Week 4 (Illumina Sequencing) Flashcards
why did cost of human genome begin to drastically decrease in cost in 2007?
illumina sequencing was created
why can most biological questions be addressed with sequencing?
DNA is the blueprint for life
sequencing is a _______ to be applied to address a question
tool
in the field of genomics, most questions involve ___________ information
sequence
why are more and more sequencing methods being developed?
to address new questions
what can the 5’ methylated cap tell you?
where transcription started
4 basic steps to illumina sequencing
0) experimental design
1) sample prep
2) cluster generation
3) sequencing
4) data analysis
template
DNA used as input to create a library
sequencing library
template DNA that has undergone all the manipulations to enable it to be sequenced
adapters
sequencing primer binding sites
indexes/barcodes/tags
a unique DNA sequence ligated to fragments within a sequencing library for downstream in silicon sorting and identification
flow cell
a glass slide with one, two, four, or eight physically separated lanes. Each lane is coated with a low (nano-well) of surface bound, adapter complimentary olives
do all flow cells have a flow?
no, is often a term that has been generalized
bridge amplification
the process where a single molecule is amplified to form a cluster
why do we need bridge amplification?
to make multiple copies of DNA in order to generate enough molecules so we can read out the fluorescent labels
cluster
a clonal grouping of template DNA bound to the surface of a flow cell
fluor
chemical structure that emits light at a certain wavelength then excited by laser
step 1 of illumina sequencing
sample prep
what does sample prep consist of?
extracting the template DNA
typical DNA extraction produces _________ DNA fragments
large (get about 50 kb)
DNA shearing
a large amount of base pairs are extracted, but illumina needs a much smaller amount of base pairs, so the DNA must be broken down into smaller fragments
how many base pairs are needed in illumina sequencing?
less than 600 bp
library fragment size has ____________ implications for analysis
downstream
the ________ is constructed by fragmenting a DNA sample and ligating specialized adapters to both fragment ends
library
label the grey parts of the library
label the grey parts of the library
why should there be less than 600 base pairs used in bridge amplification?
if you have one molecule come in and amplify around a physical location, it needs to be small enough to only amplify the target location
Step 2 of illumina sequencing
cluster generation (flow cell clustering)
what is already present in each nano well?
2 types of oligos
in the flow cell, hybridization is enabled by the first of the two types of ______ on the surface
oligos
during cluster generation, the oligo is ____________ to the adapter region on one of the fragment strands
complementary
why do we need to copy a single molecule to make a lot of clones?
need to have enough molecules for fluorescent imaging to detect it
during cluster generation, a _________________ creates a complement of the hybridized fragment
polymerase
after the complement of the hybridized strand is made by the polymerase the double stranded molecule is denatured and the original temple is __________ _________
washed away
The strands during cluster generation are colonially amplified through _________ amplification
bridge
what occurs during bridge amplification?
- the strand folds over and the adapter region hybridizes to the second type of oligo on the flow cell
- polymerase generates the complimentary strand forming a double stranded bridge
- the bridge is denatured, resulting in 2 single stranded copies of the molecule that are tethered to the flow cell
- repeat over and over
after bridge amplification the reverse strands are cleaved and washed off, leaving only the _______ strands
forward
after bridge amplification, what is blocked to prevent unwanted priming?
the free 3’ hydroxyl
what is the difference between patterned flow cells and random flow cells?
- random flow cells had scattered random clusters that needed an imager to can it
- patterned flow cells have etched nano cells (wells) to create a physical barrier
which is better: random flow cells or patterned flow cells?
patterned flow cells
name the three reasons why patterned flow cells are better than random flow cells
- faster scan times due to ordered cluster positions
- less cluster overlap
- more clusters
Step 3 of illumina sequencing
sequencing
the sequencing step in illumina sequencing begins with the extension of the ____ sequencing primer to produce the _______ read.
first; first
what is a “cycle” during the sequencing process of illumina
a new fluorescently tagged nucleotide is added to the read
in illumina sequencing, each nucleotide has its own _____________ dye
fluorescent
One fluorescent nucleotide is incorporated based on the sequence of the ___________
template
after the addition of each nucleotide, the clusters are excited by a ________ _______ and a characteristic fluorescent signal is emitted
light source
what is sequencing-by-synthesis?
after the addition of each nucleotide, the clusters are excited by a light source and a characteristic fluorescent signal is emitted
the number of cycles depends on the ________ of the read
length
what determines the base call?
the emission wave length and signal intensity
for a given cluster, all identical strands are read ________________
simultaneously
what happens to the read product after the completion of the first read? then what is introduced?
- the first read is washed away
- the index read primer is introduced
what happens after the index is read?
it is washed off and the three prime ends are deprotected so it can fold over and bind to the second oligo on the flow cell where index 2 is read creating a double stranded bridge
during sequencing, after the bridge amplification the ________ strand is cleaved off leaving the reverse stand to be read until the desired read length is achieved
forward
3’-O-blocked reversible terminator
it has the ability to cleave off the dye after it is read and then reverse its 3’ OR group to have a hydroxyl so that sequencing can continue
Sanger fluorescent dideoxynucleotide (ddNTP) vs illumina 3’-O-blocked reversible terminator
sanger’s ddNTP will stop sequencing and is not reversible while the reversible terminator can be reversed, fluorescent cleaved and sequencing continued
illumina makes _____ base pair errors
single
illumina sequencing is based on __________ terminator chemistry
reversible
sequencing by synthesis (SBS)
all 4 fluorescently labeled bases present
what is 2 color chemistry?
- instead of each nucleotide having their own color, two colors are used.
- for example: green on T, red on C, A is equal parts green and red, G is dark (no label)
step 4 of illumina sequencing
data analysis
