 MYA.