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 ===== 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 =====
@@ Line 16: / Line 16: @@
 ==== Applied Biosystems SOLiD ====
-Sequencing by Olignucleotide Ligation and Detection (SOLiD) is a next generation sequencing technique developed by Life Technologies and purchased by Applied Biosystems in 2006.<sup>4</sup> SOLiD was made available for commercial use in the same year. This method uses technology from two-base sequencing based on ligation sequencing. Sequencing by ligation is a DNA sequencing method that uses DNA ligase to identify the nucleotide present at a given position in a DNA sequence.<sup>4</sup> Ligation sequencing does not use a DNA polymerase to generate a second complimentary strand but uses mismatch sensitivity of ligase to determine the sequence of DNA.<sup>4</sup>
+Sequencing by Olignucleotide Ligation and Detection (SOLiD) is a next generation sequencing technique developed by Life Technologies and purchased by Applied Biosystems in 2006.<sup>4</sup> SOLiD was made available for commercial use in the same year. This method uses a two-base fluorescent probe, which is based on sequencing by ligation. Sequencing by ligation uses DNA ligase to identify the nucleotide present at a given position in a DNA sequence.<sup>4</sup> Ligation sequencing does not use a DNA polymerase to generate a second complimentary strand, but uses mismatch sensitivity of ligase to determine the sequence of DNA.<sup>4</sup>
 <box 30% round right | > {{:solid_sequencing.png?260|}} </box| Figure 2 - Summary of SOLiD Sequencing. (Mardis, 2008)>
-The first step in SOLiD sequencing system is library preparation.  The are two common methods of preparing a DNA library. The first involves fragmentation while the other uses a mate-paired library. In the first process, the whole genome of the target DNA is randomly fragmented and two different 25 base pairs (bp) DNA adaptors on beads are ligated at the 5’ and 3’ ends.<sup>5</sup> Mate-paired library DNA fragments are separated from another DNA fragments of known length using an internal adaptor. This process also includes DNA adaptors at the 5’ and 3’ end similar to the first step.<sup>5</sup> Next, DNA fragments on the beads are amplified by the emulsion polymerase chain reaction (PCR) technique. The beads containing clonally amplified cDNA fragments attach to the surface of a flow cell.<sup>5</sup> Sequencing of the target DNA sequence involves multiple rounds of ligation reactions. In the first round, a universal sequencing primer is annealed to the adaptor and 16 fluorescently labeled octamer oligonucleotides are added.<sup>6</sup>  The 16 possible oligonucleotides represent all the possible combinations of two nucleotides such as AT, AG, AC and so on in the first two bases while the 3rd to 5th bases are degenerate and unknown.<sup>6</sup> The last 3 bases are bound to one of the four fluorescent labels, which are different in the emission spectrum.<sup>6</sup> Each label represents four dinucleotides in each ligation reaction and only the first two nucleotides in the probe are used to help identify the sequence of the DNA fragment. Since each label represents four possible dinucleotide combinations, the first round of annealing a probe to the primer does not exactly identify the bases specifically but allows the possible sequence to be narrowed down.<sup>6,7</sup> The final three bases are then cleaved off after detection of the fluorescent label in order to provide a free 5’ end for the ligation of the next probe. This ligation process continues for approximately 5-7 cycles.<sup>7</sup> After the cycles of ligation, fluorophore detection and cleavage, the previous ligated probes are denatured and removed. A new primer that is shorter by one base (n-1) is annealed to the template and another 5-7 cycles of ligation, detection and cleavage occurs. This allows the detection of up to 75 base pairs depending on the specific SOLiD sequencing software used after multiple rounds have been completed.<sup>5</sup>
+The first step in the SOLiD sequencing system is library preparation. The are two common methods of preparing a DNA library. The first involves fragmentation while the other uses a mate-paired library. In the first process, the whole genome of the target DNA is randomly fragmented and two different 25 base pairs (bp) DNA adaptors on beads are ligated at the 5’ and 3’ ends.<sup>5</sup> Mate-paired library DNA fragments, however, are separated from another DNA fragments of known length using an internal adaptor. This process also includes DNA adaptors at the 5’ and 3’ end similar to the fragmentation method.<sup>5</sup> Next, DNA fragments on the beads are amplified by the emulsion polymerase chain reaction (PCR) technique. The beads containing clonally amplified complementary DNA fragments attach to the surface of a flow cell.<sup>5</sup> Sequencing of the target DNA sequence involves multiple rounds of ligation reactions. In the first round, a universal sequencing primer is annealed to the adaptor and 16 fluorescently labeled octamer oligonucleotides are added.<sup>6</sup>  The 16 possible oligonucleotides represent all the possible combinations of two nucleotides such as AT, AG, AC and so on in the first two bases while the 3rd to 5th bases are degenerate and unknown.<sup>6</sup> The last 3 bases are bound to one of the four fluorescent labels, which are different in the emission spectrum.<sup>6</sup> Each label represents four dinucleotides in each ligation reaction and only the first two nucleotides in the probe are used to help identify the sequence of the DNA fragment. Since each label represents four possible dinucleotide combinations, the first round of annealing a probe to the primer does not exactly identify the bases specifically, but allows the possible sequence to be narrowed down.<sup>6,7</sup> The final three bases are then cleaved off after detection of the fluorescent label in order to provide a free 5’ end for the ligation of the next probe. This ligation process continues for approximately 5-7 cycles.<sup>7</sup> After the cycles of ligation, fluorophore detection, and cleavage, the previous ligated probes are denatured and removed. A new primer that is shorter by one base (n-1) is annealed to the template and another 5-7 cycles of ligation, detection and cleavage occurs. This allows the detection of up to 75 base pairs depending on the specific SOLiD sequencing software used after multiple rounds have been completed.<sup>5</sup>
-Multiple cycles and rounds allows for the interrogation of every base in two different ligation reactions in addition to two different primers. This process is called double interrogation and it is effective at increasing accuracy and specificity of the template strand.<sup>7,8</sup> The fluorescent detection and visualization is analyzed using a color space reference sequence as opposed to a nucleotide based reference sequence.<sup>7,8</sup> In order to analyze the data from the detected fluorescence, extended algorithm software programs based on color space are used. There are multiple programs that are available in the market for analyzing SOLiD data.<sup>8</sup>
+Multiple cycles and rounds allows for the interrogation of every base in two different ligation reactions in addition to two different primers. This process is called double interrogation and it is effective at increasing accuracy and specificity of the template strand.<sup>7,8</sup> The fluorescent detection and visualization is analyzed using a colour space reference sequence as opposed to a nucleotide based reference sequence.<sup>7,8</sup> In order to analyze the data from the detected fluorescence, extended algorithm software programs based on colour space are used. There are multiple programs that are available in the market for analyzing SOLiD data.<sup>8</sup>
 <box 17% round | > {{:color_space2.png?100|}} </box| Figure 3 - Color Space reference sequence. (Mardis, 2008)>
@@ Line 28: / Line 28: @@
  {{ 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 ====
@@ Line 34: / Line 34: @@
 <box 30% round right | > {{:illumina_.png?300|}} </box| Figure 5 - Illumina: Sample Prep & Cluster Generation. (Mardis, 2008)>
-Illumina sequencing technology is one of the most successful and widely adopted next-generation sequencing (NGS) technologies worldwide. It enables a wide variety of applications, allowing researchers to ask virtually any question related to the genome, transcriptome (the full range of messenger RNA molecules expressed by an organism, or epigenome (several chemical compounds that can tell the genome what to do of any organism.<sup>9,10</sup>
+Illumina sequencing technology is one of the most successful and widely adopted next-generation sequencing (NGS) technologies worldwide. It enables a wide variety of applications, allowing researchers to ask virtually any question related to the genome, transcriptome (the full range of messenger RNA molecules expressed by an organism), or epigenome (several chemical compounds that can tell the genome what to do).<sup>9,10</sup>
  ===Sample Prep===
@@ Line 49: / Line 49: @@
 ===Sequencing===
-The method of sequencing utilized by Illumina is called sequencing by synthesis.<sup>11</sup> The first sequencing cycle begins by adding four fluorescently-labeled reversible terminator nucleotides, primers, and DNA polymerase to the flow cell. The primer attaches to the DNA being sequenced. The DNA polymerase then binds to the primer and adds the first fluorescently-labelled terminator nucleotide (base) to the new DNA strand. Terminator nucleotides have been modified in two ways. First, they are “reversible terminators”, in that, a chemically cleavable moiety at the 3’ hydroxyl position allows only a single-base incorporation to occur in each cycle. Second, one of four fluorescent labels, also chemically cleavable, corresponds to the identity of each nucleotide (A, C, G, or T).<sup>12</sup>  Once a terminator base has been added, no more bases can be added to the strand of DNA until the terminator moiety is cut from the DNA. Lasers are passed over the flow cell 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) emit a different colour. The emitted fluorescence from each cluster is captured and the first base is identified.The fluorescently-labelled terminator group is then removed from the first base. This allows for the next sequencing cycle to begin with the next fluorescently-labelled terminator base being added alongside the first base. After laser excitation, the image is captured as before, and the identity of the second base is recorded. The sequencing cycles are repeated to determine the sequence of bases in a fragment, one base at a time. The process continues until millions of clusters have been sequenced.<sup>11</sup>
+The method of sequencing utilized by Illumina is called sequencing by synthesis.<sup>11</sup> The first sequencing cycle begins by adding four fluorescently-labelled reversible terminator nucleotides, primers, and DNA polymerase to the flow cell. The primer attaches to the DNA being sequenced. The DNA polymerase then binds to the primer and adds the first fluorescently-labelled terminator nucleotide (base) to the new DNA strand. Terminator nucleotides have been modified in two ways. First, they are “reversible terminators”, in that, a chemically cleavable moiety at the 3’ hydroxyl position allows only a single-base incorporation to occur in each cycle. Second, one of four fluorescent labels, also chemically cleavable, corresponds to the identity of each nucleotide (A, C, G, or T).<sup>12</sup>  Once a terminator base has been added, no more bases can be added to the strand of DNA until the terminator moiety is cut from the DNA. Lasers are passed over the flow cell 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) emit a different colour. The emitted fluorescence from each cluster is captured and the first base is identified.The fluorescently-labelled terminator group is then removed from the first base. This allows for the next sequencing cycle to begin with the next fluorescently-labelled terminator base being added alongside the first base. After laser excitation, the image is captured as before, and the identity of the second base is recorded. The sequencing cycles are repeated to determine the sequence of bases in a fragment, one base at a time. The process continues until millions of clusters have been sequenced.<sup>11</sup>
 ===Data Analysis===
@@ Line 67: / Line 67: @@
-Firstly, the DNA sample is prepared from fragmented RNA and then linked together.<sup>13</sup>  Then they are clonally amplified by emulsion PCR onto beads known as Ion Sphere Particles. The beads are placed into proton-sensing wells on a semiconductor sequencing chip so that one bead can roughly fit into hundreds of millions of wells.<sup>14</sup>  The chip is then submerged into a nucleotide solution and results in the release of protons and a corresponding change in pH. This change in pH is documented by the PGM to determine whether the right nucleotide was used in the process or not, let alone if a nucleotide was added at all.<sup>13</sup> As sequencing occurs, each of the four bases is introduced sequentially.<sup>14</sup> A clear indication of the correct nucleotide being added is the presence of voltage. There will be no voltage found if the wrong nucleotide is added, and there will be double the voltage if two nucleotides are added.<sup>4</sup> The more nucleotides present, the greater the increase in voltage and pH.<sup>15</sup> It is important to note that Ion Torrent sequencing is very fast and allows for many more reads to be done per sequencing run as there is no optical detection necessary.<sup>13</sup>
+Firstly, the DNA sample is prepared from fragmented RNA and then linked together.<sup>13</sup>  The library is then clonally amplified by emulsion PCR onto beads known as Ion Sphere Particles. The beads are placed into proton-sensing wells on a semiconductor sequencing chip so that one bead can roughly fit into hundreds of millions of wells.<sup>14</sup>  The chip is then submerged into a nucleotide solution and results in the release of protons and a corresponding change in pH. This change in pH is documented by the PGM to determine whether the right nucleotide was used in the process or not, let alone if a nucleotide was added at all.<sup>13</sup> As sequencing occurs, each of the four bases is introduced sequentially.<sup>14</sup> A clear indication of the correct nucleotide being added is the presence of voltage. There will be no voltage found if the wrong nucleotide is added, and there will be double the voltage if two nucleotides are added.<sup>4</sup> The more nucleotides present, the greater the increase in voltage and pH.<sup>15</sup> It is important to note that Ion Torrent sequencing is very fast and allows for many more reads to be done per sequencing run as there is no optical detection necessary.<sup>13</sup>
 <box 20% round | > {{:ion_torrent_2.png?125|}} </box| Figure 8 - The process of how nucleotides are determined based on the release of protons for base calling. (Corney, 2013)>
-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 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 }}
-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 =====
@@ Line 92: / Line 143: @@
 ==== Ion Torrent PGM - Microbial Pathogens ====
-One of the more interesting applications of Ion Torrent PGM sequencer is how it can be used to identify microbial pathogens. In May and June of 2011, a toxin known as Shiga-toxin, which produces //Escherichia coli// (//E. coli//) was having an ongoing outbreak in Germany. More than 3000 people were infected. With the recent advancements in NGS systems this specific incident showcased the efficiency and progress that Ion Torrent PGM sequencer has made. The whole genome sequencing of the Ion Torrent PGM sequencer allowed for the scientists to find the specific antibiotic resistances needed to by identifying which type of E. coli was present. What was revealed was the presence of a hybrid of 2 //E. coli// strains. One being entero aggregative //E. coli// and the other being entero hemorrhagic //E. coli//. The presence of a hybrid of 2 //E. coli// strains seemed to help explain why the outbreak had behaved mostly pathogenic. Based on the sequencing results found from the //E. coli//, Ion Torrent PGM shows a potential of fast, but limited throughput sequencer for when an outbreak of a new disease occurs.<sup>4</sup>
+One of the more interesting applications of Ion Torrent PGM sequencer is how it can be used to identify microbial pathogens. In May and June of 2011, a toxin known as Shiga-toxin, which produces //Escherichia coli// (//E. coli//) was having an ongoing outbreak in Germany. More than 3000 people were infected. With the recent advancements in NGS systems this specific incident showcased the efficiency and progress that Ion Torrent PGM sequencer has made. The whole genome sequencing of the Ion Torrent PGM sequencer allowed for the scientists to find the specific antibiotic resistances needed to by identifying which type of //E. coli// was present. What was revealed was the presence of a hybrid of 2 //E. coli// strains. One being entero aggregative //E. coli// and the other being entero hemorrhagic //E. coli//. The presence of a hybrid of 2 //E. coli// strains seemed to help explain why the outbreak had behaved in a pathogenic manner. Based on the sequencing results found from the //E. coli//, Ion Torrent PGM provides a fast method of sequencing for when an outbreak of a new disease occurs.<sup>4</sup>
 ==== Direct to Consumer Genetic Testing ====
 <box 12% round right | > {{:playground:illumina_humanomniexpress-24_format_chip.jpg|}} </box| Figure 10 - Illumina HumanOmniExpress-24 Format Chip. (23andMe, 2015)>
-Advancements in terms of the scale and efficiency of genetic sequencing has decreased the cost of using next generation technologies. This decrease in cost has allowed the power of these high-throughput DNA sequencing technologies to be harnessed by companies, like 23andMe, which offer direct to consumer genetic testing.<sup>20</sup>
+Advancements in terms of the scale and efficiency of genetic sequencing has decreased the cost of using next generation technologies. This decrease in cost has allowed the power of these high-throughput DNA sequencing technologies to be harnessed by companies, like 23andMe, which offer direct-to-consumer (DTC) genetic testing.<sup>20</sup>
 andMe is a company that uses the Illumina HumanOmniExpress-24 format chip (a form of Illumina next generation sequencing), to sequence DNA, and subsequently analyze it to provide genetic profiling information directly to consumers.<sup>21</sup> One simply has to pay $199, and provide a saliva sample. The information provided is divided into four categories: carrier status, wellness, traits, and ancestry. Carrier status indicates whether a person carries genes associated with any of 36 different disorders, such as, cystic fibrosis. Wellness addresses aspects including, whether an individual is lactose intolerant, or if they tend to get red and flushed when they drink alcohol. The traits information looks at a set of nearly two dozen traits that are dictated by genes including, male-patterned baldness, eye colour, and hair colour. Finally, ancestry information is also provided, including a breakdown of the percentage, by genetic makeup, of different racial and ethnic groups in an individual’s background. Finally, ancestry information is also provided in regards to, ancestry composition (a breakdown of the percentage, by genetic makeup, of different racial and ethnic groups in an individual’s background), DNA relatives, maternal and paternal lineages, and even neanderthal percentage.<sup>21</sup>
-However, services such as those offered by 23andMe are not left unregulated. Until November 2013, 23andMe offered consumers genetic testing to estimate their risk for 240 heath conditions, such as breast cancer and heart disease, for $99.<sup>22</sup> In November 2013, the FDA shut down the company after it was determined that the company lacked the appropriate approval to give people potentially life-altering information about their health. Of particular concern were assessments such as those for BRCA-related genetic risk and drug responses (e.g., warfarin sensitivity) because of the potential health consequences that could result from false positive or false negative assessments for these high-risk indications.<sup>23</sup> For instance, "if the BRCA-related risk assessment for breast or ovarian cancer reports a false positive, it could lead a patient to undergo prophylactic surgery, chemoprevention, intensive screening, or other morbidity-inducing actions, while a false negative could result in a failure to recognize an actual risk that may exist".<sup>23</sup> The FDA’s other concern related to how people were interpreting the reported results. Some people found the information confusing misinterpreting the presence of a genetic variant that is simply linked to a disease, with a definite expectation of developing the disorder.<sup>23</sup> After implementing changes to address the FDA's concerns, nearly 2 years later in October 2015, 23andMe released the current FDA approved, previously mentioned, $199 test (a scaled back version of the original).<sup>24</sup>
+However, services such as those offered by 23andMe are not left unregulated. Until November 2013, 23andMe offered consumers genetic testing to estimate their risk for 240 heath conditions, such as breast cancer and heart disease, for $99.<sup>22</sup> In November 2013, the FDA shut down the company after it was determined that the company lacked the appropriate approval to give people potentially life-altering information about their health. Of particular concern were assessments such as those for BRCA-related genetic risk and drug responses (e.g., warfarin sensitivity) because of the potential health consequences that could result from false positive or false negative assessments for these high-risk indications.<sup>23</sup> For instance, "if the BRCA-related risk assessment for breast or ovarian cancer reports a false positive, it could lead a patient to undergo prophylactic surgery, chemoprevention, intensive screening, or other morbidity-inducing actions, while a false negative could result in a failure to recognize an actual risk that may exist".<sup>23</sup> The FDA’s other concern related to how people were interpreting the reported results. Some people found the information confusing; misinterpreting the presence of a genetic variant that is simply linked to a disease, with a definite expectation of developing the disorder.<sup>23</sup> After implementing changes to address the FDA's concerns, nearly 2 years later in October 2015, 23andMe released the current FDA approved, previously mentioned, $199 test (a scaled back version of the original).<sup>24</sup>
 The applications of next-generation sequencing seem almost endless, allowing for rapid advances in many fields including the field of consumer genetic testing.
 <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 =====
-.
+. 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.
-.
+. Grada, A., & Weinbrecht, K. (2013). Next-generation sequencing: methodology and application. //Journal of Investigative Dermatology//, //133//(8), e11.
-. 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.
+. 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.
-. Liu, L., Li, Y., Li, S., Hu, N., He, Y., Pong, R., ... & Law, M. (2012). Comparison of next-generation sequencing systems. BioMed Research International, 2012.
+. Liu, L., Li, Y., Li, S., Hu, N., He, Y., Pong, R., ... & Law, M. (2012). Comparison of next-generation sequencing systems. //BioMed Research International//, 2012.
-. Ansorge, W. J. (2009). Next-generation DNA sequencing techniques. New biotechnology, 25(4), 195-203.
+. Ansorge, W. J. (2009). Next-generation DNA sequencing techniques. //New Biotechnology//, //25//(4), 195-203.
-. Corney, D. (2013). RNA-seq Using Next Generation Sequencing. Materials And Methods, 3, 203. http://dx.doi.org/10.13070/mm.en.3.203
+. Corney, D. (2013). RNA-seq Using Next Generation Sequencing. //Materials And Methods//, //3//, 203. http://dx.doi.org/10.13070/mm.en.3.203
 .Metzker, M. L. (2010). Sequencing technologies—the next generation.Nature reviews genetics, 11(1), 31-46.
@@ Line 130: / Line 181: @@
 . Illumina. (2010). Illumina Sequencing Technology. Retrieved January 21, 2016, from https://www.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf
-. Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. Nature Biotechnology, 26, 1135-1145. Retrieved January 21, 2016.
+. Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. //Nature Biotechnology//, //26//, 1135-1145. Retrieved January 21, 2016.
 . Corney, D. C. (2013). RNA-seq Using Next Generation Sequencing. Retrieved January 17, 2016, from http://www.labome.com/method/RNA-seq-Using-Next-Generation-Sequencing.html
-. Quail, M. A., Smith, M., Coupland, P., Otto, T. D., Harris, S. R., Connor, T. R., ... & Gu, Y. (2012). A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC genomics, 13(1), 341.
+. Quail, M. A., Smith, M., Coupland, P., Otto, T. D., Harris, S. R., Connor, T. R., ... & Gu, Y. (2012). A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. //BMC Genomics//, //13//(1), 341.
-. Kremkow, B. G., & Lee, K. H. (2015). Sequencing technologies for animal cell culture research. Biotechnology letters, 37(1), 55-65.
+. Kremkow, B. G., & Lee, K. H. (2015). Sequencing technologies for animal cell culture research. //Biotechnology Letters//, //37//(1), 55-65.
-. Gagan, J., & Van Allen, E. M. (2015). Next-generation sequencing to guide cancer 	therapy. Genome medicine, 7(1), 1-10.
+. Gagan, J., & Van Allen, E. M. (2015). Next-generation sequencing to guide cancer therapy. //Genome Medicine//, //7//(1), 1-10.
-. Drilon, A., Wang, L., Arcila, M. E., Balasubramanian, S., Greenbowe, J. R., Ross, J. S., ... & Ladanyi, M. (2015). Broad, hybrid capture-based next-generation sequencing identifies actionable genomic alterations in" driver-negative" lung adenocarcinomas. Clinical Cancer Research, clincanres - 2683.
+. Drilon, A., Wang, L., Arcila, M. E., Balasubramanian, S., Greenbowe, J. R., Ross, J. S., ... & Ladanyi, M. (2015). Broad, hybrid capture-based next-generation sequencing identifies actionable genomic alterations in" driver-negative" lung adenocarcinomas. //Clinical Cancer Research//, clincanres - 2683.
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@@ Line 156: / Line 207: @@
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 === Images retrieved from: ===
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