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group_3_presentation_1_-_next_generation_sequencing [2016/01/29 20:21] domazee |
group_3_presentation_1_-_next_generation_sequencing [2018/01/25 15:18] (current) |
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| ===== What is NGS? ===== | ===== What is NGS? ===== | ||
| - | Technological advances in molecular genetics at the end of the twentieth century have established a strong foundation for genetic analysis. Shotgun sequencing has allowed for the sequencing of longer DNA sections, which has played a significant role in the Human Genome Project (cite). In this method, DNA is enzymatically broken into smaller fragments and cloned for individual sequencing prior to realignment (cite). Nearly a decade later, subsequent advances in next generation sequencing (NGS) technologies have made genomic analysis much more economically feasible and have thus enabled applications of genomics across clinical and research settings (cite). | + | Technological advances in molecular genetics at the end of the twentieth century have established a strong foundation for genetic analysis. Shotgun sequencing has allowed for the sequencing of longer DNA sections, which has played a significant role in the Human Genome Project.<sup>1</sup> In this method, DNA is enzymatically broken into smaller fragments and cloned for individual sequencing prior to realignment.<sup>1</sup> Nearly a decade later, subsequent advances in next generation sequencing (NGS) technologies have made genomic analysis much more economically feasible and have thus enabled applications of genomics across clinical and research settings.<sup>2</sup> NGS presents a platform for high-throughput sequencing of DNA.<sup>2</sup> There are various NGS technologies, namely 454 Life Sciences, Illumina, Biosystems/SOLiD and Ion Torrent which have allowed for the sequencing of whole genomes in a cost and time efficient manner.<sup>2</sup> |
| ===== Different Types of NGS Technology ===== | ===== Different Types of NGS Technology ===== | ||
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| {{ youtube>large:nlvyF8bFDwM }} | {{ youtube>large:nlvyF8bFDwM }} | ||
| - | Figure 4 - A summary of SOLiD sequencing steps are illustrated in this video. | + | <style center>Figure 4 - A summary of SOLiD sequencing steps are illustrated in this video. </style> |
| ==== Illumina Sequencing Technology ==== | ==== Illumina Sequencing Technology ==== | ||
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| However, there can be error that results from Ion Torrent sequencing. When trying to distinguish between 7-8 nucleotides, it becomes a bit more challenging which is why error rates can go as high as 1.7%.<sup>13</sup> The reason for this is because of the homopolymer sequences (a sequence of identical bases) being used. As the homopolymer length increases, the deletion error rate increases and the insertion error rate stays relatively constant. This causes in a large release in protons, and when a certain concentration is exceeded the pH readings start to become ambiguous which causes insertion or deletion. These errors could be much larger if not for the Ion Torrent’s short read lengths which mitigates the number of successive nucleotides being read by these errors.<sup>15</sup> | However, there can be error that results from Ion Torrent sequencing. When trying to distinguish between 7-8 nucleotides, it becomes a bit more challenging which is why error rates can go as high as 1.7%.<sup>13</sup> The reason for this is because of the homopolymer sequences (a sequence of identical bases) being used. As the homopolymer length increases, the deletion error rate increases and the insertion error rate stays relatively constant. This causes in a large release in protons, and when a certain concentration is exceeded the pH readings start to become ambiguous which causes insertion or deletion. These errors could be much larger if not for the Ion Torrent’s short read lengths which mitigates the number of successive nucleotides being read by these errors.<sup>15</sup> | ||
| - | ===== Advantages and Disadvantages of The 4 Types of NGS ==== | + | ===== Advantages and Disadvantages of The 4 Types of NGS ===== |
| {{ :screen_shot_2016-01-28_at_2.30.21_pm.png?400 }} | {{ :screen_shot_2016-01-28_at_2.30.21_pm.png?400 }} | ||
| - | Figure 9 - A table comparing the next generation sequencing techniques (Liu, 2012). | + | <style center> Figure 9 - A table comparing the next generation sequencing techniques (Liu, 2012). </style> |
| + | ---- | ||
| + | |||
| + | **454 Life Sciences**<sup>4</sup> | ||
| + | |||
| + | * Advantages | ||
| + | * Long read length | ||
| + | * Fast relative to other NGS technologies | ||
| + | * Low capital cost | ||
| + | * Low cost per experiment | ||
| + | * Disadvantages | ||
| + | * Error rate with polybase = more than 6 | ||
| + | * High cost per mb | ||
| + | * Low throughput | ||
| + | |||
| + | ---- | ||
| + | |||
| + | **Biosystems/SOLiD**<sup>4</sup> | ||
| + | |||
| + | * Advantages | ||
| + | * High accuracy | ||
| + | * Each lane of Flow-Chip can run independently | ||
| + | * Disadvantages | ||
| + | * Short read assembly | ||
| + | * More gaps in assemblies than Illumina data | ||
| + | * Less even data distribution than Illumina | ||
| + | * High capital cost | ||
| + | |||
| + | ---- | ||
| + | |||
| + | **Illumina Sequencing**<sup>4</sup> | ||
| + | |||
| + | * Advantages | ||
| + | * High throughput | ||
| + | * Low cost instrument and runs | ||
| + | * Low cost/Mb for a small platform | ||
| + | * Long run times | ||
| + | * Disadvantages | ||
| + | * Relatively few reads | ||
| + | * Higher cost/Mb compared to other Illumina platforms | ||
| + | |||
| + | ---- | ||
| + | |||
| + | **Ion Torrent**<sup>4</sup> | ||
| + | |||
| + | * Advantages | ||
| + | * Low-cost | ||
| + | * Instrument upgraded through disposable chips | ||
| + | * Very simple machine with few moving parts | ||
| + | * Clear trajectory to improved performance | ||
| + | * Disadvantages | ||
| + | * Higher error rate than Illumina | ||
| ===== Applications of NGS ===== | ===== Applications of NGS ===== | ||
| Line 105: | Line 156: | ||
| <box width classes round blue centre| **DID YOU KNOW? **> __**Applications **__: | <box width classes round blue centre| **DID YOU KNOW? **> __**Applications **__: | ||
| - | The first sequenced genome cost approximately $3 billion dollars, however, sequencing costs have decreased significantly. Lower costs for genome sequencing are essential in improving patient care. </box| Breakthrough Technology: In 2014, Illumina announced the introduction of HiSeq X, a new high-throughput sequencer that will allow for whole genome sequencing for $1000. The applications of the HiSeq X sequencer, however, remains for research purposes and the $1000 whole genome sequencing cost has yet to be incorporated in clinical laboratories> | + | The first sequenced genome cost approximately $3 billion dollars, however, sequencing costs have decreased significantly. Lower costs for genome sequencing are essential in improving patient care (Hayden, 2014). </box| Breakthrough Technology: In 2014, Illumina announced the introduction of HiSeq X, a new high-throughput sequencer that will allow for whole genome sequencing for $1000. The applications of the HiSeq X sequencer, however, remains for research purposes and the $1000 whole genome sequencing cost has yet to be incorporated in clinical laboratories (Hayden, 2014).> |
| ===== References ===== | ===== References ===== | ||
| - | 1. | + | 1. Wilson, B. J., & Nicholls, S. G. (2015). The Human Genome Project, and recent advances in personalized genomics. //Risk Management and Healthcare Policy//, //8//, 9–20. |
| - | 2. | + | 2. Grada, A., & Weinbrecht, K. (2013). Next-generation sequencing: methodology and application. //Journal of Investigative Dermatology//, //133//(8), e11. |
| 3. Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., … Rothberg, J. M. (2005). Genome Sequencing in Open Microfabricated High Density Picoliter Reactors. //Nature//, //437//(7057), 376–380. | 3. Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., … Rothberg, J. M. (2005). Genome Sequencing in Open Microfabricated High Density Picoliter Reactors. //Nature//, //437//(7057), 376–380. | ||
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| 20. Nature. (2015). Applications of next-generation sequencing. Retrieved January 21, 2016, from http://www.nature.com/nrg/series/nextgeneration/index.html | 20. Nature. (2015). Applications of next-generation sequencing. Retrieved January 21, 2016, from http://www.nature.com/nrg/series/nextgeneration/index.html | ||
| - | |||
| - | 22. Hayden, E. (2015). Out of regulatory limbo, 23andMe resumes some health tests and hopes to offer more. Retrieved January 21, 2016, from http://www.nature.com/news/out-of-regulatory-limbo-23andme-resumes-some-health-tests-and-hopes-to-offer-more-1.18641 | ||
| 21. 23andMe. (2015). 23andMe Canada - DNA Genetic Testing & Analysis. Retrieved January 21, 2016, from https://www.23andme.com/en-ca/ | 21. 23andMe. (2015). 23andMe Canada - DNA Genetic Testing & Analysis. Retrieved January 21, 2016, from https://www.23andme.com/en-ca/ | ||
| + | |||
| + | 22. Hayden, E. (2015). Out of regulatory limbo, 23andMe resumes some health tests and hopes to offer more. Retrieved January 21, 2016, from http://www.nature.com/news/out-of-regulatory-limbo-23andme-resumes-some-health-tests-and-hopes-to-offer-more-1.18641 | ||
| 23. FDA. (2013). 23andMe, Inc. 11/22/13. Retrieved January 21, 2016, from http://www.fda.gov/ICECI/EnforcementActions/WarningLetters/2013/ucm376296.htm | 23. FDA. (2013). 23andMe, Inc. 11/22/13. Retrieved January 21, 2016, from http://www.fda.gov/ICECI/EnforcementActions/WarningLetters/2013/ucm376296.htm | ||
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| === Images retrieved from: === | === Images retrieved from: === | ||
| - | Mardis, E. R. (2008). Next-generation DNA sequencing methods. Annu. Rev. Genomics Hum. Genet., 9, 387-402. | + | Mardis, E. R. (2008). Next-generation DNA sequencing methods. //Annu. Rev. Genomics Hum. Genet.//, //9//, 387-402. |
| Tinning, M. (2012, August 1). NGS technologies - platforms and applications. Retrieved January 20, 2016, from http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications | Tinning, M. (2012, August 1). NGS technologies - platforms and applications. Retrieved January 20, 2016, from http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications | ||
