Illumini

Question 1

What is the process for Illumini

Answer 1

1. extract DNA
2. library prep- DNA breaking and tagging
3. sequencing
4. analysis

Question 2

how is DNA extracted

Answer 2

1. mechanical fragmentation
2. end repair of staggered ends and phosphorylation at 5’
3. adenylation at 3’
4. adapter ligation
5. amplification

Question 3

How does adapter ligation work

Answer 3

P5 (top) and P7 (bottom strand)will attach to a chip

Rd1 SP and Rd2 SP are where the primers BIND (NOT THE PRIMERS)

Question 4

what are quantification and qualification methods in illumina

Answer 4

quantification = qPCR and flurescnet dye

Qualification = Bioanalyzer

Question 5

what does the bioanalyzer contain

Answer 5

polyacrylamide and flurescent dye

Question 6

T/F a broad sample peak is best

Answer 6

F - a broad peak is non specific, tight peak is best

Question 7

how to calculate library concentration in nM

Answer 7

(X x10^6) / (660 xlibrary size bp)

X = Quibit or PicoGreen

Question 8

steps of cluster generation

Answer 8

1. ssDNA hybridizes and extended by polymerase
2. dsDNA denatures and original is washed away
3. ssDNA binds P5 to P7 (bridge amplification) and polymerization
4. dsDNA bridge is formed and denatured into two ssDNA
5. bridge amplification occurs until all bound
6. linearization by denaturation of dsDNA bridges
7. Reverse strands cleaved and washed leaving identical forward stands
8. primer hybridizes to Rb1S (starting spot for DNApol)

Question 9

difference of 4 channel and 2 channel SBS

Answer 9

4 - 4 different fluoresent dye, one for each base needing 4 images per cycle

2- 2 dyes and two images for all four base

Question 10

what color goes with what base in 4 channels

Answer 10

CATG
RYGB

B - black/none

(CATG and ROYGBIV)

Question 11

what color goes with what base in 2 channel

Answer 11

CATG
R@GB

@ = both R and G
B- black/none

Question 12

steps in pair end sequencing

Answer 12

1. read product stripped off
2. ssTemplate = bridge to primer and 3’ end is extended
3. dsDNA bridge is linearized and the original strand is cleaved
4. sequencing primer binds to Rb2S

Question 13

steps in index reads

Answer 13

1. read 1 - sequence
2. index read
3. paired end turnaround - complement
4. index read 2
5. read 2 - reverse strand

Question 14

what 2 steps are data analyzed

Answer 14

1. during the run (real time)
2. after the run

Question 15

what is the data analysis real time

Answer 15

interprets intensity based on Q score

Question 16

what is the data analysis after the run

Answer 16

FASTQ and aligns read with original template

Question 17

what does the higher Q score mean

Answer 17

higher accuracy of base pairs (Q >30 = 1/1000 for a mistake)

Question 18

what is the Phred (Q) score measure

Answer 18

assess accuracy of sequencing - probability that a given base is incorrectly called by the machine

Question 19

benefit of Illumina

Answer 19

increase read depth = more reads to increase sensitivity