Week 4 (Illumina Sequencing)

Question 1

why did cost of human genome begin to drastically decrease in cost in 2007?

Answer 1

illumina sequencing was created

Question 2

why can most biological questions be addressed with sequencing?

Answer 2

DNA is the blueprint for life

Question 3

sequencing is a _______ to be applied to address a question

Answer 3

tool

Question 4

in the field of genomics, most questions involve ___________ information

Answer 4

sequence

Question 5

why are more and more sequencing methods being developed?

Answer 5

to address new questions

Question 6

what can the 5’ methylated cap tell you?

Answer 6

where transcription started

Question 7

4 basic steps to illumina sequencing

Answer 7

0) experimental design
1) sample prep
2) cluster generation
3) sequencing
4) data analysis

Question 8

template

Answer 8

DNA used as input to create a library

Question 9

sequencing library

Answer 9

template DNA that has undergone all the manipulations to enable it to be sequenced

Question 10

adapters

Answer 10

sequencing primer binding sites

Question 11

indexes/barcodes/tags

Answer 11

a unique DNA sequence ligated to fragments within a sequencing library for downstream in silicon sorting and identification

Question 12

flow cell

Answer 12

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

Question 13

do all flow cells have a flow?

Answer 13

no, is often a term that has been generalized

Question 14

bridge amplification

Answer 14

the process where a single molecule is amplified to form a cluster

Question 15

why do we need bridge amplification?

Answer 15

to make multiple copies of DNA in order to generate enough molecules so we can read out the fluorescent labels

Question 16

cluster

Answer 16

a clonal grouping of template DNA bound to the surface of a flow cell

Question 17

fluor

Answer 17

chemical structure that emits light at a certain wavelength then excited by laser

Question 18

step 1 of illumina sequencing

Answer 18

sample prep

Question 19

what does sample prep consist of?

Answer 19

extracting the template DNA

Question 20

typical DNA extraction produces _________ DNA fragments

Answer 20

large (get about 50 kb)

Question 21

DNA shearing

Answer 21

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

Question 22

how many base pairs are needed in illumina sequencing?

Answer 22

less than 600 bp

Question 23

library fragment size has ____________ implications for analysis

Answer 23

downstream

Question 24

the ________ is constructed by fragmenting a DNA sample and ligating specialized adapters to both fragment ends

Answer 24

library

Question 25

label the grey parts of the library

Answer 25

Question 26

label the grey parts of the library

Answer 26

Question 27

why should there be less than 600 base pairs used in bridge amplification?

Answer 27

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

Question 28

Step 2 of illumina sequencing

Answer 28

cluster generation (flow cell clustering)

Question 29

what is already present in each nano well?

Answer 29

2 types of oligos

Question 30

in the flow cell, hybridization is enabled by the first of the two types of ______ on the surface

Answer 30

oligos

Question 31

during cluster generation, the oligo is ____________ to the adapter region on one of the fragment strands

Answer 31

complementary

Question 32

why do we need to copy a single molecule to make a lot of clones?

Answer 32

need to have enough molecules for fluorescent imaging to detect it

Question 33

during cluster generation, a _________________ creates a complement of the hybridized fragment

Answer 33

polymerase

Question 34

after the complement of the hybridized strand is made by the polymerase the double stranded molecule is denatured and the original temple is __________ _________

Answer 34

washed away

Question 35

The strands during cluster generation are colonially amplified through _________ amplification

Answer 35

bridge

Question 36

what occurs during bridge amplification?

Answer 36

1. the strand folds over and the adapter region hybridizes to the second type of oligo on the flow cell 2. polymerase generates the complimentary strand forming a double stranded bridge 3. the bridge is denatured, resulting in 2 single stranded copies of the molecule that are tethered to the flow cell 4. repeat over and over

Question 37

after bridge amplification the reverse strands are cleaved and washed off, leaving only the _______ strands

Answer 37

forward

Question 38

after bridge amplification, what is blocked to prevent unwanted priming?

Answer 38

the free 3' hydroxyl

Question 39

what is the difference between patterned flow cells and random flow cells?

Answer 39

- 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

Question 40

which is better: random flow cells or patterned flow cells?

Answer 40

patterned flow cells

Question 41

name the three reasons why patterned flow cells are better than random flow cells

Answer 41

1. faster scan times due to ordered cluster positions 2. less cluster overlap 3. more clusters

Question 42

Step 3 of illumina sequencing

Answer 42

sequencing

Question 43

the sequencing step in illumina sequencing begins with the extension of the ____ sequencing primer to produce the _______ read.

Answer 43

first; first

Question 44

what is a "cycle" during the sequencing process of illumina

Answer 44

a new fluorescently tagged nucleotide is added to the read

Question 45

in illumina sequencing, each nucleotide has its own _____________ dye

Answer 45

fluorescent

Question 46

One fluorescent nucleotide is incorporated based on the sequence of the ___________

Answer 46

template

Question 47

after the addition of each nucleotide, the clusters are excited by a ________ _______ and a characteristic fluorescent signal is emitted

Answer 47

light source

Question 48

what is sequencing-by-synthesis?

Answer 48

after the addition of each nucleotide, the clusters are excited by a light source and a characteristic fluorescent signal is emitted

Question 49

the number of cycles depends on the ________ of the read

Answer 49

length

Question 50

what determines the base call?

Answer 50

the emission wave length and signal intensity

Question 51

for a given cluster, all identical strands are read ________________

Answer 51

simultaneously

Question 52

what happens to the read product after the completion of the first read? then what is introduced?

Answer 52

- the first read is washed away - the index read primer is introduced

Question 53

what happens after the index is read?

Answer 53

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

Question 54

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

Answer 54

forward

Question 55

3'-O-blocked reversible terminator

Answer 55

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

Question 56

Sanger fluorescent dideoxynucleotide (ddNTP) vs illumina 3'-O-blocked reversible terminator

Answer 56

sanger's ddNTP will stop sequencing and is not reversible while the reversible terminator can be reversed, fluorescent cleaved and sequencing continued

Question 57

illumina makes _____ base pair errors

Answer 57

single

Question 58

illumina sequencing is based on __________ terminator chemistry

Answer 58

reversible

Question 59

sequencing by synthesis (SBS)

Answer 59

all 4 fluorescently labeled bases present

Question 60

what is 2 color chemistry?

Answer 60

- 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)

Question 61

step 4 of illumina sequencing

Answer 61

data analysis