Genes & Genomes - Lec 3 - NGS sequencing Flashcards
learn about the next generation of sequencing
What is the goal of genome sequencing
Determine the order of nucleotides across a genome
What are the problems associated with Genome sequencing?
1) Genomes are huge
2) Large amounts of DNA are needed for sequencing
3) Current DNA sequencing methods can handle only short stretches of DNA
What is the solution to the problems facing genome sequencing?
Sequence millions of pieces of DNA and use computer to assemble the small pieces
What are the limitations of Sanger sequencing?
1) Must have a colony for every reaction - Must have 1 tube & gel per reaction
2) Must do DNA prep for every reaction
3) Low throughput
4) Expensive
5) Requirement for large-scale cloning & robotics
What are the advantages of Sanger sequencing?
1) Long reads (600-1000Bp)
2) Low error rate (99.999% accuracy)
3) suitable for small projects
4) Data tractable with PC and free software
What are the present ways of genome sequencing?
Next-generation sequencing (Illumina dominant)
What is Illumina sequencing?
Takes advantage of miniaturization to engage in massive parallel analysis.
Carries out millions/billions of sequencing reactions simultaneously in each of millions/billions of tiny wells/clusters
How is DNA prepared for Illumina sequencing?
1) extract DNA
2) fragment DNA using sonication
3) Attach adapters to sequence
What is the Illumina flow cell coated with?
Two types of oligos
Explain Illumina DNA cloning amplification on a flow cell? (6 steps, 4 has 3 substeps)
1) Hybridisation is first enabled by the two types of oligos on the flow cell which bind to the complementary adaptor region on one end of the fragmented DNA strands
2) DNA polymerase creates a complement of the hybridised fragment
3) The double-stranded molecule is then denatured and the original template is washed away
4) the new strands are clonally amplified through bridge amplification
a) The strand bends over and adaptor region that’s on top of the strand hybridises to the second type oligo on the flow cell
b) polymerase forms a DNA double-stranded bridge
c) Bridge is denatured
5) process is the repeated again
6) reverse strands are washed away leaving only forward strands, 3 prime ends are blocked.
How is the DNA sequenced on the Illumina flow cell?
1) The extension of the first sequencing primer produces the first read
2) With each cycle, fluorescently tagged bases compete for addition to the growing chain
3) Only one is incorporated based on the sequence of the template
4) After the addition of a nucleotide the clusters are excited by a light source and a fluorescent signal is emitted (sequencing via synthesis)
5) Number of cycles determines the length of reading
6) Emmition wave length among with signal intensity, determine the base call
7) All strands are read simultaneously
8) After completion of reading the read product is washed away and the same process is done for 3 prime end
How are Flow cell readings analysed?
1) reads with similar base calls are locally clustered.
2) Forward and reverse reads are paired creating contiguous sequences and are aligned to the reference genome for identification
What is De-novo assembly?
When NGS reads are used to sequence and then assemble a new genome
What is the disadvantage of De-novo assembly?
1) Difficult due to short reads
2) Particularly different if there is low coverage
What is resequencing?
If a reliable sequence is already available, reads can be compared to identify differences without the problem of assembly
