DNA Sequencing Flashcards
overview of Illumina sequencing
sample preparation: sample extracted and purified for the next stage in the process
library preparation: prepares the sample for sequencing by adding special adapters that are recognized by the sequencer
sequencing: reads the sequence of the sample and creates the raw data
analysis: analysis of sequencing data has three stages: primary, secondary analysis and interpretation
what is the aim of library prep?
obtain nucleic acid fragments with adapters attached on both ends
- indexes used to tag indiv samples to allow for pooling
- RD1 and RD2 SP are primers used to initiate sequencing
library validation (2)
- quantification: how much DNA do we have?; qPCR and fluorometric methods
- qualification: check that we have fragments of the same size; bioanalyzer or fragment analyzer
what is a flow cell?
cluster generation occurs on a flow cell
flow cell is a thick glass slide with channels or lanes
each lane is coated with a lawn of oligos complementary to library adapters
library molar conversion
660 * library size bp (BioAnalyzer)
= library concentration in nM => normalize to 4 nM and use 5uL and follow the “denature and dilute” MiSeq guide
what is a cluster?
group of DNA strands positioned closely together
each cluster represents thousands of copies of the same DNA strand in a 1-2 micron spot
cluster generation
hybridize fragment and extend
- single-stranded DNA libraries are hybridized to primer lawn
- bound libraries are then extended by polymerases
denature dbl-stranded DNA
- dbl stranded molecule is denatured
- original template washed away
- newly synthesized strand is covalently attached to flow cell surface
what happens in bridge amplification?
ss molecule flips over and forms a bridge by hybridizing to adjacent, complementary primer (P5 flips over to P7 and P7 starts synthesizing)
hybridized primer extends by polymerases
dbl sranded bridge formed then …
dbl stranded bridge is denatured = copies of covalently bound single-stranded templates
then bridge amplification starts again as ss molecules flip or to hybridize adjacent primers; hybridized primers extends by polymerase = multiple bridges formed
what happens after multiple bridges are formed (after bridge amp)?
linearization = dsSNA bridges are denatured
reverse strand cleacage = reverse strands are claved and washed away = laving a cluster with forward srands only
what happens after linearization & reverse strand cleavage?
read 1 primer hybridization = sequencing primer is hybridized to read 1 sequencing primer binding site
then flood flow cell with fluor-labeled nucleotide one at a time (dideoxynucleotide that lacks 3’ OH)
4-channel SBS chemistry
each of the four DNA bases emits an intensity of a unique wavelength
collects four images = during each cycle, each cluster appears in only one of four images
2-channel chemistry
only 2 dyes
takes less time = red and green
- green = T
- red = C
- both images = A
- clusters not present/dark = G
what is DNA sequencing?
the determination of the order of nucleotides ina DNA molecules, read in a 5’ to 3’
DNA sequencing is used in the medical lab for a variety of purposes:
- detecting mutations
- typing microorganisms
- identifying human haplotypes
- designating polymorphisms
define pyrosequencing
- based on sequencing by synthesis principle
- stepwise synthesis of DNA by the addition of dNTPs
- a light signal when a dNTP is added
- generating chain of DNA by sequencing processes just like Sanger
- difference is = no fluorophore; we are generating light every time we add nucleotide; chemiluminescent (inert chemical oxidized and then chemical rxn generates light)!!
four enzymes present in the system of pyroseuqnecing at all times
- DNA polymerase (want high fidelity 5-3’ exonuc and 3-5’ exonuclease)
- ATP sulfurylase (used in first step)
- luciferase (chemilum rxn)
- apyrase
dNTPs added one at a time
three primers required for pyrosequencing
- fwd primer
- sequencing primer downstream of the fwd primer but just upstream of target sequence
- reverse primer that is biotinylated
DNA sequencing is used in the medical laboratory for a variety of purposes:
detecting mutations
typing microorganisms
identifying human haplotypes
designating polymorphisms
this represents the most common method of sequencing DNA
dideoxynucleotide sequencing
- least expensive and easy to do
- using ddNTPs = attach to 3’ ribose; have a hydrogen atom attached to 3’ rather than an OH group
these molecules terminate DNA chain elongation because they cannot form a phosphodiester bond with the next deoxyribonucleotide
dideoxyribonucleotides
dye terminator chemistry
- each of 4 dideoxynucleotides is tagged with a different fluorescent dye
- one reaction is performed
> contains the DNA polymerase, primer nucleotides, and all four dye-labeled dideoxynucleotides - products = injected into a single capillary
nucleotides colours
C = blue
T = red
G = black
A = green
template DNA components
mixed with labeled dideoxynucleotides and regular nucleotides
Sanger Sequencing Workflow
- extraction of DNA (PURE)
- amplification of DNA fragment to be sequences (PCR)
- sample preparation (removal of PCR reagent, quantitation of DNA)
- Sanger sequencing reaction
- clean up sequencing of reaction - removal of primer, dNTPs, ddNTPs
- capillary electrophoresis
- data analysis
Sanger sequencing denaturation
sequencing reaction started by heating the mixture, which causes the 2 complementary strands of the DNA template to separate
Sanger Sequencing Annealing
sequence of newly created copy of the template DNA is complementary to the original DNA template
Sanger Sequencing Extension
in the last step, the temperature is raised so that the enzyme can bind to the DNA and create a copy of the template
Sanger sequencing - chain termination
while making a copy of the DNA template, the enzyme incorporates both nucleotides and the fluorescently tagged dideoxynucleotides
however, whenever a ddNTP is incorporated, the reaction is terminated
NOTE: ddNTP can be incorporated at any position so each strand can be terminated at any position
since there are billions of copies of DNA template, there will be a mixture of strands at many different lengths
capillary electrophoresis
- after Sanger sequencing
- injected DNA molecules are separated through array according to size (smaller fragments are faster)
- flowable polymer minimizes electroendosmosis
- DNA migrates from cathode to anode
- DNA fragments labeled with fluorophores by PCR
- after electrophoresis = analysis
what happened if no signal shows up on the electropherogram?
insufficient template
thermal cycler malfunction
loss of product during clean up
reagents not added or deteriorated
what happened if there are low signals on the electropheogram?
insufficient sample volume
failed injection
old buffer
broken or blocked capillary
what happened if there are large peaks or blobs in the first 120 bases
poor clean up of sequencing rxn
- precise location of the blobs varies according to the dye used and the specific configuration
what happened if there are shoulders on all peaks of the electropherogram ?
capillary array needs to be replaced
overloaded sample
homopolymeric region in sample or STUTTER
what happened if there are double peaks at the beginning of the sequence on the electropherogram?
seen when a PCR product is used for sequencing
more than one PCR product is present in the rxn
summary of Sanger sequencing
- primer annealing and chain extension
- ddNTP binding and chain termination
- fluorescently labelled DNA sample
- enzymatic clean-up of sequencing reactions
- capillary gel electrophoresis and fluorescence detection
- electropherogram generation and bioinformatics
how many primers does pyrosequencing have?
3,
one on 5’ and another at 3’(biotinylated) = PCR primers ???
sequencing primer is downstream of forward primer
pyrosequencing steps
isolate DNA then PCR w two primers
- run PCR w one of the primers biotinylated
- immobilize biotinylated PCR producys onto streptavidin coated beads
- separate strands by denaturation in NaOH
- wash/neutralized the immobilized srand
- anneal sequencing primer
(steps 2-5 = 10-15 mins)
pyrosequencing workflow
extraction
PCR
sample preparation
pyrosequencing
analysis
a method to determine a DNA sequence without having to make a sequencing ladder
pyrosequencing
this method relies on the generation of luminescence when nucleotides are added to a growing strand of DNA
pyrosequencing
describe th pyrosequencing rxn
step 1:
- generation of light after nucleotide addition (release pyrophosphate or PPi)
step 2:
- PPi used to generate ATP from adenosine phosphosulfate (APS)
- ATP and luciferase generate light by conversion of luciferin -> oxyluciferin (luminometer)
step 3:
- system regenerated with apyrase that degrades residual free dNTP & dATP
what is pyrosequencing used for?
most useful for short-to-moderate sequence analysis
> mutation or single nucleotide polymorphism (SNP) detection
- commonly used for infectious disease and HLA typing
- use for bisulfite sequencing
also referred to as methylation-specific sequencing
bisulfite DNA sequencing
what is bisulfite DNA sequencing?
a modification of chain termination sequencing designed to detect methylated nucleotides
methylated DNA involved in:
- gene expression regulation
- chromatin structure regulation
- cell differentiation
- implicated in a number of diseases, including several types of cancer
Bisulfire DNA sequencing procedure
- cut genome with restriction enzymes
- run an agarose gel & fragments of interest are purified from the gel (DNA from fixed tissue can be used directly)
- DNA denatured by hear and exposed to bisulfite solution
- Bisulfite incubation = deamination of cytosines; turning them into uracils
NOTE: 5-methyl cytosines are unchanged - treated template is used for PCR amplification
- PCR amplicons are sequences
- methylated detected by comparing treated sequence with an untreated sequence, noting where the treated sequence CG base pairs are not changed to TA
