DNA based NGS technologies

What has improved the ability to interrogate genome function using NGS?

The ability to use sequencers as sophisticated counting machines has been key developments in the areas of interrogating DNA and genome function

What techniques can be used to study chromatin?

MNase-seq

What does MNase-seq do>

determine where the positions of nucleosomes are in the genome

What does understanding chromosome function require? what technique has shown the presence of chromosome territories?

• Understanding chromosome function requires understanding of higher order structures

  • Eg. if you do DNA FISH on a cell, you observe chromosome territories - organisation within the nucleus

    • suggests a higher order organisation within the nucleus

What are TADs?

regions of the chromosome interact with eachother but are insulated from other regions of the chromosome

Process of chromosome conformation capture?

• Cell of interest is cross linked via a cross linking agent

• this causes proteins to be cross linked

• this cross linking links distinct regions of the genome together through proteins

• chromatin is then frgamented

• this fragmentation maintains regions of the chromosome that are in close proximity, capturing and freezing interactions

• ends of the sonicated fragments must then be repaired - do this by adding a base containing biotin in - this blunts ends, enabling ligation tp produce circular DNA

• at the centre of the ligation there are the inserted biotinylated nucleotides

• we then further fragment the circular DNAs, using streptavidin to pull down and isolate the biotinylated DNA junctions

• this can then be used to make libraries

• we sequence the ligation fragments, such that you reach the internal junction

What does sequencing across ligation junctions in HiC do>

• identifies sequences of DNA that are non-contigious in the genome

• these can then be positioned on the chromosome

How is data describing the interactions of non-contiguous regions of the genome interrogated?

• create a matrix that positions the ligation junctions on an axis moving away from the chromosome

  • we can then fill in dots corresponding to whoere the interactions occur on the chromosome

• deviations from randomness can then describe where interactions occur in the cell, and show the presence of TADs

What do dips between TADS correspond to?

dips between the TADs corespond to insulated regions of the chromosome

what can we see when looking at TADs on a much larger scale?

Looking at a much larger scale, this pattern extends across the breath of the entire chromosomes

Shows that some TADs tend to interact with eachother more frequently, and others interact with themselves more frequently

this indicates the presence of compartments

what does the presence of compartments indicated by hi-c indicate? how did ngs enable this?

• shows how chromosomes are structured at a much higher level, this likley has a role in function

• only with using NGS counting approaches that interactions sampled over billions of events have been possible to sequence at the scale required

what is determined when the presence of TAD structures is tested with single cell hiC

• indicates that there is much more heterogeneity in TAD structure than expected

• therefore, the average observed with many cell HiC is likely the probability that you see a given cell in a defined state

• therefore, although many NGS approaches are done on ensembles, they may not properly indicate what is happening at the single cell level

what js the problem of many cell hi-c?

• these use an average of cells, and so represents the average behaviour across an entire population - this may not be representative of individual cells

historically, what low res techniques have been used to probe DNA replication?

• historically, have used low res techniques based on labelling replicating regions of the genome

  • analogues of DNA bases with attached groups to allow detection - eg. BrdU, EdU

  • pulse into cells, and use nucleotide features to visualise (eg. immunofluorescence in cells)

  • can see early/middle/late s-phase cells have different positions of nt incorporations

  → told us a lot about timing of replication. but it is very low res

what can DNA combing assays show about DNA replication?

• first pulse label with one nt analogue, and then a second - both in different colours

• take DNA from cells, stretch onto slides and visualise to provides hgih res information

• indicates rate of replication progression across the forl and how frequenctly it is initiated acros the chromosome

problem with DNA combing assays?

• does not provide sequence level information

• need a technology to understand DNA replication at a genome scale in relation to sequences

Wha can repli-seq be used to study?

• DNA replication

what does repli-seq rely on to study genomes?

relies on pulse labelling with a nt analogue - specifically label regions of DNA during a short pulse of BrdU

process of repli-seq?

• Firstly, specifically label regions of DNA during a short pulse of BrdU

• We then tae asynchronously growing cells and separate them based on their cell cycle stage usingg FACs

• Fragment DNA from cells, add adaptors, use Ab against BrdU to IP DNA replicated specifically during pulse labelling (and therefore undergoing active replication)

• We use this DNA to make libraries for NGS sequencing.

• Sequencing can then be mapped back to the genome

  • allows identification of the initiation zones

what does repli-seq tell you?

can see the speed and positon during the cell cycle of the initiation and elongation events occuring

what has repli-seq allowed us to determine about initiation of DNA replication?

• has indicated where initiation occurs, how frequently it occurs and the rates of intiiation

• furthermore, this has shown that replication timing is related to underlying chromosome organisation and structure - for example, replication timing zones van be compared to A and B compartments from hiC maps - these correspond very well, indicating that the position in the nucleus and underlying chromosome functionality have major roles in dictating which regions of the genome are susceptible to initiation and replication.

What does OK-seq allow?

• mapping of initiation zones

What does OK-seq sequence?

• Okazaki fragments

What does ok-seq rely on?

• relies on the ability to mark actively replicating regions of the chromosome with a pulse labelling of a nt analogue - EdU used

process of ok-seq?

• pulse label cells with EdU → isolate DNA

• Run isolated DNA through a density gradient

  • short fragments are yet to be ligated, and so migrate to the top as they are very small

  • this allows isolation of Okazaki fragments from the rest of the gDNA

• Edulabelling makes the fragments amenable to click chemistry.

• this allows addition of biotin → we can then isolate with streptavidin to create libraries for seqeuncign

• this produces strand specific information for Okazaki fragments and directionality

• this can be used to make initiation zones

what is SCAR-seq?

sister chromatids after replication by DNA seqeubncing

what can SCAR-seq be used to study?

• whether old and new histones are deposited equally on leading and lagging strands

how does SCAR-seq work?

• pulse label with a nt analogue

• this allows isolation of newly replicated DNA

• we then use ChIP to separate old and new nucleosomes (due to their different modifications)

  • this isolates newly replicated region using click them from pulse labelling → add biotin and capture on streptavidin beads

• denature DNA - keeps labelled DNA (provides info on directionality

• Add adaptors and seqeunce

• this can create a ratio for comparisons between leading and lagging strands

in studying deposition of nucleosomes, what does SCAR-seq take advantage of?

• take advantage of all histones on chromatin having a specific modifications

  • old → have h4k20me2

  • new → have h4k5ac

what did scar-seq show when studying old and new nucleosomes?

shows there is no bias in where these go - indviates a biochemical mechnaims to enable this equal segregation

what did knockouts of MCM2A residues show in the scar-seq profiles of old and new nucleosomes?

• mutation of MCM2A residues that interact with H3/4 → ratios are very different

  • without this binding, can no longer equally segregate parental nucleosomes between the strands

  • old histones were being deposited towards leading

  • new to lagging

  → mechanisms to ensure segregation of the histones equally

What does CHOR-seq stand for?

chromatin occupancy after replication

how do we do CHOR-seq?

• time course labelling with nucleotide added where the region of the genome is being replicated during the pulse

• chase with unlabelled nucleotides, allowing for replisome progression

• we can then study how the replisome looks after a defined period of time

• we study this via a chIP based approach - immunoprecipitate for modified histones - then make libraries, isolate biotinylated DNA, amplify libraries and sequence

How can DNA sequencing be useful in screening of compound libraries?

• adding DNA could allow screening in a much smaller format

  • DNA sequences are ligated onto individual compound libraries

  • then create POI on magnetic beads

  • put compound onto beads

  • anything interacting with POI is retained on the bead

  • can sequence to identify the nature of those molecules that were inhibitory - bound to the molecule

    • this is done by NGS