L20 - Nucleic acid sequencing 2 Flashcards
(14 cards)
what do chain terminator ddNTPs have and not have
Do have 5’ phosphate so can be incorporated into DNA
do NOT have 3’ OH so cannot form new bond with what would be the next nucletide
what are next generation sequencing approaches
modern techniques that allow sequencing of millions of fragments at the same time
= instead of 1 at a time - Sanger
describe the Illumina sequencing method to form a cluster
Next generation method
- randomly break the DNA into different sized fragments
- attach/ligate adaptor sequences to BOTH ends of BOTH strands of the fragments
= adaptor has universal primer binding site AND attachment sequence to bind to flow cell/surface
- library of DNA fragments with adaptor sequences attached to surface in flow cell
- attached fragments are copied to produce a ‘cluster’ via bridge amplification
= each cluster is different but all the DNA WITHIN a cluster is identical
= the clusters are now ready to be sequenced
how does illumina sequencing actually sequence the fragments once they are clusters on the surface
- universal primers anneal to the same adaptor sequence that was added to all fragments
- all 4 fluorescently labelled ddNTPS are added with polymerase
- the correct ddNTP is Incorporated into the strand GROWING from the primer and flourescence is detected
- chemical catalyst removes fluorophore from the ddNTP and creates 3’OH
- all 4 ddNTPs and polymerase are added again
= proscess continues until fragment is fully sequenced
= all the clusters on the surface are doing the same thing at the same time and flourescence is picked up in REAL TIME = allows millions of fragments to be sequenced at the same time
what are the 2 things that happen once a ddNTP has bound and flourescnce detected
chemical catalyst added:
- removes fluorophore
- creates 3’OH on the ddNTP to allow next nucleotide in sequence to be added
what is illumina sequencing best for
huge sequences - whole genomes
= great for shotgun sequencing
compare sanger to next gen sequencing
sanger:
isolate and sequence each fragment individually
= adding of ddNTPs to have differnt coloured and sized fragments
= run through gel and use of detector
slow + expensive BUT very accurate
Next Gen:
sequence all fragments simultaneously and the put together no need to put in plasmids
= fast and less expensive BUT slightly less accurate
in Next gen as soon as you have the DNA fragments you can begin to construct sequence –> in sanger there are more steps with peparation of clone library and then obtaining the end sequences by reading in from the known ends and addition of ddNTPs
= and THEN you can construct whole sequence
name one problem with assembly of overlaps/contigs when sequencing whole geomes
genome wide repeats or tandem DNA repeats
= incorrect fragments put together in sequence
what is ‘mapping’ and ‘resequencing’
Mapping:
comparing sequence to an already known sequence for reference
= finds variants or anomalies
Resequencing:
sequencing an already known genome to compare to map
what do ChIP-seq and RNA-seq tell us
ChIP-seq:
Chromatin Immunoprecipitation
= where do proteins bind in the genome
RNA-seq:
= measure expression/how tuned on genes are in genome
describe the method of RNA-seq
- extract RNA
- convert to cDNA
- randomly fragment
- add adaptor sequences to end
- sequence via Next-gen
- map to reference genome = more reads of specific fragments = higher expression of that gene
describe the method ChIP-seq
- add proteins to bind/crosslink to DNA
- fragment DNA
- use specific antibody to bind to added cross-linking proteins
- remove the protein
- next-gen sequencing = map the sequences to the genome to find where the proteins binded
what can we add to analyse different samples in a single next-gfen sequencing reaction
index sequences within the added adaptor sequences
= show which experiment/patient that particular fragment/cluster is from
how does nanpore sequencing work
pore has hole in middle big enough for a single strand of DNA
= depending on which base is inside pore depends how well ions can flow though
= reading electric current you can work out which base is inside pore at a time
can read really long sequences but error prone
