Shane 2: Sanger Sequencing vs NGS

Question 1

Give the read length, no of reads/run, throughput, SNP error rate, Indel error rate and costs of Sanger Sequencing.

Answer 1

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

Question 2

Give the read length, no of reads/run, throughput, SNP error rate, Indel error rate and costs of Illumina

Answer 2

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

Question 3

Talk about the efforts to sequence the first human genome

Answer 3

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

Question 4

What is mean tby SNP error rate?

Answer 4

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

Question 5

What are indels?

Answer 5

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

Question 6

How large is the human genome?

Answer 6

3 billion base pairs of DNA on a single chromosome -> x2 copies => 6 billion base pairs in the whole genome

Question 7

Why is Illumina NextSeq sequencing not done anymore and what is used instead?

Answer 7

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

Question 8

Why is Illumina NextSeq sequencing not done anymore and what is used instead?

Answer 8

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

Question 9

What are the four main next gen sequencing technologies available?

Answer 9

Illumina -> most prevalent
SOLID (life technologies)
Ion Torrent (life technologies0
Pacific Biosciences

Question 10

What are the two main 3rd generation approaches to sequencing

Answer 10

Oxford Nanopore (commercially available)

Illumina Nanopore (licensed an alternative Nanopore technology)

Question 11

What is the basis of ion torrent technology?

Answer 11

Uses semi-conductors to tell when hydrogen ions are released

Question 12

Why are there so many different sequencing technologies?

Answer 12

Companies have to keep making new sequencing technology as you cant get a patent for Sanger etc

Question 13

What are the two main sequencing template approaches to sequencing?

Answer 13

Clonal amplification of single molecules

Single DNA Molecule as a squencing template

Question 14

What is meant by the clonal amplification of single molecules as an aproach to sequencing, give two examples

Answer 14

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

Question 15

What is meant by the single DNA molecule as a sequencing template as an aproach to sequencing, give two examples

Answer 15

— Challenge of keeping single molecules stable during sequencing
— Avoid amplification biases
— Pacific Biosciences, Oxford Nanopore

Question 16

How does clonal amplification signaling work?

Answer 16

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

Question 17

What is the main benefit of single DNA molecule sequencing

Answer 17

This allows us to sequence much longer continuous strands of DNA

Question 18

What is the history behind the Illumina: Flow Cell method of sequencing?

Answer 18

Illumina: Flow Cells with “Molecular Colonies”

Originally research done in Cambridge, was known as Solexa -> chemistry department of Cambridge

Sold to Illumina in 2006

Question 19

How does the Illumina Flow cell sequencing method works?

Answer 19

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

Question 20

What are the main pros and cons of the illumina sequincing

Answer 20

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

Question 21

What kind of nucleotides are used in Illumina sequencing

Answer 21

Fluorescently labelled reversible terminater nucleotides

Question 22

How are the fluorescent nucleotides used in Illumina reversible?

Answer 22

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

Question 23

What is the main con of the Illumina sequencing

Answer 23

Takes a lot of time due to many washing steps

To sequence 150bp sequence there will be 150 wash cycles -> add reversible nucleotide, block 3’OH, read fluorescent signal, cleave off/wash signal off, add next nucleotide

150bp length can take 12 hours or longer

Question 24

What kind of elongation is used on the illumina?

Answer 24

Illumina Paired End Sequencing

Question 25

What is illumina paired end sequencing?

Answer 25

A method of sequencing two strands of DNA at the one time Genomic DNA, purified, denatured with heat, fragmented into small sequences using enzymes or sonicaters Ligate on adapter sequences onto the forward and reverse short sequences using DNA ligase Each sequence now has an adapter 1 site (A1 site) and priming site (SP1), the complementary reverse has an A2 and SP2 sites The A1 site allows binding onto a flow cell The priming site is a sequence for which you can design primers for i.e. it allows you to prime the sequencing reaction, complementary design for reverse i.e. use primer 1 for forward sequence and primer 2 for reverse etc

Question 26

Give a brief run down of illumina paried end sequencing

Answer 26

Fragment genomic DNA Ligate adapters Generate clusters - bind to flow cell Sequence first end Regenerate clusters and sequence paired end

Question 27

Give a brief run down of illumina paried end sequencing

Answer 27

Fragment genomic DNA Ligate adapters Generate clusters - bind to flow cell Sequence first end Regenerate clusters and sequence paired end

Question 28

Talk a little about the illumina flow cells

Answer 28

There are like several hundred million flow cells Hundreds of millions of reactions on the one plate Clusters are only a micron in size - smalled than a bacterial cell Sequencing one nucleotide at a time -> creates a sequence about 150bp in length => 150 images put together to determine sequence

Question 29

Give some examples of illumina sequencers

Answer 29

MiniSeq System -> for targeted sequencing MiSeq series -> small genome and targeted sequencing HiSeq X Series ->population and production scale whole genome sequencing NovaSeq Series -> population and production scale genome, exome, transcriptome sequencing and more -> can cost up to a million euros -> none in Ireland

Question 30

Illumina Sequencing (2 pros + 3 cons)

Answer 30

Pros: - Very high throughput - can do millions of clusters per cell - Best price/bp but machines can be very expensive/some are affordable Cons: - relatively long run time -12 hours plus for a run of 150bp - Sequencing quality decreases towards the end -> polymerase struggles to incorporate large fluorescent molecules near 150bp - Imaging interference in low diversity libraries -> fluorescence interference in highly repetitive sequences

Question 31

How does ion torrent sequencing work

Answer 31

Developed in 2010 by Life Technologies A form of emulsion PCR using magnetic beads

Question 32

How does Ion torrent work

Answer 32

Uses magnetic beads coated in short oligonucleotides Each bead is in an oil droplet along with DNA polymerase ad nucleotides Chips have thousands of wells each with ion sensors Each bead fits into a well Semi conductor detect the release of hydrogen ions released when any nucleotide is icorporated by polymerase

Question 33

Why do we need to amplify the signal for ion torrent

Answer 33

Its done to increase the amouont of H+ signal prodced so that its release can be detected by an ion sensor

Question 34

How do ion sensors work?

Answer 34

They detect H+ released upon incorporation of nucleotides by polymerase They borrow the technology used in semi-conductors

Question 35

Ion torrent sequencing is based on what kind of sequencing?

Answer 35

Semi-conductor sequencing The sequencing is carried out on the chip, no imaging is required

Question 36

How does H+ detection work in ion torrent sequencing?

Answer 36

H+ is released with the formation of phosphodiester bonds This brings about a pH change Slightly acidic pH is measured with a sensor

Question 37

Explain in your own way how ion torrent sequencing works?

Answer 37

Cycle 1: add an A, if the A is not encorporated i.e. no H+ released then wash away Cycle 2: add a G, if not encorporated wash away Cycle 3: add a C etc etc Cycle 4: add a T, if encorporated then H+ released Therefore we know there is a T at position 1, then we go onto the next cycle and keep repeating until you get a desired length

Question 38

If the signal detected is twice as strong upon adding of a nucleotide in ion torrent sequencing, what does this indicate?

Answer 38

If the signal is twice as strong you know two of the same nucleotide have been added on in a row

Question 39

What are two examples of ion torrent sequencers?

Answer 39

Ion PGM Ion Proton

Question 40

Talk about the Ion PGM Ion torrent sequencer

Answer 40

Personal genome machine 3 different types of chips Can do 200 or 400bp reads Can run up to 5.5million reads/Ion 318 chip i.e. over 5 million wells per chip 4-7 hour run time Much quicker than sanger, no need for fluorescence etc

Question 41

Talk about the Ion proton ion torrent sequencer

Answer 41

Newer ion torrent sequencer Up to 200bp reads Up to 60-80 million reads (way more than ion PGM) 2-4 hour run time -> short run time hence its use in hospital labs Useful for targetted gene sequencing e.g. for cancers or genetic disorders

Question 42

What are the three main pros of ion torrent sequencing and the two main cons

Answer 42

Pros: - fast - relatively cheap - scalable (can buy different chips depending on need) Cons: - relatively high error rate - emulsion PCR

Question 43

Talk about the high error rate of ion torrent PCR

Answer 43

High error rate seen where there are repetitive sequences e.g. 3 As in a row -> you would think the signa would be three times higher but this is not always the case there was a very high error rate associated with this in the beginning but it has since gotten better

Question 44

Talk about the difficulties of emulsion PCR in Ion torrent sequencing

Answer 44

Emulsion PCR is very technically challenging and can take a while to get it to work in the lab

Question 45

What is PacBio Sequencing and who set it up?

Answer 45

Single Molecule Real Time Sequencing (SMRT) Set up in 2011 by Pacific Biosciences Developed in Standord University California

Question 46

How does PacBio Sequencing Work

Answer 46

Single Molecule Real Time Sequencing SMRT Chips have individual wells One copy of DNA sequence in each well DNA in single strand form Inside each well there is an imobilised DNA polymerase i.e. stuck to bottom of well There are fluorescently labelled nucleotides in well floating around Polymerase will incorporate complemenary nucleotides A fluorescence pulse occurs everytime a nucleotide is added on The fluorescent pulse is measured in real time - this happens very quickly The polymerase cannot move hence how we know the exact position where the nucleotide is bing added on

Question 47

Why is the DNA polymerase immobilised in PacBio Sequencing, and how

Answer 47

Immobilised by fixing it to the bottom of the well This stops the polymerase randomly inserting nucleotides Allows us to know the start point of transcription

Question 48

Talk about the wells used in PacBio Seuencing, why is this done

Answer 48

The wells in the chips used are very shallow This stops any fluorescent nucleotides from floating out

Question 49

Talk about the pros and cons of PacBio Sequencing

Answer 49

No PCR ie dont need to amplify DNA prior to sequencing Can do very long reads - read lengths averaging 10-15kb and a max of 40kb Can be used to observe DNA modifications Throughput per run is low -> approximately 1 million reads Run time is short Error rate is high - same nucleotide repeats cause issues

Question 50

Talk about Oxford Nanopore, what is the principle behind it

Answer 50

Makes use of bacterial nanopore proteins which drag DNA through small perforations in a chip 'nanopore' DNA is 'sequenced' as it is dragged through the nanopore

Question 51

How does PacBio sequencing allow for observation of DNA modifications

Answer 51

Anytime a cytosine is methylated a different pulse is seen This allows us to identify any points of DNA cytosine methylation

Question 52

Explain how we identify base pairs using Oxford Nanopore sequencing

Answer 52

A flow of ions flows through the nanopore constantly Each base blocks the current to a different degree Each different nucleotide it blocks the ion current a very specific amount

Question 53

Talk about nanopore proteins and explain how we use them

Answer 53

Nanopore proteins play a role in bacterial cells - normally they take up DNA They transport DNA into the bacterial cell from outside the well They take up double stranded DNA Motor protein and motor enzyme transports DNA across the nanopore one strand at a time - forward strand first then the reverse strand -> can sequence both strands this way

Question 54

What are two commercially available oxford nanopore sequencers?

Answer 54

MinION GridION

Question 55

Talk about some of the portable Oxford Nanopore technologies

Answer 55

Flongle and SmidgION For use in the field Often used in microbioogy e.g. for covid detection