next-generation DNA sequencing

cab week 2

Sanger sequencing

• di-deoxy variants of the nucleotide bases ATCG and radiolabelled them to find out

next generation sequencing

high throughput: NGS enables simultaneous sequencing of millions of DNA fragments, drastically increasing the vol of data generated compared to traditional methods like Sanger sequencing

clonal amplification: techniques like bridge PCR or emulsion PCR are used to amplify DNA fragments, ensuring sufficient signal for detection during sequencing

parallel processing: DNA fragments are sequenced in parallel, allowing for rapid analysis of entire genomes or transcriptomes

wide applications: NGS used in diverse fields, including medical diagnostics, evolutionary biology, microbiome analysis and personalised medicine

cost and accuracy: while initial equipment costs are high, NGS provides relatively low per-base sequencing costs and high accuracy, especially with deep sequencing

high throughput

• simultaneous sequencing: NGS can sequence millions of DNA fragments at the same time, sunlike Sanger, which processes one fragment at a time

• large data output: generates massive amouynts of sewqunecing data in a single run, ideal for whole genome

• efficiency: shorter time

• scalable: can be used for small scale or large scale

clonal amplification

emulsion PCR: DNA fragments attach to beads and are amplified with oil droplets, creating identical copies of each fragment

bridge PCR: DNA fragments bind to primers on glass slide, forming clusters of DNA through PCR repetitions

signal detection: amplification ensures a detectable signal from sequencing reactions as unamplified single molecules are too faint to read

accuracy boost: amplified DNA clusters reduce sequencing errors by generating stronger and clearer signals

parallel processing

flow cells or chips: NGS spreads DNA fragments across a flow cell or chip, enabling simultaneous sequencing of each fragment

millions of reactions: multiple sequencing reactions occur in parallel, increasing speed and throughput

automated analysis: machines handle most of the sequencing process, allowing researchers to focus on interpreting data

real time monitoring: advanced detectors, like cameras or pH sensors track sequencing reactions in real time

applications

medical diagnostics: used for identifying genetic mutations, tumour profiling and personalised medicine

evolutionary studies: helps trace genetic relationships and evolutionary history by comparing genomes

microbiome analysis: enables study of microbial diversity and function within ecosystems or the human body

forensic biology: assists in solving crimes by identifying individuals through DNA evidence

cost and accuracy

lower cost per base: sequencing costs have dropped significantly, making genome sequencing affordable for routine use

deep sequencing: reads each part of the genome multiple times, ensuring high accuracy by reducing random errors

error detection: dual strand sequencing and improved chemistry help minimise mistakes

initial investment: while equipment is expensive, high throughput and scalability reduce long term costs for researchers

dideoxynucleosides are the key to Sanger sequencing

• DNA polymerase joins nucleotides by a condensation reaction between one phosphate and one OH group

Sanger method

• primer can be used to direct DNA polymerase to begin synthesising DNA strands from a specific location in target DNA

• if di-deoxy nucleotides were incorporated into the reaction mix (dTTP) the polymerase would stall and fall off when it reaches T in the sequence

dNTPs cause chain termination

• DNA polymerase stalls and falls off whenever it incorporates a dNTP, releasing a short, aborted chain

• if you run 4 separate reactions, (one with a dATP, one with dCTP and one with dGTP), you can find out where As, Cs, Ts and Gs in short sequence by separating these aborted chains by electrophoresis

Sanger sequencing products were first run on gels

• reaction products run on agarose/polyacrylamide gels

• each band represents one point at which chain has terminated

• band pattern indicates the sequence

• good compared to earlier techniques

radiolabelled nucleotides are used to visualise the gel band patterns

• dNTPs are radiolabelled with radioactive phosphorus (32P)

• toxic and difficult to work with

• gels need to be overlaid with x-ray film overnight

using 4 different fluorescent tags enable single lane sequencing

• eventually realised that nucleotides could be fluorescently labelled instead of radiolabelled

• non toxic and faster to read

• no requirement for radiation protection or X-ray film

• if 4 different fluorescent tags were used for ATCG, the sequencing reaction products could be separated in just one lane (rather than 4)

the separation of sequencing products moved on from gels to capillaries

• companies soon built machines that separated DNA in capillaries, rather than gels and used lasers to detect the terminated chains as they moved past detector in real time

• accelerated progress, up to 1000 bp per reaction and multiple reactions can be measured at once on the same machine (multiple capillaries)

sequencing the first human genome

• started in 90s

• 3 billion bases: too long to sequence directly

• genome broken down into bacterial artificial chromosomes (BACS)

• each BAC: 150,000 bp

• BAC sequences then aligned based on overlap

• called shotgun sequencing

• requires lots of cloning

amplification: how emulsion PCR works

1. genomic DNA is sheared randomly to create mixture of short genome fragments

2. short DNA pieces are called adapters which are like primers and are ligated to each fragment end

3. 2 different adapters are ligates to each end of the fragment

how emulsion PCR works

1. billions of tiny plastic beads are mixed with labelled DNA fragments

2. each bead is coated in primer matching adaptor sequence

3. mixture is dilutes to the point where there is just one DNA fragment per bead

4. mixture is vibrates in oil to form emulsion of tiny droplets (each containing 1 bead and 1 molecule of DNA0

5. mixture containing droplets is subjected to standard PCR thermal cycling

6. causes each bead to become covered in thousands of copies of same DNA sequence

7. each of these segments is 400-700 bp long

8. this amplification is required to generate sufficient DNA molecules so that they can be detected by fluorescence which is basis of most high throughput sequencers

how bridge PCR works

• amplification can also be achieved on a glass slide using bridge PCR

• short adapter sequences ligated to both ends of the DNA fragments bind to corresponding primers on a slide

• PCR cycling is used to form dsDNA bridges with nearby adaptors for the other end of the target DNA

• this process is repeated until localised clusters form on glass slide, each cluster containing hundreds of copies of same DNA sequence

HTS sequences millions of clonal clusters at the same time

• once the millions pf clusters have been formed on glass slides/beads, they are spread evenly over glass slide or chip

• every bead or cluster is then monitored by a sensitive camera/pH detecting chip

• single type of nucleotide is washed over the whole chip causing a signal to be released from each cluster, then repeated with a different nucleotide

• millions of beads or clusters can be sequenced in parallel (therefore, term: high throughput)

current high throughput DNA sequencing (HTS) technologies

1. Illumina: reversible dye terminator sequencing

2. Roche 454: pyrosequencing

3. Ion torrent: pH detection

4. Oxford Nanopore: single molecule real time sequencing

5. Pacific Bioscience: single molecule real time sequencing

Illumina sequencing by synthesis

• bridge PCR using 2 adapters is used to prepare clusters on glass slides

• primer is added to bind to the first adaptor sequence to enable DNA polymerase extension

• fluorescently labelled nucleotides (A T C or G) are flowed across flow cell

• modified so only one nucleotide can be added to growing chain at a time

• DNA polymerase adds one nucleotide to each chain

Illumina photographs 40 mill clusters per flow cell

• camera photographs entire flow cell and records colour of each cluster under fluorescence excitation

• colour indicates what type of nucleotide (A T C G) was last to bind to that cluster

• if next base in sequence was T, whole cluster would be red (since red T was latest nucleotide added)

• another solution is flowed across cell, causing fluorescent tags to be cleaved from nucleotides

• removes all colour from each cluster, enables another nucleotide to be added in next round

Illumina sequences each strand once in each direction

• one the read from the first adaptor is finished, the complementary strand is washed away

• process is then repeated in the same way, but starting from the other primer (second adaptor)

• Illumina sequencing checks the sequence in both directions

Roche 454 sequencing

• Roche 454 sequencing begins with cluster formation on plastic beads which are then spread onto a flow cell

• 1 type of nucleotide is flowed over the flow cell at a time

• when DNA polymerase incorporates a nucleotide, pyrophosphate is released

• converted to ATP, which is then used by an enzyme to produce a flash of light

• camera records the flash of light

• magnitude of flash of light indicates how many nucleotides were added

• unbound nucleotides are degraded by apyrase and washed away

• next type of nucleotide is washed over the flow cell and the process repeats

• method is also called pyrosequencing

Ion Torrent sequencing works

• ion torrent sequencing also uses beads trapped in tiny wells of a flow cell

• at the bottom of the flow cell, silicon chip acts as a tiny pH meter

• when a nucleotide binds, H+ is released and this changes the pH in each well

• magnitude of the change in pH indicates how many of that type of nucleotide were added each time

• unbound nucleotides are washed away and the next type of nucleotide is flowed across the flow cell

• repetition of this ATCG cycle reveals changes in pH and sequence of the cluster on each bead in each well

• this method struggles to accurately sequence long repeats of the same nucleotide (like pyrosequencing)

pros and cons of each method

the 3 HTS methods discussed to produce read lengths of 400-700 bp

• these must then be aligned with a reference genome to figure out where they fit in

• they are not very useful for genomes that have not been sequenced before

resequencing vs. de novo sequencing

resequencing: resequencing is the term for sequencing a member of a species of which other members have already been sequenced. Reads need only be aligned to a reference genome, so need only be several hundred bp long

de novo sequencing: is the term used to sequence a genome from scratch. It is much more time-intensive and costly, and requires reads at least 1,000 bp long, which is partly why the first human genome was so expensive.

computers are used to re-assemble the reads against a reference genome

• most high throughput sequencing technologies require lots of computer time to re-assemble a genome sequence from many thousands of short reads by alignment with a reference genome

definitions of depth and coverage

• most high throughput sequencing platforms generate millions of short reads

• aligned toa reference genome by homology

• the same part of a genome can be sequenced many times by different fragments

• referred to as the depth

• a genome may be sequenced to 30x depth

• increasing depth increases the sequence accuracy enormously

depth and coverage

• not all of the genome is equally easy to sequence, some regions are easy and some are hard

• only 90-95% of genome is fully sequenced and to good depth

• called coverage

why genome sequencing is useful

• medical diagnosis

  • rare mutations, tumour profiling, personalised med

• biotechnology

  • discovering new genes, developing useful constructs

• forensic biology

  • identifying suspects from DNA samples

• virology

  • new viruses, diagnosis, monitoring of recombination

• biological systematics

  • enormously useful for studies of evolution and relatedness

• biomedical research

  • new gene discovery, microbiome

GWAS for diseases

• common variants have been identified which are associated with risk of numerous common diseases

• useful to be able to sequence the genomes of 1000s of people

• improves personalised med

RNA sequencing

• RNA req uses HTS to monitor a cell’s transcriptome

• can give us unparalleled insight into exactly what type of program a cell is running

bisulphite sequencing for epigenetics

• epigenetics marks on DNA control cell differentiation

• methyl-cytosine is a key epigenetic mark

• treatment of DNA with bisulphite converts cytosine residues to uracil, but leaves 5 methylcytosine resides unaffected

• HTS of bisulphite treated DNA can be used to discover where cytosine has been methylated in the genome