This summary, or should I say syllabus, covers all the content of the lectures that were given during Functional Genomics at the Radboud University. The syllabus explains everything in great detail, almost as if a teacher was talking to you.
The human genome project aimed to sequence all of
the 3 billion base pairs in the human genome.
Two major inventions in the field of sequencing were
done in 1977 and 1985. First Sanger Sequencing was
discovered and then PCR was invented. Most of the
human genome was sequenced by means of Sanger
Sequencing. This technique works as follows:
DNA chains are normally elongated at the OH-group
on the ribose.
In Sanger sequencing, DNA is elongated in four
compartments, each compartment contains a
different ddNTP. A ddNTP is a chain terminator,
lacking the prior mentioned OH-group (nowadays
containing a fluorescent tag). Eventually all of the
synthesized DNA fragments are placed on the same
gel, resulting in separation on size.
Sanger Sequencing is still the gold standard in
diagnostics. One drawback of SS is the limited
throughput: #of samples/DNA molecules
Next generation sequencing techniques have been
developed. A potent concept is sequencing by
synthesis:
Illumina Sequencing
DsDNA is fragmented (by sonication or restriction
digestion) into fragments of 200-300 bp
Adapter sequences are ligated to
the ends of the DNA.
These adapters attach to a glass
surface (to complementary small
DNA oligonucleotides)
Then the molecules are locally
amplified (solid phase PCR) to form
clusters.
The ssDNA fragments become
dsDNA by DNA polymerase,
incorporating reversible fluorescent
chain terminators. After each cycle
the glass surface is imaged and the
fluorophore is cleaved off.
, By Kayleigh S.
The short read sequences are then aligned to the reference genome, that was created by the human
genome project.
Sequencing by Ligation (SOLiD)
With this technique eventually every base position is measured twice, resulting in a higher accuracy,
however each sequencing run takes about 1-2 weeks. Due to its accuracy it is better at detecting
insertions, deletions and SNPs.
Preparation starts with the fragmentation of DNA by means of
nebulization (forcing DNA through hole creating mechanically
sheared fragments), sonication (using ultrasonic waves to create
gas bubbles in the sample and shear DNA by resonance vibration)
or digestion (with restriction enzymes). Then adapter sequences
are ligated to the ends of the fragment. These fragments are then
hybridized to beads and multiplied on the bead by PCR. (these are
now called polonies: polymerase colonies). Filled beads are
captured by polystyrene beads that attach to the second adapter.
In the next step, a probe will be annealed and measured, which is repeated several times.
Eventually the whole fragment has been covered, but that does not mean the sequencing is
completed. You end up now with a sequencing coverage that looks like the one below:
This whole process is repeated four times, but each time the primers start with an offset of 1
base. Even though there are two nucleotides in the beginning, only one of the nucleotides is
determined.
This technique, as many others, has problems with palindromic sequences because they
tend to anneal to themselves, forming hairpin structures.
Again in pictures:
The first base is
known, since it
belongs to the
primer.
, By Kayleigh S.
Ion Torrent
This sequencing technique is based on the fact that for every base that is incorporated, a H+ ion is
released. Each cycle of base addition only contains one type of base. When more than one nucleotide
is inserted the signal increases proportionally. This proportional increase stays accurate to about 6
nucleotides in a row. The advantage of this technique is that it is very fast, however the amount of
reads per chip is small.
Single Molecule Real Time sequencing of PacBio
SMRT uses mixed dNTPs attached to fluorophores. A DNA polymerase is attached to the bottom of a
well and incorporates the dNTPs. Incorporation of a true hit takes longer than the rejection of one.
The disadvantage of this technique is that it is expensive, however it is therefore fast.
Nanopore sequencing
Sequences the DNA by measuring current deviations through the nanopore upon base
incorporation. This technique allows for long read lengths, but is still under development, so only has
moderate accuracy until now.
Targeted genome sequencing is applied mostly when there is already a suspicion to which gene is
mutated.
Depending on what you want to sequence, you should use a different reference genome. In case you
wish to find a germline mutation, you should use the standard reference genome. In case you want
to find a de novo mutation, as is present in cancer, you should use the genome of healthy cells of
that individual.
RNA-sequencing
Can be used to identify the amount of transcription in a cell at a certain moment of time. It could
also easily indicate a mutation in a certain protein. With RNA sequencing the whole exome (of a
given cell type) could be sequenced. The introns are of course not sequenced. The RNA is converted
to DNA by reverse transcriptase.
ChIP sequencing
Chromatin Immunoprecipitation sequencing is used to identify where a certain protein is contacting
the chromatin. This could be useful to identify interactions of transcription factors, RNA
polymerases, histone modifiers etc.
You precipitate the protein of interest by attaching it to a specific antibody. Next, the protein and the
attached DNA is purified. The DNA is then sequenced and placed on the reference genome. Now you
know where the protein was performing its action.
ATAC/DNaseI sequencing
This technique is used to determine which regions in the genome are most accessible. ATAC is short
for Assay for Transposase-Accessible Chromatin. It works as follows: the transposase inserts a piece
of DNA into the genome, but can only do so at positions where the chromatin is accessible. These
pieces of DNA are sequencing adaptors, the accessible DNA can therefore easily be amplified and
sequenced.
, By Kayleigh S.
Epigenomics 1
Genes that are transcribed by RNA polymerase II are regulated by DNA binding factors that activate
or repress transcription. However, in eukaryotic cells the DNA is wrapped around histones and
packed into chromatin. This structure allows for another layer of DNA transcription regulation.
Epigenomics: study of the complete set of epigenetic
modifications on the genetic material of a cell, which is
called the epigenome. Epigenetic modifications are
reversible modifications on the DNA or histones that
affect gene expression but do not alter the DNA
sequence.
Nucleosomes consist of an octamer of histones wrapped
with 142 base pairs of DNA. These nucleosomes form the
repeating unit of chromatin and are made up of 4
different histone types:
H3 + H4 form a dimer + dimer
H2a + H2B form a dimer + dimer, together octamer.
There are also some histone variants, these are believed to
add diversity and possibly have a function in gene
regulation. Some histone types are involved in centromere
formation.
Histones are chemically modified in order to regulate the
gene expression. There are about three categories of
proteins involved in histone regulation.
For each type of protein there are multiple proteins encoded in the genome: about 55 HMTs and 19
HATs. This increases the complexity of variation even more.
Histones can be modified on different amino acids, one of the main modified amino acids are Lysines.
These lysines can be methylated in different extends: mono- di- or trimethylation. This is done by
the KMT, every specific type of lysine methylation requires a special enzyme. The methylation of
lysine forms a binding scaffold for other proteins.
In contrast to lysine methylation, Histone Acetyl Transferases are less specific for an exact lysine
residue. Lysine acetylation again provides a scaffold for binding of other proteins.
All of these chemical modifications play roles in gene expression, DNA replication and DNA repair.
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