FU: Les 1 - Sequencing & Genomics

What is functional genomics

A field in molecular biology that attempts to describe gene (and protein) functions & interactions by answering questions about DNA function at the level of gene, RNA transcript and protein products (at a genome-wide approach)

aka: when is a gene expressed, in which tissue, celltype and under what conditions

Shortly describe the principle of Sanger Sequencing

DNA is isolated, fragmented and denatured into ssDNA. Then primers are annealed to begin synthesis. a mix of dNTPs en fluorescently labeled ddNTPs is added to the DNA. After the addition of a ddNTP, elongation is terminated. bc this happens randomly, fragments of diff size are synthesized. These are separated based on their length using electrophoresis; using a detector, the colour of the fluorescent label can be detected.

What is a limition of sanger sequencing

The throughput (number of samples) is low, cuz each reaction sequencing only 1 DNA fragment

Name some extra limitations of Sanger sequencing

1. high costs per basepair

2. Limited read length for complex structures (kan wel tot 800-1000 bp reads, maar dan wordt accuracy minder)

3. low data output

4. time consuming & labour intensive

5. less sensitive to heterogenous samples (In heterogeneous samples (e.g., tumor tissues with multiple mutations), detecting variations can be challenging.)

List the sequencing by synthesis workflow

1. DNA fragmentation

2. Adapter ligation (these are complementary to the primers on the flow cell, will hybridize)

3. Solid phase amplification (bridge amplification) forming clusters

4. Sequencing, for this you add primers, DNA pol, fluorescent chain terminations (chemically reversible)

5. After each cycle the glas surface is imaged

6. chain terminators are reversed (cleaving off the fluorophore)

7. next cycle can be initiated

How do we know which part of the genome each piece of DNA came from

Map you sequenced fragments to the reference genome (i.e., look for overlap)

How do you calculate how many cycles you need to make sure that your

sequence read can be placed uniquely

je weet: a stretch of nucleotides can repeat itself every 256 nt (want 4^4 =256), so stel you want to sequence a fragment of 1 million bp: 4^15 =1.07×10^9 (=1×10^9) which exceeds 1 million. dus dan is het goed (slide 13 pp)

Shortly describe ion torrent sequencing

Occurs on beads in microwell. Whenever the right dNTP flows by and is incorporated into the complementary strand, an H+ is released, changing the pH, changing the charge in the ion sensing plate. This creates a voltage across the ion-sensitive transistor, sending a signal to the pc to record the dNTP species.
(dus als T is recorded → this is the complementary sequence, dus target sequence heeft op die plek een A)

Maak af: during ion semiconductor sequencing, the timing of the peak determines … and the height of the peak determines…

the timing of the peak determines which nt wa added and the height of the peak determines how many (back to back)

Name the advantage and disadvantage of ion semiconductor sequencing

+: very fast, 2 - 4 hour run

-: limited number of reads per semiconductor chip (each well can contain 1 read)
(can also only detect up to 7 identical nucleotides, beyond this the signal becomes unreliable)

Single molecule real time sequencing (SMRT)

Uses mixed dNTPs attached to fluorophores, has a DNA polymerase fixed at the bottom of the well, pulling template DNA through. fluorescent pulse is measured, Retention time of the fluorescent dNTP serves as the read out

wordt vooral gebruikt in diagnostics.

Advantage & disadvantage SMRT

+ very long reads (8-20 kb)

+ fast

+ can directly detect DNA modifications
- moderate throughput (wordt met de tijd steeds beter though)

- expensive

Nanopore sequencing

a modified nanopore unzips the DNA and measure electrical current of each base, so the base sequence is determined using changes in the current over the membrane

advantage and disadvantage Nanopore

+ very long reads of native DNA (no amplification required)

+ you can directly sequence modified RNA (ss molecules)

+ you can detect DNA modifications (e.g., methylation) cuz these have a diff effect on electrical current

- moderate accuracy (wordt met de tijd steeds beetr though)

name the sequencing application you would use for each goals

• Mutation discovery -

• mutation discovery or diagnostics -

• expression profiling -

• Genomic localisation mapping -

• DNA accessibility profiling -

• Genome folding mapping -

mutation discovery/diagnostics - WGS &V targeted sequences (exome fo cancer genes bijv)

expression profiling - RNA sequencing

Genome localisation mapping - ChIP sequencing

DNA accessibility profiling - DNAseqI/ATAC sequencing

Genome folding mapping - 4C/HiC

main trick of these applications is how you prepare your sample, cuz its all sequencing of short fragments of DNA (& all are bulk assays)

Workflow RNA sequencing

1. isolate RNA

2. Reverse transcription (kan door target/start at polyA tail, or of use random hexamers)

3. produce 2nd strand of cDNA

4. fragment ds cDNA

5. Ligate adapters

6. PCR amplify

7. Sequence

8. Map of the genome

ChIP sequencing

used to identify where a protein is contacting the chromatin, can be used for DNA binding transcription factors, histone modifications, basal transcription machinery like RNA pol II (basically anything that can bind/sit on the DNA)

workflow ChIP sequencing

1. cross-link protein to DNA (anything that can bind the DNA could also leave agains so thats why)

2. Fragment DNA

3. Add bead-attached antibodies, this immunoprecipitates (pulls out) the protein

4. leaves you with relatively pure short DNA fragments, you can sequence these (after librar prep)

5. map sequence fragments to the genome

DNaseI/ATAC (assay for transposes-accessible chromatin) sequencing

used if you want to determine which regions in the genome are most accessible (where proteins sit on the genome, bijv; where nucleosomes are displaced to allow transcription factors to bind).

concept ATAC sequencing

relies on Tn5, a transposon that inserts a piece of DNA into the genome. This can only occur where chromatin is accessible (naked DNA). Using Tn5, adapters are added to the genome (heb je deze stap van library prep meteen gedaan). amplification with PCR provides fragments of the right size, these can be sequenced

how is DNAseI-seq diff from ATAC seq

DNAseI-seq uses DNAseI to cut accessible regions of the genome, which are then isolated and prepared for sequencing, bij ATAC gebruik je transposon to cut and directly prep DNA

Wat betekenen de pieken op deze foto/hoe lees je dit af

sharp peaks due to specific location

Intensity of peaks = number of sites that were open across the population of cells

Where u see DNase, is where the DNA was open