Sequencing Flashcards
what did Sanger first sequence and won Nobel prize for?
1958 AA structure of insulin
1980 developed first DNA sequencing technology
describe Sanger sequencing
uses DNA polymerase and a template strand with a primer (DNA pol needs primer).
DNA pol can attach 5’ hydroxyl of a new NT to 3’ of primer and bases.
- use mixtures including all nucleoSides and also one type of ddNTP (didepoxynucleoside triphosphate). this stops the chain from extending, because it has no OH, just H.
- ddNTPs get randomly incorporated, forming a mixture of fragments of different lengths, each ending in ddNTP. this is done for each ddNTS (4 mixtures).
- the correct proportions of ddNTPs must be added cover just enough of corresponding base. too much - all fragments are short. too little - doesn’t capture every complementary NT.
- polyacrylamide gel electrophoresis - separates oligonucteltides. originally used radioactive label on ddNTPs
Fragments are nested so fragments overlap and can determine order.
What is the Maxam Gilbert sequencing method?
- developed near time of Sanger sequencing
- ssDNA labelled with radioactivity at 5’ end (rather than NT as in Sanger)
- base specific reagents cleave DNA.
Dimethyl sulfate (+NaOH) - A and G (+HCl, just A)
Hydrazine and Piperidine (T and C)
Hydrazine, piperidine and NaCl (C) - separated on gel, read sequence straight from gel.
- doesn’t use primed DNA synthesis so is limited to sequences near restriction sites.
-toxic reagents.
what was a major advancement in Sanger sequencing?
- using fl dyes rather than radioactivity
- can attach different dye to each ddNTP and do one reaction mixture instead of 4. Run on same gel lane.
what allowed automation of DNA sequencing?
Capillary systems replace flat gels.
1986
can have 96 capillaries at once.
how long a sequence can Sanger reaction produce?
1000bp
automated machines can produce 1Mb per day
what is a shred score?
Sequence quality/ accuracy
q
q=20 means probability of <1% error per base.
Now, usually q range is 80 - 90 (1 base in 100000000 wrong)
What is a common unit of sequencing cost?
USD per 1,000 bases determined to q=20 accuracy
when did price of sequencing drop?
2008 - invention of next gen sequencing technology
work flow of illumination sequencing
- library generation
- library fragments bind to complimentary oligos in nanowells on flow cell.
- bridge amplification - amplifies a single molecule into a cluster of replicated fragments. 1 cluster per well.
- Cycles exposing clusters to nucleoside triphosphate mixtures. polymerase adds a NT each time. only one NT added each stem due to blocking group in reagents.
Laser pulses in each cycle excite newest added NT which has fl tag. - distribution of colors in the image shows which base added to cluster.
- remove fl tags and blocking groups after imaging. cycle repeats.
what is involved in DNA library preparation prior to illuminating sequencing?
- fragment DNA (300-800bp)
- ligate adapter sequence to both ends.
adapter = a sequencing primer binding site, an index, and a flow cell attachment site.
adaptors on each end of fragment are different.
what is one of the most popular set machines today?
Illumina HiSeq 4000
what amount of DNA sequences can illumine flow cell produce?
720Gb in 48-60 hours.
What is the primary error in SBS?
Phase error:
in a cluster, 1000 identical sequences. all produce same signal together in each cycle.
possible for some to get out of sync, eg if 2 bases incorporated instead of 1(incomplete blocking).
more cycles, more will get out of phase. eventually unphased signal will swamp in phase signal.
examples of NGS technologies
Illumina - very versatile, only one to survive
Roche 454 - pyrosequencing
ABI SOLiD - seq by oligoNT ligation and detection
