Chapter 3

Question 1

Frederick Sanger and the development of DNA sequencing

Answer 1

• Challenges in the early 1970s
   Preparing pure samples of ssDNA (single-strand DNA)
   Restriction Enzymes – new discovery; not entirely understood.
   Bacteriophage ΦX-174 – a natural source of 5,386 bp of purifiable ssDNA; genome still too long; sequence assembly challenge
   Enabled sequencing of relatively longer fragments, making unambiguous assembly possible.
• Requirements
   DNA polymerase – replication enzyme, i.e., DNA synthesis
   Primer: a short stretch of the complementary strand to be extended by successive addition of nucleotides. Provides free 3’OH group.
   Deoxynucleoside triphosphates (dNTPs) – nucleotides used to form a newly synthesized strand.
   Dideoxynucleoside triphosphates (ddNTPs) – dNTPs that lack free 3’OH.

 Read gels from the bottom (5’), top = 3’

Question 2

the concerns (6) and positives around the applicability of the WGS method.

Answer 2

Concerns:

• First, it was believed that the WGS method will not work in genomes that have many repetitive sequences (i.e., complex eukaryotic genomes) since these regions are known to create problems during assembly.
   It was argued that the technique worked smoothly in prokaryotes because they contain relatively less internal repetitive sequence.
   However, the WGS method was subsequently used successfully to sequence the D. melanogaster genome, which by a counterargument, still had fewer repeats than mammalian genomes and thus contributed to its successful sequencing by shotgun methods.
• Second, it was also believed that genomes that contain highly skewed base compositions will also complicate the application of WGS methods, e.g., Plasmodium falciparum – contains approximately 80% AT in its genome. However, many genomes have since been sequenced despite this fact.

Positives – WGS approach
* It may be possible to identify genes in a partly assembled genome with many gaps, provided that the genes are contained within contigs.
* Fruit fly WGS sequencing by Celera – ‘proof of principle’; completed the ‘commercial’ human genome project using the academic sequence as reference.

Question 3

two main (conceptual) differences between BAC-to-BAC and WGS methods.

Answer 3

• BAC-to-BAC methods are more robust than WGS methods.
   In diploids, fragments arising from homologous regions of two chromosomes of a pair may have sequence differences.
   The correct assembly must place them at the same location while noting the discrepancies, thus, the assembly must not split these reads into different contigs because of the imperfect matches.
• Highly inbred laboratory strain vs outbred population or pooled DNA
   Would present a more severe assembly challenge (considering the point above)

Question 4

general workflow for NGS methods (e.g., Illumina)

Answer 4

• Library generation – DNA is fragmented, followed by adaptor ligation to both ends (same sequence!); fragment lengths are 300 - 800 bp.
• Library fragments are attached (via adaptors) to oligos on a flow cell.
   Flow-cell (a glass slide with lanes) can contain billions of nanowells.
   Each well comprises a ‘lawn’ of attached oligonucleotides that are complementary to the adaptor sequence.
• Bridge amplification in situ (i.e., ‘bridge PCR’)
   Generates a monoclonal cluster of replicated fragments around each original fragment; one cluster per nanowell.
• Sequencing by synthesis
   Polymerase adds a base – each base has a different fluorescent tag and a blocking group (to ensure only 1 base is added at a time)
   The distribution of colours, 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 colours, one frame per position.