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Next Generation Sequencing

Next-generation sequencing (NGS) is a modern DNA sequencing technology that offers advantages over traditional Sanger sequencing in terms of speed, cost, sample size, and accuracy. NGS allows for massive parallel sequencing, drastically reducing the time and financial resources required for genomic studies while enabling high-throughput analysis of genetic material. Technologies such as Illumina sequencing and Ion Torrent have revolutionized the field, allowing for the sequencing of entire human genomes at unprecedented speeds and lower costs.

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What is Next Generation DNA Sequencing Next generation sequencing (NGS), also known as highthroughput sequencing, is the catchall term used to describe a number of different modern sequencing technologies including: • Illumina (Solexa) sequencing • Roche 454 sequencing • Ion torrent: Proton / PGM sequencing • SOLiD sequencing These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing Dr. Shiny C Thomas, Department of Biosciences, ADBU
The four main advantages of NGS over classical Sanger sequencing are: • speed • cost • sample size • accuracy • NGS is significantly cheaper, quicker, needs significantly less DNA and is more accurate and reliable than Sanger sequencing. Let us look at this more closely. • For Sanger sequencing, a large amount of template DNA is needed for each read. • In NGS, a sequence can be obtained from a single strand
• NGS is quicker than Sanger sequencing in two ways. Firstly, the chemical reaction may be combined with the signal detection in some versions of NGS, whereas in Sanger sequencing these are two separate processes. • Secondly and more significantly, only one read (maximum ~1kb) can be taken at a time in Sanger sequencing, whereas NGS is massively parallel, allowing 300Gb of DNA to be read on a single run on a single chip.
• The reduced time, manpower and reagents in NGS mean that the costs are much lower. The first human genome sequence cost in the region of £300M. Using modern Sanger sequencing methods, aided by data from the known sequence, a full human genome would still cost £6M. Sequencing a human genome with Illumina today would cost only £6,000.
Evolution of Technologies Gene Transcription
Sequencing for Transcriptomics? • Sample cells • Purify mRNA* • mRNA ⇒ cDNA • Cleaving into 35-400nt fragments • Sequence fragments in parallel • PCR amplification * or DNA for genomic sequencing
Next Generation Sequencing Technology Overview 1. (c)DNA is fragmented 2. Adaptors ligated to fragments 3. Several possible protocols yield array of PCR colonies. 4. Enzymatic extension with fluorescently tagged nucleotides. 5. Cyclic readout by imaging the array.
• Fragmentation of DNA • Primers are attached to the surface of a bead Technology 1. (emulsion PCR)
2. Bead preparation • Fragments, with adaptors, are PCR amplified within a water drop in oil. • One primer is attached to the surface of a bead • 3’ modification of fragments, to covalently bind bead to chip surface.
3. Cycles of elongation
4. Cycles of imaging
Sequence of images Sequence read over multiple cycles Repeated cycles of sequencing to determine the sequence of bases in a given fragment, a single base at a time.
A single transect image
• While these “first-generation” instruments were considered high throughput for their time, the Genome Analyzer emerged in 2005 and took sequencing runs from 84 kilobase (kb) per run to 1 gigabase (Gb) per run. • The short read, massively parallel sequencing technique was a fundamentally different approach that revolutionized sequencing capabilities and launched the “next-generation” in genomic science. • From that point forward, the data output of next- generation sequencing (NGS) has—more than doubling each year
Figure 1: Sequencing Cost and Data Output Since 2000—The dramatic rise of data output and concurrent falling cost of sequencing since 2000. The Y-axes on both sides of the graph are logarithmic.
• In 2005, with the Genome Analyzer, a single sequencing run could produce roughly one gigabase of data. • By 2014, the rate climbed to a 1.8 terabases of data in a single sequencing run—an astounding 1000× increase. • It is remarkable to reflect on the fact that the first human genome, famously co published in Science and Nature in 2001, required 15 years to sequence and cost nearly 3 billion dollars. • In contrast, the HiSeq X™ Ten, released in 2014, can sequence over 45 human genomes in a single day for approximately $1000 each.
Human Genome Sequencing Over the Decades—The capacity to sequence all 3.2 billion bases of the human genome (at 30× coverage) has increased exponentially since the 1990s. In 2005, with the introduction of the Illumina Genome Analyzer System, 1.3 human genomes could be sequenced annually. Nearly 10 years later, with the Illumina HiSeq X Ten fleet of sequencing systems, the number has climbed to 18,000 human genomes a year.
• Beyond the massive increase in data output, the introduction of NGS technology has transformed the way scientists think about genetic information. • The $1000 dollar genome enables population-scale sequencing and establishes the foundation for personalized genomic medicine as part of standard medical care. • Researchers can now analyze thousands to tens of thousands of samples in a single year. • The rate of progress is stunning.
• As costs continue to come down, we are entering a period where we are going to be able to get the complete catalog of disease genes. • This will allow us to look at thousands of people and see the differences among them, to discover critical genes that cause cancer, autism, heart disease, or schizophrenia.
The Basics of NGS Chemistry • In principle, the concept behind NGS technology is similar to CE (Capillary Electrophoresis) sequencing— DNA polymerase catalyzes the incorporation of fluorescently labeled deoxyribonucleotide triphosphates (dNTPs) into a DNA template strand during sequential cycles of DNA synthesis. • During each cycle, at the point of incorporation, the nucleotides are identified by fluorophore excitation. • The critical difference is that, instead of sequencing a single DNA fragment, NGS extends this process across millions of fragments in a massively parallel fashion.
• Illumina sequencing by synthesis (SBS) chemistry is the most widely adopted chemistry in the industry and delivers the highest accuracy, the highest yield of error- free reads, and the highest percentage of base calls above Q30. • The Illumina NGS workflows include 4 basic steps
1. Library Preparation— • The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5’ and 3’ adapter ligation. • Alternatively, “tagmentation” combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. • Adapter-ligated fragments are then PCR amplified and gel purified.
2. Cluster Generation— • For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. • Each fragment is then amplified into distinct, clonal clusters through bridge amplification. • When cluster generation is complete, the templates are ready for sequencing.
3. Sequencing— • Illumina SBS technology utilizes a proprietary reversible terminator–based method that detects single bases as they are incorporated into DNA template strands. • As all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies. • The result is highly accurate base-by-base sequencing that virtually eliminates sequence- context-specific errors, even within repetitive sequence regions and homopolymers.
4. Data Analysis— • During data analysis and alignment, the newly identified sequence reads are then aligned to a reference genome. • Following alignment, many variations of analysis are possible such as single nucleotide polymorphism (SNP) or insertion-deletion (indel) identification, read counting for RNA methods, phylogenetic or metagenomic analysis, and more.
Next-Generation Sequencing Chemistry Overview
Advances in Sequencing Technology Paired-End Sequencing • A major advance in NGS technology occurred with the development of paired-end (PE) sequencing. • PE sequencing involves sequencing both ends of the DNA fragments in a sequencing library and aligning the forward and reverse reads as read pairs. • In addition to producing twice the number of reads for the same time and effort in library preparation, sequences aligned as read pairs enable more accurate read alignment and the ability to detect indels, which is simply not possible with single-read data.
• Analysis of differential read-pair spacing also allows removal of PCR duplicates, a common artifact resulting from PCR amplification during library preparation. • Furthermore, paired-end sequencing produces a higher number of SNV calls following read-pair alignment. • While some methods are best served by single-read sequencing, such as small RNA sequencing, most researchers currently use the paired-end approach.
Paired-End Sequencing and Alignment—Paired-end sequencing enables both ends of the DNA fragment to be sequenced. • Because the distance between each paired read is known, alignment algorithms can use this information to map the reads over repetitive regions more precisely. This results in much better alignment of the reads, especially across difficult-to-sequence, repetitive regions of the genome.
What is the Illumina method of DNA sequencing? Illumina sequencing has been used to sequence many genomes and has enabled the comparison of DNA sequences to improve understanding of health and disease. Illumina sequencing generates many millions of highly accurate reads making it much faster and cheaper than other available
How does Illumina DNA sequencing work? 1. The first step in this sequencing technique is to break up the DNA into more manageable fragments of around 200 to 600. 2. Short sequences of DNA called adaptors, are attached to the DNA fragments. 3. The DNA fragments attached to adaptors are then made single stranded. This is done by incubating the fragments with sodium hydroxide. 4. Once prepared, the DNA fragments are washed across the Flow cell. The complementary DNA binds to primers on the surface of the flow cell and DNA that doesn’t attach is washed away.
5. The DNA attached to the flow cell is then replicated to form small clusters of DNA with the same sequence. • When sequenced, each cluster of DNA molecules will emit a signal that is strong enough to be detected by a camera. 6. Unlabelled nucleotides and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flow cell. • This creates ‘bridges’ of double-stranded DNA between the primers on the flow cell surface. 7. The double-stranded DNA is then broken down into single stranded DNA using heat, leaving several million dense clusters of identical DNA sequences.
8. Primers and fluorescently labelled terminators (terminators are a version of nucleotide base – A, C, G or T - that stop DNA synthesis) are added to the flowcell. 9. The primer attaches to the DNA being sequenced. 10. The DNA polymerase then binds to the primer and adds the first fluorescently-labelled terminator to the new DNA strand. • Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA.
11. Lasers are passed over the flowcell to activate the fluorescent label on the nucleotide base. • This fluorescence is detected by a camera and recorded on a computer. Each of the terminator bases (A, C, G and T) give off a different colour. 12. The fluorescently-labelled terminator group is then removed from the first base and the next fluorescently- labelled terminator base can be added alongside. • And so the process continues until millions of clusters have been sequenced.
13. The DNA sequence is analysed base-by-base during Illumina sequencing, making it a highly accurate method. • The sequence generated can then be aligned to a reference sequence, this looks for matches or changes in the sequenced DNA.
➢ In Illumina sequencing, 100150bp reads are used. ➢ Somewhat longer fragments are ligated to generic adaptors and annealed to a slide using the adaptors. ➢ PCR is carried out to amplify each read, creating a spot with many copies of the same read. ➢ They are then separated into single strands to be sequenced. ➢ The slide is flooded with nucleotides and DNA polymerase. ➢ These nucleotides are fluorescently labelled, with the colour corresponding to the base. ➢ They also have a terminator, so that only one base is added at a time.
➢ An image is taken of the slide. In each read location, there will be a fluorescent signal indicating the base that has been added.
➢ The slide is then prepared for the next cycle. The terminators are removed, allowing the next base to be added, and the fluorescent signal is removed, preventing the signal from contaminating the next image. ➢ The process is repeated, adding one nucleotide at a time and imaging in between. ➢ Computers are then used to detect the base at each site in each image and these are used to construct a sequence.
➢ All of the sequence reads will be the same length, as the read length depends on the number of cycles carried out.
Ion Torrent • The sequencing chemistry itself is remarkably simple. Naturally, a proton is released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH. • Each micro-well of the Ion Torrent semiconductor sequencing chip contains approximately one million copies of a DNA molecule. • The Ion Personal Genome Machine (PGM™) sequencer sequentially floods the chip with one nucleotide after another.
• • If a nucleotide complements the sequence of the DNA molecule in a particular micro-well, it will be incorporated and hydrogen ions are released. • The pH of the solution changes in that well and is detected by the ion sensor, essentially going directly from chemical information to digital information. • If there are two identical bases on the DNA strand, the voltage is double, and the chip records two identical bases. • If the next nucleotide that floods the chip is not a match, no voltage change is recorded and no base is called.
• Because this is direct detection—no scanning, no cameras, no light—each nucleotide incorporation is measured in seconds enabling very short run times. • Naturally, a proton is released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH.
Ion Torrent: Proton / PGM sequencing • Unlike Illumina and 454, Ion torrent and Ion proton sequencing do not make use of optical signals. • Instead, they exploit the fact that addition of a dNTP to a DNA polymer releases an H+ ion. • As in other kinds of NGS, the input DNA or RNA is fragmented, this time ~200bp. Adaptors are added and one molecule is placed onto a bead. • The molecules are amplified on the bead by emulsion PCR. • Each bead is placed into a single well of a slide. Like 454, the slide is flooded with a single species of dNTP, along with buffers and polymerase, one NTP at a time.
• The pH is detected is each of the wells, as each H+ ion released will decrease the pH. • The changes in pH allow us to determine if that base, and how many thereof, was added to the sequence read. • The dNTPs are washed away, and the process is repeated cycling through the different dNTP species.
The pH change, if any, is used to determine how many bases (if any) were added with each cycle.
Pyrosequencing • Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis“ principle. • It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides.
• The desired DNA sequence is able to be determined by light emitted upon incorporation of the next complementary nucleotide • Only one out of four of the possible A/T/C/G nucleotides are added and available at a time • So that only one letter can be incorporated on the single stranded template (which is the sequence to be determined).
• The intensity of the light determines if there are more than one of these "letters" in a row. • The previous nucleotide letter (one out of four possible dNTP) is degraded before the next nucleotide letter is added for synthesis: allowing for the possible revealing of the next nucleotide(s) via the resulting intensity of light (if the nucleotide added was the next complementary letter in the sequence). This process is repeated with each of the four letters until the DNA sequence of the single stranded template is determined.
Procedure
• "Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. • The pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. • Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.
• The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. • The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.
• The single strand DNA (ssDNA) template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin. 1. The addition of one of the four deoxynucleoside triphosphates (dNTPs) (dATPαS, which is not a substrate for a luciferase, is added instead of dATP to avoid noise) initiates the second step. • DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi).
2. ATP sulfurylase converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. • This ATP acts as a substrate for the luciferase mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. • The light produced in the luciferasecatalyzed reaction is detected by a camera and analyzed in a pyrogram. 3. Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another Nucleotide.
Limitation • Currently, a limitation of the method is that the lengths of individual reads of DNA sequence of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain termination methods (e.g. Sanger sequencing).
Pyrosequencing cycle • Add dATP. If light is emitted, your sequence starts with A. If not, the dATP is degraded (or elutes past immobilized primer). • Add dGTP. If light is emitted, the next base must be a G. • Then add T, then C. You now know at least one (maybe more) base of the sequence. • Repeat!
Pyrosequencing output Runs of bases produce higher peaks – for instance, the sequence for (a) is GGCCCTTG. Sample (c) comes from a heterozygous individual (hence the heights in multiples of ½)
Roche 454 Roche 454 sequencing system is the first commercial platforms for the next generation sequencing technology. Its main principle of sequencing is illustrated as follows.
a. Preparation of DNA Library • DNA Library construction in 454 sequencing system is different from that of Illumina. • It uses spray method to break DNA samples into small fragments of 300-800bp, and adds different adapters at both ends. • Otherwise, use primers for amplification after DNA denaturation, clone into specific vectors, and finally constructing single stranded DNA library
b. Emulsion PCR • These single stranded DNAs would be fixed by 28um beads which are buried in emulsion. • The biggest feature of emulsion PCR is the formation of a large number of independent reaction space for DNA amplification. • The key technology is to separate different beats using the characteristics of emulsion.
• The basic process is as follows. • Before sample DNA amplification, aqueous solution with all components of PCR reaction will be infused into the surface of mineral oil with high-speed rotation, and it forms numerous small water droplets wrapped by mineral oil. • One small droplet forms an independent PCR reaction space. Ideally, each small drop of water contains only one DNA template and one bead.
• On the surface of beads, which are wrapped by small water droplets, there are complementary oligos to match those adapters, so the single stranded DNA can specifically bind to the beads. • At the same time, incubation system contains PCR reagents to ensure that each small DNA fragment fixed on the bead can be the unique template for amplification. • Moreover, PCR products can be also combined with magnetic beads. • After the reaction accomplishment, emulsion system can be destroyed and target DNAs would be accumulated.
• Finally, each small fragment will be amplified about 1 million times, so as to achieve the amount level required by the sequencing process.
c. Pyrosequencing • A polymerase and single strand DNA binding protein are needed to process beads with DNAs before sequencing. • Then these beads are put on PTP plate. • This plate has many special nanopores of 44um diameters. • Each nanopore can only accommodate one bead, which can fix the position of each bead through this method, in order to be convenient for sequencing. • The method used in this sequencing process is pyrosequencing.
• Put a smaller bead into the nanopore, and start the sequencing reaction. • DNA sequencing reaction is based on the single stranded DNAs which have been amplified and fixed. • If one dNTP can pair with the template DNA, the pyrophosphate group will be released after synthesis. • The released pyrophosphate group reacts with ATP sulfuric acid chemical enzymes to produce ATP. • CO-oxidation of ATP and luciferase makes the fluorescein molecule triggered and fluoresce, and the CCD camera on the other side of the PTP board records the signal of fluorescent.
• Finally the results are processed by computer software. • Because each kind of dNTP produces unique fluorescence color in the reaction, DNA sequence can be measured according to the fluorescence colors. • After the reaction, ATP are degraded by diphosphatase, leading to fluorescence quenching, so that sequencing reaction goes into the next cycle.
454 sequencing • Roche 454 sequencing can sequence much longer reads than Illumina. • Like Illumina, it does this by sequencing multiple reads at once by reading optical signals as bases are added. • As in Illumina, the DNA or RNA is fragmented into shorter reads, in this case up to 1kb. • Generic adaptors are added to the ends and these are annealed to beads, one DNA fragment per bead. • The fragments are then amplified by PCR using adaptor specifc primers. • Each bead is then placed in a single well of a slide. So each well will contain a single bead, covered in many PCR copies of a single sequence.
• The wells also contain DNA polymerase and sequencing buffers. • The slide is flooded with one of the four NTP species. Where this nucleotide is next in the sequence, it is added to the sequence read. • If that single base repeats, then more will be added. • So if we flood with Guanine bases, and the next in a sequence is G, one G will be added, however if the next part of the sequence is GGGG, then four Gs will be added.
• The addition of each nucleotide releases a light signal. • These locations of signals are detected and used to determine which beads the nucleotides are added to.
• This NTP mix is washed away. The next NTP mix is now added and the process repeated, cycling through the four NTPs.
• This kind of sequencing generates graphs for each sequence read, showing the signal density for each nucleotide wash. • The sequence can then be determined computationally from the signal density in each wash.
• All of the sequence reads we get from 454 will be different lengths, because different numbers of bases will be added with each cycle.
SOLiD • An open source sequencer that utilizes emulsion PCR to immobilize the DNA library onto a solid support and cyclic sequencing-by-ligation chemistry. Sequencing Library Preparation and Immobilization • The in vitro sequencing library preparation for SOLiD involves fragmentation of the DNA sample to an appropriate size range (400–850 bp), end repair and ligation of “P1” and “P2” DNA adapters to the ends of the library fragments
Used in
• Emulsion PCR is applied to immobilize the sequencing library DNA onto “P1” coated paramagnetic beads. • High-density, semi-ordered polony arrays are generated by functionalizing the 3 ¢ ends of the templates and immobilizing the modified beads to a glass slide. • The glass slides can be segmented up to eight chambers to facilitate up scaling of the number of analyzed samples.
• Sequencing by Ligation The SOLiD sequencing chemistry is based on ligation (Fig) • A sequencing primer is hybridized to the “P1” adapter in the immobilized beads. • A pool of uniquely labeled oligonucleotides contains all possible variations of the complementary bases for the template sequence
• SOLiD technology applies partially degenerate, fluorescently labeled, DNA octamers with dinucleotide complement sequence recognition core. • These detection oligonucleotides are hybridized to the template and perfectly annealing sequences are ligated to the primer. • After imaging, unextended strands are capped and fluorophores are cleaved. • A new cycle begins 5 bases upstream from the priming site. • After the seven sequencing cycles first sequencing primer is peeled off and second primer, starting at n-1 site, is hybridized to the template.
• In all, 5 sequencing primers (n, n-1, n-2, n-3, and n-4) are utilized for the sequencing. • As a result, the 35-base insert is sequenced twice to improve the sequencing accuracy.

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Next Generation Sequencing

  • 1.
    What is NextGeneration DNA Sequencing Next generation sequencing (NGS), also known as highthroughput sequencing, is the catchall term used to describe a number of different modern sequencing technologies including: • Illumina (Solexa) sequencing • Roche 454 sequencing • Ion torrent: Proton / PGM sequencing • SOLiD sequencing These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing Dr. Shiny C Thomas, Department of Biosciences, ADBU
  • 2.
    The four mainadvantages of NGS over classical Sanger sequencing are: • speed • cost • sample size • accuracy • NGS is significantly cheaper, quicker, needs significantly less DNA and is more accurate and reliable than Sanger sequencing. Let us look at this more closely. • For Sanger sequencing, a large amount of template DNA is needed for each read. • In NGS, a sequence can be obtained from a single strand
  • 3.
    • NGS isquicker than Sanger sequencing in two ways. Firstly, the chemical reaction may be combined with the signal detection in some versions of NGS, whereas in Sanger sequencing these are two separate processes. • Secondly and more significantly, only one read (maximum ~1kb) can be taken at a time in Sanger sequencing, whereas NGS is massively parallel, allowing 300Gb of DNA to be read on a single run on a single chip.
  • 4.
    • The reducedtime, manpower and reagents in NGS mean that the costs are much lower. The first human genome sequence cost in the region of £300M. Using modern Sanger sequencing methods, aided by data from the known sequence, a full human genome would still cost £6M. Sequencing a human genome with Illumina today would cost only £6,000.
  • 5.
  • 6.
    Sequencing for Transcriptomics? •Sample cells • Purify mRNA* • mRNA ⇒ cDNA • Cleaving into 35-400nt fragments • Sequence fragments in parallel • PCR amplification * or DNA for genomic sequencing
  • 7.
    Next Generation Sequencing TechnologyOverview 1. (c)DNA is fragmented 2. Adaptors ligated to fragments 3. Several possible protocols yield array of PCR colonies. 4. Enzymatic extension with fluorescently tagged nucleotides. 5. Cyclic readout by imaging the array.
  • 8.
    • Fragmentation ofDNA • Primers are attached to the surface of a bead Technology 1. (emulsion PCR)
  • 9.
    2. Bead preparation •Fragments, with adaptors, are PCR amplified within a water drop in oil. • One primer is attached to the surface of a bead • 3’ modification of fragments, to covalently bind bead to chip surface.
  • 10.
    3. Cycles ofelongation
  • 11.
    4. Cycles ofimaging
  • 12.
    Sequence of images Sequenceread over multiple cycles Repeated cycles of sequencing to determine the sequence of bases in a given fragment, a single base at a time.
  • 13.
  • 14.
    • While these“first-generation” instruments were considered high throughput for their time, the Genome Analyzer emerged in 2005 and took sequencing runs from 84 kilobase (kb) per run to 1 gigabase (Gb) per run. • The short read, massively parallel sequencing technique was a fundamentally different approach that revolutionized sequencing capabilities and launched the “next-generation” in genomic science. • From that point forward, the data output of next- generation sequencing (NGS) has—more than doubling each year
  • 15.
    Figure 1: SequencingCost and Data Output Since 2000—The dramatic rise of data output and concurrent falling cost of sequencing since 2000. The Y-axes on both sides of the graph are logarithmic.
  • 16.
    • In 2005,with the Genome Analyzer, a single sequencing run could produce roughly one gigabase of data. • By 2014, the rate climbed to a 1.8 terabases of data in a single sequencing run—an astounding 1000× increase. • It is remarkable to reflect on the fact that the first human genome, famously co published in Science and Nature in 2001, required 15 years to sequence and cost nearly 3 billion dollars. • In contrast, the HiSeq X™ Ten, released in 2014, can sequence over 45 human genomes in a single day for approximately $1000 each.
  • 17.
    Human Genome SequencingOver the Decades—The capacity to sequence all 3.2 billion bases of the human genome (at 30× coverage) has increased exponentially since the 1990s. In 2005, with the introduction of the Illumina Genome Analyzer System, 1.3 human genomes could be sequenced annually. Nearly 10 years later, with the Illumina HiSeq X Ten fleet of sequencing systems, the number has climbed to 18,000 human genomes a year.
  • 18.
    • Beyond themassive increase in data output, the introduction of NGS technology has transformed the way scientists think about genetic information. • The $1000 dollar genome enables population-scale sequencing and establishes the foundation for personalized genomic medicine as part of standard medical care. • Researchers can now analyze thousands to tens of thousands of samples in a single year. • The rate of progress is stunning.
  • 19.
    • As costscontinue to come down, we are entering a period where we are going to be able to get the complete catalog of disease genes. • This will allow us to look at thousands of people and see the differences among them, to discover critical genes that cause cancer, autism, heart disease, or schizophrenia.
  • 20.
    The Basics ofNGS Chemistry • In principle, the concept behind NGS technology is similar to CE (Capillary Electrophoresis) sequencing— DNA polymerase catalyzes the incorporation of fluorescently labeled deoxyribonucleotide triphosphates (dNTPs) into a DNA template strand during sequential cycles of DNA synthesis. • During each cycle, at the point of incorporation, the nucleotides are identified by fluorophore excitation. • The critical difference is that, instead of sequencing a single DNA fragment, NGS extends this process across millions of fragments in a massively parallel fashion.
  • 21.
    • Illumina sequencingby synthesis (SBS) chemistry is the most widely adopted chemistry in the industry and delivers the highest accuracy, the highest yield of error- free reads, and the highest percentage of base calls above Q30. • The Illumina NGS workflows include 4 basic steps
  • 22.
    1. Library Preparation— •The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5’ and 3’ adapter ligation. • Alternatively, “tagmentation” combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. • Adapter-ligated fragments are then PCR amplified and gel purified.
  • 23.
    2. Cluster Generation— •For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. • Each fragment is then amplified into distinct, clonal clusters through bridge amplification. • When cluster generation is complete, the templates are ready for sequencing.
  • 24.
    3. Sequencing— • IlluminaSBS technology utilizes a proprietary reversible terminator–based method that detects single bases as they are incorporated into DNA template strands. • As all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies. • The result is highly accurate base-by-base sequencing that virtually eliminates sequence- context-specific errors, even within repetitive sequence regions and homopolymers.
  • 25.
    4. Data Analysis— •During data analysis and alignment, the newly identified sequence reads are then aligned to a reference genome. • Following alignment, many variations of analysis are possible such as single nucleotide polymorphism (SNP) or insertion-deletion (indel) identification, read counting for RNA methods, phylogenetic or metagenomic analysis, and more.
  • 26.
  • 30.
    Advances in SequencingTechnology Paired-End Sequencing • A major advance in NGS technology occurred with the development of paired-end (PE) sequencing. • PE sequencing involves sequencing both ends of the DNA fragments in a sequencing library and aligning the forward and reverse reads as read pairs. • In addition to producing twice the number of reads for the same time and effort in library preparation, sequences aligned as read pairs enable more accurate read alignment and the ability to detect indels, which is simply not possible with single-read data.
  • 31.
    • Analysis ofdifferential read-pair spacing also allows removal of PCR duplicates, a common artifact resulting from PCR amplification during library preparation. • Furthermore, paired-end sequencing produces a higher number of SNV calls following read-pair alignment. • While some methods are best served by single-read sequencing, such as small RNA sequencing, most researchers currently use the paired-end approach.
  • 32.
    Paired-End Sequencing andAlignment—Paired-end sequencing enables both ends of the DNA fragment to be sequenced. • Because the distance between each paired read is known, alignment algorithms can use this information to map the reads over repetitive regions more precisely. This results in much better alignment of the reads, especially across difficult-to-sequence, repetitive regions of the genome.
  • 33.
    What is theIllumina method of DNA sequencing? Illumina sequencing has been used to sequence many genomes and has enabled the comparison of DNA sequences to improve understanding of health and disease. Illumina sequencing generates many millions of highly accurate reads making it much faster and cheaper than other available
  • 34.
    How does IlluminaDNA sequencing work? 1. The first step in this sequencing technique is to break up the DNA into more manageable fragments of around 200 to 600. 2. Short sequences of DNA called adaptors, are attached to the DNA fragments. 3. The DNA fragments attached to adaptors are then made single stranded. This is done by incubating the fragments with sodium hydroxide. 4. Once prepared, the DNA fragments are washed across the Flow cell. The complementary DNA binds to primers on the surface of the flow cell and DNA that doesn’t attach is washed away.
  • 35.
    5. The DNAattached to the flow cell is then replicated to form small clusters of DNA with the same sequence. • When sequenced, each cluster of DNA molecules will emit a signal that is strong enough to be detected by a camera. 6. Unlabelled nucleotides and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flow cell. • This creates ‘bridges’ of double-stranded DNA between the primers on the flow cell surface. 7. The double-stranded DNA is then broken down into single stranded DNA using heat, leaving several million dense clusters of identical DNA sequences.
  • 36.
    8. Primers andfluorescently labelled terminators (terminators are a version of nucleotide base – A, C, G or T - that stop DNA synthesis) are added to the flowcell. 9. The primer attaches to the DNA being sequenced. 10. The DNA polymerase then binds to the primer and adds the first fluorescently-labelled terminator to the new DNA strand. • Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA.
  • 37.
    11. Lasers arepassed over the flowcell to activate the fluorescent label on the nucleotide base. • This fluorescence is detected by a camera and recorded on a computer. Each of the terminator bases (A, C, G and T) give off a different colour. 12. The fluorescently-labelled terminator group is then removed from the first base and the next fluorescently- labelled terminator base can be added alongside. • And so the process continues until millions of clusters have been sequenced.
  • 38.
    13. The DNAsequence is analysed base-by-base during Illumina sequencing, making it a highly accurate method. • The sequence generated can then be aligned to a reference sequence, this looks for matches or changes in the sequenced DNA.
  • 39.
    ➢ In Illuminasequencing, 100150bp reads are used. ➢ Somewhat longer fragments are ligated to generic adaptors and annealed to a slide using the adaptors. ➢ PCR is carried out to amplify each read, creating a spot with many copies of the same read. ➢ They are then separated into single strands to be sequenced. ➢ The slide is flooded with nucleotides and DNA polymerase. ➢ These nucleotides are fluorescently labelled, with the colour corresponding to the base. ➢ They also have a terminator, so that only one base is added at a time.
  • 40.
    ➢ An imageis taken of the slide. In each read location, there will be a fluorescent signal indicating the base that has been added.
  • 41.
    ➢ The slideis then prepared for the next cycle. The terminators are removed, allowing the next base to be added, and the fluorescent signal is removed, preventing the signal from contaminating the next image. ➢ The process is repeated, adding one nucleotide at a time and imaging in between. ➢ Computers are then used to detect the base at each site in each image and these are used to construct a sequence.
  • 43.
    ➢ All ofthe sequence reads will be the same length, as the read length depends on the number of cycles carried out.
  • 44.
    Ion Torrent • Thesequencing chemistry itself is remarkably simple. Naturally, a proton is released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH. • Each micro-well of the Ion Torrent semiconductor sequencing chip contains approximately one million copies of a DNA molecule. • The Ion Personal Genome Machine (PGM™) sequencer sequentially floods the chip with one nucleotide after another.
  • 45.
    • • If anucleotide complements the sequence of the DNA molecule in a particular micro-well, it will be incorporated and hydrogen ions are released. • The pH of the solution changes in that well and is detected by the ion sensor, essentially going directly from chemical information to digital information. • If there are two identical bases on the DNA strand, the voltage is double, and the chip records two identical bases. • If the next nucleotide that floods the chip is not a match, no voltage change is recorded and no base is called.
  • 46.
    • Because thisis direct detection—no scanning, no cameras, no light—each nucleotide incorporation is measured in seconds enabling very short run times. • Naturally, a proton is released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH.
  • 52.
    Ion Torrent: Proton/ PGM sequencing • Unlike Illumina and 454, Ion torrent and Ion proton sequencing do not make use of optical signals. • Instead, they exploit the fact that addition of a dNTP to a DNA polymer releases an H+ ion. • As in other kinds of NGS, the input DNA or RNA is fragmented, this time ~200bp. Adaptors are added and one molecule is placed onto a bead. • The molecules are amplified on the bead by emulsion PCR. • Each bead is placed into a single well of a slide. Like 454, the slide is flooded with a single species of dNTP, along with buffers and polymerase, one NTP at a time.
  • 53.
    • The pHis detected is each of the wells, as each H+ ion released will decrease the pH. • The changes in pH allow us to determine if that base, and how many thereof, was added to the sequence read. • The dNTPs are washed away, and the process is repeated cycling through the different dNTP species.
  • 55.
    The pH change,if any, is used to determine how many bases (if any) were added with each cycle.
  • 57.
    Pyrosequencing • Pyrosequencing isa method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis“ principle. • It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides.
  • 58.
    • The desiredDNA sequence is able to be determined by light emitted upon incorporation of the next complementary nucleotide • Only one out of four of the possible A/T/C/G nucleotides are added and available at a time • So that only one letter can be incorporated on the single stranded template (which is the sequence to be determined).
  • 59.
    • The intensityof the light determines if there are more than one of these "letters" in a row. • The previous nucleotide letter (one out of four possible dNTP) is degraded before the next nucleotide letter is added for synthesis: allowing for the possible revealing of the next nucleotide(s) via the resulting intensity of light (if the nucleotide added was the next complementary letter in the sequence). This process is repeated with each of the four letters until the DNA sequence of the single stranded template is determined.
  • 60.
  • 61.
    • "Sequencing bysynthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. • The pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. • Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.
  • 62.
    • The templateDNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. • The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.
  • 63.
    • The singlestrand DNA (ssDNA) template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin. 1. The addition of one of the four deoxynucleoside triphosphates (dNTPs) (dATPαS, which is not a substrate for a luciferase, is added instead of dATP to avoid noise) initiates the second step. • DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi).
  • 65.
    2. ATP sulfurylaseconverts PPi to ATP in the presence of adenosine 5´ phosphosulfate. • This ATP acts as a substrate for the luciferase mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. • The light produced in the luciferasecatalyzed reaction is detected by a camera and analyzed in a pyrogram. 3. Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another Nucleotide.
  • 66.
    Limitation • Currently, alimitation of the method is that the lengths of individual reads of DNA sequence of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain termination methods (e.g. Sanger sequencing).
  • 67.
    Pyrosequencing cycle • AdddATP. If light is emitted, your sequence starts with A. If not, the dATP is degraded (or elutes past immobilized primer). • Add dGTP. If light is emitted, the next base must be a G. • Then add T, then C. You now know at least one (maybe more) base of the sequence. • Repeat!
  • 68.
    Pyrosequencing output Runs ofbases produce higher peaks – for instance, the sequence for (a) is GGCCCTTG. Sample (c) comes from a heterozygous individual (hence the heights in multiples of ½)
  • 69.
    Roche 454 Roche 454sequencing system is the first commercial platforms for the next generation sequencing technology. Its main principle of sequencing is illustrated as follows.
  • 70.
    a. Preparation ofDNA Library • DNA Library construction in 454 sequencing system is different from that of Illumina. • It uses spray method to break DNA samples into small fragments of 300-800bp, and adds different adapters at both ends. • Otherwise, use primers for amplification after DNA denaturation, clone into specific vectors, and finally constructing single stranded DNA library
  • 72.
    b. Emulsion PCR •These single stranded DNAs would be fixed by 28um beads which are buried in emulsion. • The biggest feature of emulsion PCR is the formation of a large number of independent reaction space for DNA amplification. • The key technology is to separate different beats using the characteristics of emulsion.
  • 73.
    • The basicprocess is as follows. • Before sample DNA amplification, aqueous solution with all components of PCR reaction will be infused into the surface of mineral oil with high-speed rotation, and it forms numerous small water droplets wrapped by mineral oil. • One small droplet forms an independent PCR reaction space. Ideally, each small drop of water contains only one DNA template and one bead.
  • 74.
    • On thesurface of beads, which are wrapped by small water droplets, there are complementary oligos to match those adapters, so the single stranded DNA can specifically bind to the beads. • At the same time, incubation system contains PCR reagents to ensure that each small DNA fragment fixed on the bead can be the unique template for amplification. • Moreover, PCR products can be also combined with magnetic beads. • After the reaction accomplishment, emulsion system can be destroyed and target DNAs would be accumulated.
  • 75.
    • Finally, eachsmall fragment will be amplified about 1 million times, so as to achieve the amount level required by the sequencing process.
  • 76.
    c. Pyrosequencing • Apolymerase and single strand DNA binding protein are needed to process beads with DNAs before sequencing. • Then these beads are put on PTP plate. • This plate has many special nanopores of 44um diameters. • Each nanopore can only accommodate one bead, which can fix the position of each bead through this method, in order to be convenient for sequencing. • The method used in this sequencing process is pyrosequencing.
  • 77.
    • Put asmaller bead into the nanopore, and start the sequencing reaction. • DNA sequencing reaction is based on the single stranded DNAs which have been amplified and fixed. • If one dNTP can pair with the template DNA, the pyrophosphate group will be released after synthesis. • The released pyrophosphate group reacts with ATP sulfuric acid chemical enzymes to produce ATP. • CO-oxidation of ATP and luciferase makes the fluorescein molecule triggered and fluoresce, and the CCD camera on the other side of the PTP board records the signal of fluorescent.
  • 78.
    • Finally theresults are processed by computer software. • Because each kind of dNTP produces unique fluorescence color in the reaction, DNA sequence can be measured according to the fluorescence colors. • After the reaction, ATP are degraded by diphosphatase, leading to fluorescence quenching, so that sequencing reaction goes into the next cycle.
  • 79.
    454 sequencing • Roche454 sequencing can sequence much longer reads than Illumina. • Like Illumina, it does this by sequencing multiple reads at once by reading optical signals as bases are added. • As in Illumina, the DNA or RNA is fragmented into shorter reads, in this case up to 1kb. • Generic adaptors are added to the ends and these are annealed to beads, one DNA fragment per bead. • The fragments are then amplified by PCR using adaptor specifc primers. • Each bead is then placed in a single well of a slide. So each well will contain a single bead, covered in many PCR copies of a single sequence.
  • 80.
    • The wellsalso contain DNA polymerase and sequencing buffers. • The slide is flooded with one of the four NTP species. Where this nucleotide is next in the sequence, it is added to the sequence read. • If that single base repeats, then more will be added. • So if we flood with Guanine bases, and the next in a sequence is G, one G will be added, however if the next part of the sequence is GGGG, then four Gs will be added.
  • 81.
    • The additionof each nucleotide releases a light signal. • These locations of signals are detected and used to determine which beads the nucleotides are added to.
  • 82.
    • This NTPmix is washed away. The next NTP mix is now added and the process repeated, cycling through the four NTPs.
  • 83.
    • This kindof sequencing generates graphs for each sequence read, showing the signal density for each nucleotide wash. • The sequence can then be determined computationally from the signal density in each wash.
  • 84.
    • All ofthe sequence reads we get from 454 will be different lengths, because different numbers of bases will be added with each cycle.
  • 85.
    SOLiD • An opensource sequencer that utilizes emulsion PCR to immobilize the DNA library onto a solid support and cyclic sequencing-by-ligation chemistry. Sequencing Library Preparation and Immobilization • The in vitro sequencing library preparation for SOLiD involves fragmentation of the DNA sample to an appropriate size range (400–850 bp), end repair and ligation of “P1” and “P2” DNA adapters to the ends of the library fragments
  • 87.
  • 91.
    • Emulsion PCRis applied to immobilize the sequencing library DNA onto “P1” coated paramagnetic beads. • High-density, semi-ordered polony arrays are generated by functionalizing the 3 ¢ ends of the templates and immobilizing the modified beads to a glass slide. • The glass slides can be segmented up to eight chambers to facilitate up scaling of the number of analyzed samples.
  • 92.
    • Sequencing byLigation The SOLiD sequencing chemistry is based on ligation (Fig) • A sequencing primer is hybridized to the “P1” adapter in the immobilized beads. • A pool of uniquely labeled oligonucleotides contains all possible variations of the complementary bases for the template sequence
  • 93.
    • SOLiD technologyapplies partially degenerate, fluorescently labeled, DNA octamers with dinucleotide complement sequence recognition core. • These detection oligonucleotides are hybridized to the template and perfectly annealing sequences are ligated to the primer. • After imaging, unextended strands are capped and fluorophores are cleaved. • A new cycle begins 5 bases upstream from the priming site. • After the seven sequencing cycles first sequencing primer is peeled off and second primer, starting at n-1 site, is hybridized to the template.
  • 94.
    • In all,5 sequencing primers (n, n-1, n-2, n-3, and n-4) are utilized for the sequencing. • As a result, the 35-base insert is sequenced twice to improve the sequencing accuracy.