12 - Whole genome sequencing

Question 1

Describe chain terminator sequencing (Sanger sequencing).

Answer 1

Using polyacrylamide gel (better resolution)

• Single stranded template
• Primer
• Polymerase
• All four dNTPs with one of four ddNTPs (in low concentration) in each tube

Thermal cycle sequencing using PCR technology came out which allowed this to all be done in one tube.

Question 2

Describe automated DNA sequencing

Answer 2

An extension of Sanger’s method.

• Uses fluorescently labeled ddNTPs
• Thermal cycle sequencing used to produce chain terminated products
• Capillary gel electrophoresis used to separate DNA fragments faster
• Sample runs past laser beam to cause fluorescence

Limit for individual reaction remained at about 800 bp :(

Question 3

Describe the sequencing of large DNA molecules

Answer 3

Shotgun sequencing

• Breaking DNA in a random fashion (Eg. NO RESTRICTION ENZYMES)
• Purify the fragments that are at a certain range of sizes
• Close in a plastid and prepare wells (one per clone)
• Do sanger sequencing on each fragment and do them individually, then the computer tries to put together fragments by finding overlaps.

Question 4

What are some methods to work around genome size or complexity to generate fragments you want to work with?

Answer 4

Genomic DNA is broken into large pieces and cloned into BACs, YACs or fosmids and cosmids. Each cloned DNA is THEN subject to shotgun sequencing.
sized for sequencing.

Question 5

Describe two common approaches to whole genome sequencing

Answer 5

In whole genome shotgun sequencing, the entire genome is sheared randomly into small fragments and then reassembled.

In hierarchical shotgun sequencing, the genome is first broken into larger segments. After the order of these segments is deduced, they are further sheared into fragments appropriately (extra step) and then put back together

Question 6

What is next generation sequencing?

Answer 6

Massively-parallel DNA sequencing (based on pyrosequencing)

It’s main steps are:

• Library preparation where genomic DNA is fragemented and adaptors are ligated to the fragments (700 - 800 bp)
• Library fragments are attached to micro beads. Emulsion based PCR amplification turns each bead into a fuzzy ball with millions of copies of the original DNA fragment.
• Machine takes picture of wells with one bead in each and detects a pulse of light when a replication event happens. The signal strength is proportional to the number of nucleotides incorporated. Will eventually produce a sequence for each well (each fragment)!

There is no cloning necessary, and no colony picking!

Question 7

When does next gen sequencing fail?

Answer 7

When you have multiple nucleotides of the same type next to each other.

The polymerase will incorporate both and make 2 pulses of light at the same time, computer will recognize summed intensity up to 8 or 9 consecutive base pairs, but no more.

Question 8

What is the most used technology for DNA sequencing?

Answer 8

Made by Illumina, and utilizes a different principle than next gen sequencing based on reversible terminators.

• Genomic DNA is broken into fragments
• Adhered to surface of a glass slide called a flow-cell
• Clusters are spots on the flowcell’s surface that are made up of millions of copies of a DNA fragment
• Sequencing is done in two passes, the first reads from one of the ends and the second pass reads from the other end
• This is done by a series of washes and addition of more terminating nucleotides (labeled with different dyes) in cycles

Question 9

What is a major problem with personal genome projects?

Answer 9

J. Craig Venter had his genome assembled de novo with automated Sanger.

Aaaall the other personal genomes have been aligned to Venter’s…