3. next generation sequencing

what are dideoxy chain terminators

labelled (coloured) ddNTPs

what does DNA polymerization with dideoxy chain terminators create

creates fragments with different lengths and colours

how does DNA polymerization stop

when a ddNTP is incorporated, which terminates the growth

how are the DNA fragments arranges during electrophoresis

they are arranged by size, so the longest ones will travel the least, the shortest ones will travel the furthest

what is Sanger sequencing

• a serial process

• if the human genome is broken up into 500 base pair chunks for sequencing, there would have to be 6 million sequencing reactions to perform to complete the human genome

what is second generation sequencing

massive parallel sequencing via spatially separated, clonally amplified DNA template in a flow cell

where does sequencing happen

happens in the flow cell

what is a limitation of 2nd generation sequencers

• expensive

• bulky equipment

what are the pros of third generation sequences

• lightweight and portable

• cheaper

what does a nanopore sequencer do

single molecule sequencing

what is the process of single molecule sequencing

changes in electrical signal across the nanopore when different bases pass through

what can genetic information do

• can guide treatment and prevention

  • genetic information can guide cancer treatment

  • genetic information can predict risk factor

what are bioinformatics

the field of science that uses computers. software and mathematics to store, analyze and interpret large sets of biological data, such as DNA and protein sequences

why is bioinformatics needed

• DNA contains millions to billions of letters (A, C, T, G)

• a biologist cannot read this sheer volume of raw data manually

  • bioinformatics uses computers to find patterns, spot genetic diseases and speed up research

what is the scoreboard rule of smith-waterman algorithm

match: +1 point

mismatch: -1 point

gap: -2 points

• no negative scores are allowed in the entire grid

• if a cell’s calculated value drops below zero, it immediately resets to 0

what is the traceback process

1. find the start → locate the cell with the highest numerical score

2. move backwards → at each cell, determine which neighbour (plus its respective match bonus or gap penalty) mathematically evaluates to the current cell’s score

• diagonal (match/mismatch): move diagonally up-left: aligns 1 character from sequence 1 from one from sequence 2

• up (deletion): move straight up: this introduces a gap in sequence 1

• left (insertion): move straight left: this introduces a gap in sequence 2

3. stop → continue tracing the path backward until it lands on a cell with the score

4. reconstruction → read the path in reverse to generate the matches sequence and gaps