Illumina

Question 1

Describe the dye labelled nucleotides present in illumina sequencing

Answer 1

Illumina uses dNTPs that have been modified with a fluorescent dye in the place of the 3’OH group

Question 2

What are dye labelled nucleotides called

Answer 2

Reversible dye terminators

Question 3

Describe one “cycle of chemistry” in illumina sequencing

Answer 3

1) the DNA synthesis reaction incorporates a single dye labelled nucleotide and as a result the DNA synthesis reaction stops
2) you then wash the flowcell surface, leaving the dye incorporated
3) a laser is shone on the flowcell and the colours of the fluorescence are logged by the imaging system
4) a chemical is run through the machine so that the dye labelled nucleotide is washed off and the DNA synthesis reaction can occur again

Question 4

Describe one key limitation of illumina sequencing

Answer 4

Illumina sequencing produces many short reads. illumina produces reads of around 300bp. In contrast a nanopore sequencer can manage 30,000 bp reads

Question 5

How accurate is illumina sequencing

Answer 5

Illumina is consistently 98% accurate

Question 6

What is the cost per million base pairs for illumina sequencing

Answer 6

£0.01

Question 7

How many reads per run can you generate with illumina sequencing

Answer 7

5 billion

Question 8

Describe how DNA is prepared for illumina sequencing

Answer 8

• DNA is sonicated into very small fragments (100-800bp)
• add Y shaped illumina adaptors and perform a PCR type reaction to end up with different adaptors on each end
• DNA is attached to flowcell surface and then sequencing can occur

Question 9

Describe the 3 main functions of an illumina adaptor

Answer 9

1) PCR type reaction means you end up with two different adaptors on each end
2) flowcell binding
3) sequencing primer binding

Question 10

Describe flowcell binding

Answer 10

• Randomly fragment genomic DNA and legate adaptors to both ends
• Bind single stranded fragments to the inside of a flowcell
• A polymerase will then create a double stranded molecule and the original strand is removed
• The single strand then folds over and binds to another oligo on the flowcell surface
• Polymerase generates a double stranded bridge
• This is denatured resulting in two single stranded copies of the DNA tethered to the flowcell
• This is repeated for millions of clusters resulting in colonial amplification of all of the fragments
• After bridge amplification the reverse strands are washed away and then remaining ones are used as a template for DNA synthesis
• Dye labelled nucleotides are then used to sequence each cluster
• Remember that DNA has been diluted so that only a few molecules bind every few um resulting in clusters of DNA