Sequencing by Synthesis

Question 1

Describe Illumina workflow (4)

Answer 1

1. Sample prep - extraction and purification; requires a lot of DNA
2. Library prep - adds special adapters recognized by sequencer
3. Sequencing - reads sequence and creates raw data
4. Analysis

Question 2

How is DNA prepped for Illumina ?

Answer 2

1. Mechanical fragmentation of genomic DNA; via acoustic sonication
2. End Repair & Phosphorylation
   - starts with staggered ends
   - enzymatic treatment = adds 5’ phosphate and blunts ends
3. Adenylation; to 3’ end = overhang
4. Adapter Ligation
   - uses poly A overhang
   - adapter attaches to either end of DNA

Question 3

Aim of library prep

Answer 3

To obtain nucleic acid fragments with adapters attached on both ends

Question 4

Which part of the adapter hybridizes to the lawn of the chip ?

Answer 4

P5 and P7

Question 5

Which part of the adapter binds primers ?

Answer 5

Rd1 SP and Rd2 SP

Question 6

How are libraries of Illumina quantified ?

Answer 6

• qPCR
• fluorometric methods

Question 7

How are libraries of Illumina validated for quality ?

Answer 7

Bioanalyzer/ fragment analysis: should have a small, tight peak at the desired bp, in between lower and upper markers

Question 8

Calculate library concentration

Answer 8

nM = ([Qubit or PicoGreen]* x10⁶) / (660 x library bp)

*NOTE: in ng/uL

Question 9

What is a flow cell ?

Answer 9

• thick glass slide with channels or lanes
• each lane is coated with lawn of oligos complementary to library adapters

Question 10

What is a cluster ?

Answer 10

thousands of copies of the same DNA strand positioned closely together

Question 11

Describe DNA Cluster generation in Illumina

Answer 11

1. ssDNA library hybridizes to primer lawn of oligos on flow cell
2. DNA polymerase extends from 3’ oligo
3. dsDNA molecule is denatured and original strand is washed away

REPEAT UNTIL LAWN OF OLIGOS ARE FULL:
4. Bridge amplification from P5 to P7
- DNA polymerase extends from 3’ oligo
5. Denature double-stranded bridge = doubles copies of ss templates

6. In order to sequence, reverse strands are cleaved off = only forward strands left (P5)
   NOTE: reverse strands (P7) are re-generated and sequenced after

Question 12

Describe Sequencing by Synthesis (4) in Illumina

Answer 12

1. Read 1 Primer hybridizes at the top
2. Adds complementary fluorescent ddNTP = detected
3. Chemically changes ddNTP to dNTP and cleaves fluorophore
4. Continues synthesis one nucleotide at a time

Question 13

Differentiate 4-channel vs 2-channel detection in

Answer 13

4-channel:
- uses one image
- each DNA base (GCAT) emits a different wavelength = 4 colours

2-channel:
- uses two images
G = no color (Gone)
C = red (Sea)
A = yellow (All)
T = green (Tea)

Question 14

Explain Paired End Sequencing

Answer 14

Reads both ends of DNA:
- Forward strand = Read1 seq > cleave > i7 index seq
- Bridging and extension from P5 to P7
- Reverse strand = Read2 seq > cleave > i5 index seq

Question 15

Differentiate the DNA insert and Index in Illumina adapters

Answer 15

DNA insert = different targets
- one long segment
Index = specific to patients
- Forward: i7 index seq AND Reverse: i5 index seq

Question 16

Define Q Score

Answer 16

• “Phred quality score”
• assesses accuracy
• the unlikely probability that a given base is called incorrectly by the sequencer

Question 17

T or F: a higher Q score is associated to better accuracy

Answer 17

TRUE; a higher Q score is associated to better accuracy

NOTE: Q score > 30 is ideal

Question 18

What kind of output files is produced by Illumina ?

Answer 18

FASTQ

• Q scores encoded using ASCII characters (#?<:=)

Question 19

Differentiate sequencing depth vs breadth

Answer 19

Depth: multiple reads in the same location of a sequence
- increased cost, but increased sensitivity & confidence
Breadth: more sequences in different locations are read

Question 20

How is data analyzed in Illumina ?

Answer 20

• FastQ files produced
• reads are aligned to reference sequence = variants can be identified