Next gen sequencing tech Flashcards
chemical sequencing
when DNA is subjected to control chemical treatments that generate breaks of the molecules at specific nucleotides
enzymatic sequencing
when DNA is sequenced by polymerization with dideoxynucleotide triphosphates as DNA chain terminators
sanger sequencing for genome steps:
- extract DNA
- shear
- ligate into library
- pick clones
- grow clones
- extract DNA vector
- sequence using ddNTP
- read fragments with gel or capillary
with next gen sequencing
- can have an array of amplified molecules
- increases sequencing productivity
- cheaper
the first step in sequencing is
fragmentation of DNA
what are the limits of sanger sequencing
- one fragment is sequenced at a time
- time consuming
- costly
how do we fix the limitations of sanger sequencing
- parallelization and volume miniaturization : less reagents = less cost
the first next gen machine is
454 Roche
- is an emulsion PCR
- pyrosequencing
- reads lengths 400bp
steps for nest gen sequencing
- library prep
- clonal amplification
- sequencing
- Data analysis
bridge/cluster PCR
- oligonucleotides are bound to P5 P7 covalently
- primers are annealed with the DNA to be sequenced
- primers are extended by addition of dNTP and Taq DNA polymerase
- denaturation
- end products: the slide will contain clusters of amplified sequences
illumina reversible terminators
- all 4 nucleotides are labelled
- higher accuracy
- no problems with homopolymer repeats
ion torrent uses - to detect sequencing pattern
an ion semiconductor sensor that detects variations in H+ ions, when a nucleotide is incorporated the rxn releases an H+
3rd gen sequencing: SMRT
single molecule real time DNA sequencing tech
advantages of SMRT
- has zero mode wave guides
- phospholinked nucleotides are added so a light is giver off, these are linked with much higher accuracy
nanopore sequencing
a small voltage is sent through a nanopore in a membrane separating 2 chambers containing aqueous electrolytes, the ion current can be measured
