Notes

Dear Dr. Jauch,

I am Edison, a year 1 MBBS student who has just finished the IAS08 part 3 lecture concerning the techniques of modern DNA sequencing.
Below is my understanding of the process of sequencing-by-synthesis. It would be highly appreciated if you could correct any misconceptions and gaps in my understanding concerning the matter.After it is proof-read, I would happily share my understanding on the Moodle page for everyone’s reference.

The process of ‘sequencing by synthesis’
1. Sequencing libraries (single stranded DNA) are prepared from fragmented DNA samples
2. Adaptors (short DNA sequences) are attached to the ends of fragmented DNA samples to aid in the attachment to the ‘oligos’ (complementary sequences to the Adaptors, allowing for annealing) of the ‘lawns’ of the flow cell, stabilizing the fragmented DNA samples for bridge amplification
3. Process of bridge amplification:
1. The DNA sample anneals to one of the 2 types of ‘oligo’ complementary to the Adaptor sequence on the lawn, then a polymerase creates a complement of the hybridised fragment, creating double stranded DNA. The original DNA sample (template) is washed away
2. Bridging begins in the second round of amplification and thereafter. The new single- stranded DNA now anneals to another ‘oligo’, forming the bridge. Polymerases then create a complement of that bridge again. After that, the double stranded bridge is denatured to form 2 single-stranded DNA fragments that both stem from the 2 ‘oligos’
3. The reverse strands (3’ → 5’) are washed off, leaving only the forward strands. The 3’ ends of the forward strands are blocked by ddNTPs to prevent unwanted priming
4. Now the flow cell ‘lawn’ is adequately prepared to start sequencing by synthesis
4. Process of sequencing by synthesis:
1. Fluorescently-labelled nucleotides anneal to the sequence via complementary base pairing beginning after the ‘oligo’ at the start of the DNA chain
2. After the nucleotide binds to the chain, the nucleotide is excited by a light source and a characteristic fluorescent signal is emitted. The whole DNA chain is read in this way, creating a first read of the sequence
3. After the first read is finished, the now double-stranded DNA is denatured, and the fluorescently labelled DNA chain is washed away
5. Bridge amplification starts again, but this time the reverse strand is kept, and sequencing by synthesis starts again. The process is repeated many times to generate millions of reads.
6. Barcodes allow the pooling of multiple sequencing samples in a sequencing run. It means that similar base sequences generated by the reads can be locally clustered, and that forward and reverse reads can be paired, and aligned back to the reference genome. This resolves ambiguous alignments.

Moreover, I would also like to know whether the explicit details of long and short read sequencing methods such as sequencing-by-synthesis is required in our syllabus, or whether it is outside of the field of MBBS study. Thank you very much for your time.

Yours sincerely,
Edison Wu
HKUMed