Sanger Sequencing vs NGS Flashcards
Give the read length, no of reads/run, throughput, SNP error rate, Indel error rate and costs of Sanger Sequencing.
Read Length: 800bp
No of reads/run: 96 [<1 day]
Throughput: 6MB/day
SNP error rate: low
Indel error rare: low
Costs: 500 euro/Mb
Give the read length, no of reads/run, throughput, SNP error rate, Indel error rate and costs of Illumina
Read Length: 2x150bp
No of reads/run: 400,000,000 [<1 day]
Throughput: 120GB/day
SNP error rate: high (aprrox 0.5%)
Indel error rare: low
Costs: <0.05 euro/Mb
Talk about the efforts to sequence the first human genome
The human genome was first sequenced in 2003
At this time many different institutes were all working at the same time to sequence different chromosomes
It took about 10 years and about a billion euros to do
Sanger -> multiple sanger sequencing ran at once, all day everyday
Private industry looking to patent the human genome vs public industry
What is mean tby SNP error rate?
This is the ability of a system to correctly identify Snps/incorrect bases
A low error rates means a high likelihood that the sequence is correct
What are indels?
Insertions and deletions
Everyone has these, they are a part of normal variation of the human genome, the majority of these are harmless but some can be pathogenic
How large is the human genome?
3 billion base pairs of DNA on a single chromosome -> x2 copies => 6 billion base pairs in the whole genome
Why is Illumina NextSeq sequencing not done anymore and what is used instead?
Illumina can only do short reads
Paired end sequencing is now done -> sequencing the forward and reverse strand at a time
-> if you used Sanger you would have to design reverse and forward primers to do this etc
Why is Illumina NextSeq sequencing not done anymore and what is used instead?
Illumina can only do short reads
Paired end sequencing is now done -> sequencing the forward and reverse strand at a time
-> if you used Sanger you would have to design reverse and forward primers to do this etc
What are the four main next gen sequencing technologies available?
Illumina -> most prevalent
SOLID (life technologies)
Ion Torrent (life technologies0
Pacific Biosciences
What are the two main 3rd generation approaches to sequencing
Oxford Nanopore (commercially available)
Illumina Nanopore (licensed an alternative Nanopore technology)
What is the basis of ion torrent technology?
Uses semi-conductors to tell when hydrogen ions are released
Why are there so many different sequencing technologies?
Companies have to keep making new sequencing technology as you cant get a patent for Sanger etc
What are the two main sequencing template approaches to sequencing?
Clonal amplification of single molecules
Single DNA Molecule as a squencing template
What is meant by the clonal amplification of single molecules as an aproach to sequencing, give two examples
Single molecule only briefly needed as a template
— Thousands of identical molecules boost signal
— Two different methods:
• Bridge amplification of molecules immobilized on surface - Illumina
• Emulsion PCR — Ion Torrent
What is meant by the single DNA molecule as a sequencing template as an aproach to sequencing, give two examples
— Challenge of keeping single molecules stable during sequencing
— Avoid amplification biases
— Pacific Biosciences, Oxford Nanopore
How does clonal amplification signaling work?
Makes use of amplification (by PCR) to amplify up a sequence before its fluorescence detection
Used in Ion torrent and bridge aplication methods
-> generally if you detect fluorescence you have to amplify up prior e.g. in ion torrent you have to increase the number of DNA molecules to burst the signal so we can detect enough hydrogen ions
What is the main benefit of single DNA molecule sequencing
This allows us to sequence much longer continuous strands of DNA
What is the history behind the Illumina: Flow Cell method of sequencing?
Illumina: Flow Cells with “Molecular Colonies”
Originally research done in Cambridge, was known as Solexa -> chemistry department of Cambridge
Sold to Illumina in 2006
How does the Illumina Flow cell sequencing method works?
Makes use of flow cells, a type of slides
Short oligonucleotide sequences only a few nucleotides long are spread across the entire surface of flow cell
These are used to bind DNA onto flow cell
Clusters on flow cell are formed of the same sequence of DNA -> each cluster started of as one DNA molecule that first bound and then is amplified by PCR to produce many copies in close proximity to it
What are the main pros and cons of the illumina sequincing
Can sequence millions of cluster reactions at once i.e. on the same flow cell
You have to measure (take an image) every cycle i.e. the sequence is built up one nucleotide at a time
It requires specially designed chemistry using reversible dye-terminators and a polymerase
Termination is a reversable process unlike Sanger -> this alloows us to stop the reaction and on another nucleotide at any point, image it, and then continue the reaction
What kind of nucleotides are used in Illumina sequencing
Fluorescently labelled reversible terminater nucleotides
How are the fluorescent nucleotides used in Illumina reversible?
You can chemically cleave of the fluorescent group of the nucleotide and wash it away at any point -> have to wash away to prevent background fluorescence when you go to add next nucleotide
You can then block the 3’OH group until your read to add the next nucleotide group - gives us time to image the last previous nucleotide that was added (temporarily block 3’ OH)
When the OH group is freed you can add the next nucleotide
