Summary Genome, proteome, metabolome analysis

Laura van den End



1. Genome sequencing
Why sequencing a genome? AUTOMATED BASE CALLING
- Genetic ancestry: relate to parents or not - Used after electrophoresis
- Genetic disorders and health predictions - Automated sequencer reads the signal from each
- Cancer studies: what mutations occur in tumors → fragment as they pass through a detection point.
predictability of the cancer This signal is converted into a digital format
- Conservation: want to capture the diversity that’s - Assigns a specific base to each peak in
out there to understand how its related to the chromatogram, resulting in sequencing data
health of the population - Remarks/problems:
- Reviving species from extinction - Stretch identical bases are problematic
- 96% of Tasmanian tiger genome is sequenced - Further on in the electrophoresis, bases are
- EQ: can you explain why this final 4% is so harder to call.
tricky to sequence? Repetitive parts genome
- EQ: approach to sequence 100%? CYCLE SEQUENCING
- DNA replications in separate tubes for each base.
First generation (SANGER) Each tube contains mix of normal dNTPs and
ddNTPs ⇒ fragments of different lengths, each
Basic sequencing tools ending in specific ddNTPs → gel electrophoresis
- Dideoxy chain-termination sequencing (Sanger) - Taq is heat stable
- You need: - PCR method allows to repeat the process (linear
- A template (replicated thousands of times) amplification in this case, because only one primer
- A short complementary sequence (primer/ is used). Repeating improves the quality of the
read out (25x times stronger signal).
oligonucleotide)
- DNA building blocks - PCR is also used to obtain high amounts of
- DNA polymerase that can extend the primer template. Before this, comprehensive cloning
- (one type) of dideoxynucleotide (ddATP, methods were needed.
ddGTP, ddCTP, ddTTP) in which the 3’
hydroxyl group in the ribose is replaced by
hydrogen (and which can be fluorescently
labeled (in the past with radioactive P).
- The incorporation of ddNTPs in the reaction used
to terminate the synthesis of a growing DNA
strand, resulting in partially replicated DNA
fragments. This is because DNA polymerase
requires the 3' OH group of the growing chain and
the 5' phosphate group of the incoming dNTP to
create a phosphodiester bond. DNA CLONING
- Still used today - used to sequence the first human - Sequencing via forward and reverse reaction (2
genome primers, match vector sequence) → overlap will
indicate where the sequence continues




Genome, proteome, metabolome analysis



 

, Laura van den End

SEQUENCING LARGER DNA MOLECULES - Generation of second strand sequence reads for all
- Walking method single-stranded areas
- Start sequencing from one end and “walk - Confirmation of final assembly by comparing exp
“ along it base by base and electronic restriction fingerprints (cloned
- New primers are synthesized every 500 nt fragment versus assembled sequence)
- In two directions - Generation of sequence reads for missing areas
(“gaps”)




- Shotgun method
- Random fragmentation of the DNA = smaller
overlapping fragments ⇒ sequence these →
assembly: search largest overlap + set
minimum nt overlap
- Only 1 or 2 universal primers needed
- Sequence more than you need to → get full
coverage


GENOME MAPS AS GUIDES
- Genetic map (linkage map): ordering of
polymorphic genes and molecular markers based
on genetic recombination frequencies ~ cM
- Physical map: ordering of physically identifiable
sequences (restriction analysis, DNA hybridi-
zation) ~ bp




Whole genome sequencing strategies




Physical map = ordered restriction Physical map = “clone contig”
sites


- Building clone contig by chromosome walking

SEQUENCE FINISHING
- Finishing is a manual, time-consuming process!
- Inspection of sequence reads at positions with
poor automated base calling
- Generation of new sequence reads for poor quality
areas


Genome, proteome, metabolome analysis



 

, Laura van den End

Whole genome sequencing analysis 1. DNA preparation: denaturation to produce ss
DNA templates
SEQUENCE ANNOTATION 2. Primer hybridization: short DNA primers are
- Add as much information as possible about the hybridized to the template
genome content → tools (predictions right tool for 3. Enzymatic DNA synthesis: DNA pol → PPi is
organism) released during each nucleotide incorporation
- Find ORF (simple in prokaryotes bc no introns), 4. Apyrase treatment: apyrase is added to degrade
localize exons/introns ⇒ ORF finder unincorporated nucleotides, preventing them
- Find promoter (hard) → experimental approach form interfering with the reaction
- Homology search against databases (BLAST) 5. Luciferase reaction: the released PPI is used in a
series of enzymatic reactions → light generation
GENE IDENTIFICATION IN EUKARYOTES ~ light produced is proportional to nr of
- Splice sites have a distinct mark, but hard to nucleotides incorporated
identify automatically 6. Detection and analysis: emitted light is recorded
- GPC islands: high GC content, often found in in real-time, DNA sequence is determined based
promoter. Their methylation state can cause on the order of nucleotide incorporation events
changes in gene regulation
- Easier to sequence transcribed genes
- Codon usage: certain tRNA’s may be more
“expensive”. If frequency (counts/100) deviates
from expected, the protein might not be real

EST/cDNA SEQUENCING
- Gives experimental confirmation of what protein
is coding
- EST: expressed sequence tag (single-pass sequence
read ⇒ may contain errors!)
- EST libraries contain a lot of redundancy ⇒ EST ADVANTAGES
clustering into “UniGenes” - Real time monitoring → enable dynamic and
- Non-transcribed regions are not included ⇒ gene continuous measurements
regulatory information is missing - High throughput → large scale projects
- Short read length

DISADVANTAGES
- Homopolymer errors: prone to errors in regions
with consecutive identical nucleotides
- Read length limited
- Expensive

454 sequencing
- Uses pyrosequencing technique

GENOME BROWSERS 1. Clonal amplification: emulsion PCR: isolating
- Ensembl tool at EBI → visualized genomes and individual DNA fragments in water droplets
their annotations within an oil emulsion
- Alternative: NCBI Genome data Viewer 2. Template (bead) separation: each fragment was
attached to a DNA capture bead within a water
Second generation (NGS) droplet — sequencing on PicoTiterPlate, where
- Platforms = companies that sell machines that uses each well contained a single DNA-carrying bead
a certain sequence technology or chemistry 3. Pyrosequencing reactions
4. Reading and data analysis: The emitted light
signals during the sequencing reaction were
Pyrosequencing
captured by a camera and used to determine the
- Relies on the detection of released pyrophosphate
DNA sequence — long reads
(PPi) during DNA synthesis

Genome, proteome, metabolome analysis