DNA Sequencing Part 1 Flashcards
how to make a genomic library
- DNA isolated from cells
- restriction enzymes to cleave DNA or cleave from vector
- insert into recombinant plasmid
- transform bacteria
- grow transformed bacteria to make a genomic library containing all DNA fragments in the genome
Maxim-Gilbert Chemical sequencing
- does not involve DNA synthesis
- uses chemical treatment that breaks DNA chain after G, A+G, C+T, C
- different chemicals to get cleavage after different sites
- label fragments at 5’ end. separate out on gel
both Maxam-Gilbert and Sanger methods depend on
- separation of labeled DNA fragments by electrophoresis
- limits sequencing long stretches of DNA
Sanger sequencing
- need primer binding upstream of the region of interest
(template) - DNA polymerase will add on the complementary nucleotide
- to determine the exact sequence, the reaction can be stopped using terminators
- dideoxy sequencing or chain termination
Sanger Dideoxy Chain termination
- ssDNA, DNA pol, all ddNTP’s, labeled primer, template DNA
- 4 tubes containing all the components needed to polymerize DNA - adds a small amount of ddNTP to each tube
- each tube also contains one ddNTP at 1/100 the concentration of dNTPs
- these have no hydroxy at the 3’ end and thus another NTP cannot be added to them - chain terminators
when are smaller fragments produced
- when ddNTP added closer to primer
- chains are smaller and migrate faster
how to measure Sanger
- creates a pool of DNA sequences of different length all ending with that specific nucleotide
- terminates reaction at every A, T, G, and C nucleotide in each tube.
- run on 4 lanes and visualize
- shortest bands travel furthest
- will emit light at different wavelengths
- camera detects DNA
Depend on DNA synthesis
- Sanger
- Pyro
- Illumina
Depends on Chain termination
- Sanger
- Illumina
Depends on Eelctrophoresis of DNA fragments
- Maxam-Gilbert
- Sanger
Requires making genomic library in a cloning vector
- Maxam-gilbert
- sanger
NGS
- next generation sequencing
common features of NGS
- sample preparation
- sequencing machines - solid surface
- data output
key of NGS
- massively parallel sequencing reactions
- capable of analyzing millions, even billions of reactions at a time
All NGS platforms require
- a library
a library can be obtained by
- amplification
- ligation with custom linkers
each library amplified on a
- solid surface with covalently attached adapters that hybridize the library adapter
amplification followed by
- direct step-by-step detection of nucleotide base incorporated by each amplified library fragment set
length compared to capillary sequencers
- shorter read length
library construction and amplification
- shear high molecule weight DNA with signification
- polish ends to make blunt
- ligate synthetic DNA adapters via PCR
- produce size fractions via PCR
- quantitate
- amplify library fragments on flow cell surface using PCR
- denature clusters to single-stranded
- hybridize sequence primers to linearized ss cluster DNAs
- proceed to sequencing or hybrid capture
problem with little DNA in a clinical setting
- polymerase errors problems early
- PCR amplify high/low GC content less well than 50% GC content
hybrid capture
- fragments from whole genome library are selecting by combining with probes that correspond to most human exons or gene targets
probe DNAs
- biotinylated
- selected with streptavidin magnetic beads to purify
exome
- exons of all genes annotated in the reference genome
how hybrid capture works
- target part of genome of interest
- probes hybridize to exons
- magnetic field to capture biotinylated DNA beads pull down hybridized fragments and get rid of rest of library
- denature away DNA you want
- library you’re sequencing is much reduced in complexity
multiplex PCR amplification of Targets
- if you want a very small subset of a genome
- amplify genes of interest first
- then make sequencing library
- then sequence
what sequencing requires library construction and amplification
- Illumina
3rd generation sequencing
- Pac Bio
Pyrosequencing 454
- reaction monitored by the release of a pyrophosphate during each nucleotide incorporation
- the released pyrophosphate is used in a series of chemical reactions in the generation of light
- light emission detected by a camera which records the appropriate sequences
how pyrosequencing proceeds
- incubating one base at a time
- measuring the light emission
- degrading unincorporated bases
- addition of next base
advantages of pyrosequencing
- large read lengths
- comparable to sanger sequencing
disadvantages of pyrosequencing
- high reagent costs
- high error rate over strings of 6+ homopolymers
sequencing by synthesis
- utilizes the step incorporation of reversibly fluorescent and terminated nucleotides for DNA sequencing
- all 4 labeled nucleotides are added to the sequencing chip at the same time and one sticks
- remaining washed away
- fluoro signal read
- then cleaved and washed away
- repeated until process complete
example of sequencing by synthesis
- Illumina
advantage to sequencing by synthesis
- overcomes homopolymers issue due to terminated nucleotides
disadvantage to sequencing by synthesis
- increased error rate with increased read lengths
- failure to completely remove fluorescence
- increasing background noise
- chemistry is never 100%
Ion semiconductor sequencing
- utilizes the release of H+ ions from the sequencing reaction to detect the sequence of a cluster
- each cluster located directly above a semiconductor transistor which is capable of detecting changes in pH in the solution
- During nucleotide incorporation, a single H+ ion is released into the solution and detected by a semiconductor
advantages of ion semiconductor sequencing
- more cost effective and time efficient
- low substitution error rate
- improved analysis
disadvantages of ion semiconductor sequencing
- not paired-end
- insertion/deletion
- homopolymer problems
Pac Bio sample prep
- shearing
- polish ends
- SMRTbell ligation
- sequencing primer annealing
Pac Bio Library/Polymerase complex
- DNA pol binding
- load library/pol energetics onto SMRT
Pac Bio Sequencing
- raw reads
- post filter reads
- mapped reads
- each of four nucleotides is labeled with a different colored fluorophore
- diffuse in and out
- if they dwell long enough will get detected.
Advantages to Pac Bio
- true single molecule sequencing rather than clusters
- polymerase adhered to bottom of well to pinpoint active site with objects of machine to detect sequencing reaction
- allow for longer read lengths
Nanopore
- when a small voltage is imposed across a nano pore in a membrane separating two chambers containing aqueous electrolytes, the ionic current through the pore can be measured
- molecules gong through the nano pore cause disruption of the ionic current
- by measuring the disruptions molecules can be identified.
advantage of nanopore
- use small amount of DNA
- sequence on site rapidly
- true reagentless sequencing
disadvantage of nanopore
-challenge to get uniform pores
