Chapter 3 Lec6

Question 1

Briefly describe illumina(solexa) system.

Answer 1

It involves parallel simultaneous sequencing of a library of fragments, by synthesis.

Question 2

Discuss what is involved in the workflow of illumina.

Answer 2

1. Library generation- DNA is fragmented, followed by adaptors ligation(same sequence) to both ends; typical fragment lengths are 300-800 bp.
2. (A) Library fragments are attached (via adaptors) to oligonucleotides on flow cell

• flow-cell (a glass slide with lens) can contain billions of nanowells
• each well comprises a ‘lawn’ of attached logos that are complementary to adaptor sequence.

(B) bridge amplification in situ( called ‘bridge PCR’)

• generates a monoclonal cluster of replicated fragments around each original fragment; one cluster per nanowell.

3. Sequencing by synthesis

• polymerase adds a base to adaptor-fixed primer-each base has a different fluorescent tag and a blocking group (to ensure that only 1 base is added at a time)
• the distribution of colors, in an image of the field, identifies which base was added to each cluster
• the fluorescent tag and blocking groups are removed; repeat the process
• overall, a kaleidoscopic movie of shifting colors, one frame per position