Difference between revisions of "Team:Uppsala/Transcriptomics/Sequencing"

 
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         #MinION {
 
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             <div id="toctitle"></div>
 
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             <ul>
 
             <ul>
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                 <li class="toclevel tocsection"><a href="#Project_Description" class="scroll"> <span id="whereYouAre"> Project Description  </span> </a>
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                 <li class="toclevel tocsection"><a href="#Project_Description" class="scroll"> <span id="whereYouAre"> Transciptomics</span> </a>
 
                         <ul>
 
                         <ul>
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                             <li class="toclevel nav-item active"><a href="#top" class="nav-link scroll"> Overview </a></li>
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                             <li class="toclevel nav-item active"><a href="#Seq" class="nav-link scroll"> Sequencing</a></li>
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                             <li class="toclevel nav-item"><a href="#Problem" class="nav-link scroll">  Problem  </a></li>
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                             <li class="toclevel nav-item"><a href="#Exp" class="nav-link scroll">  Expermient</a></li>
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                             <li class="toclevel nav-item"><a href="#Solution" class="nav-link scroll">  Solution </a></li>
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                             <li class="toclevel nav-item"><a href="#Res" class="nav-link scroll">  Result</a></li>
 
                             <li class="toclevel nav-item"><a href="#References" class="nav-link scroll"> References </a></li>
 
                             <li class="toclevel nav-item"><a href="#References" class="nav-link scroll"> References </a></li>
 
                         </ul>
 
                         </ul>
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                 <!-- FROM THIS POINT DOWNWARDS YOU START ADDING YOUR STUFF -->
 
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<h1>Sequencing</h1>
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<h1 id="Seq">Sequencing</h1>
 
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                             <p>Sequencing is the final step of the journey and generally makes up a lot of different and varying methods with the common purpose of determining the primary atomic-level structure of what you’re interested in, resulting in sequences of bases making it all up. Most often this includes proteins, polymers or as in our case the keys to life: genetic material; enabling us to read it like a book of sorts.<br><br>
 
                             <p>Sequencing is the final step of the journey and generally makes up a lot of different and varying methods with the common purpose of determining the primary atomic-level structure of what you’re interested in, resulting in sequences of bases making it all up. Most often this includes proteins, polymers or as in our case the keys to life: genetic material; enabling us to read it like a book of sorts.<br><br>
 
   
 
   
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                                 Oxford Nanopore’s MinION third generation sequencing device makes use of nano-sized holes called nanopores with an applied voltage across them. In essence: when you’re material of choice passes through one of this pores, each base fluctuates this current by a given amount which acts as a fingerprint to identify the given base [1].<br><br></p>
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                                 Oxford Nanopore’s MinION third generation sequencing device make use of nano-sized holes called nanopores with an applied voltage across them. In essence: when your material of choice passes through one of these pores, each base fluctuates this current by a given amount which acts as a fingerprint to identify the given base [1].<br><br></p>
  
  
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                           <img src="https://static.igem.org/mediawiki/2018/3/38/T--Uppsala--MinION.jpeg" id="MinION" >     
 
                           <img src="https://static.igem.org/mediawiki/2018/3/38/T--Uppsala--MinION.jpeg" id="MinION" >     
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<p><b>Figure 1:</b> The MinION device.</p>  
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<p><b>Figure 1.</b> The MinION device.</p>  
 
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<h2>Experiment</h2>
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<h2 id="Exp">Experiment</h2>
 
<p> Sequencing the final library-prepped RNA includes three primary steps: Priming, loading and sequencing.</p><br>
 
<p> Sequencing the final library-prepped RNA includes three primary steps: Priming, loading and sequencing.</p><br>
  
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<h3>Loading the Flowcell</h3>
 
<h3>Loading the Flowcell</h3>
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<p>Loading the flow cell is a bit tricky, as the wrong action can completely destroy the whole cell. By sucking up the primer mixture carefully with a pipette through a disposal vent, a kind of suction is created over the actuall pore-membrane area which lets you (with some quick manouvering skills) drop by drop load your sample into that chamber with the help of a pipette which sucks it all down.<br><br>
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<p>Loading the flow cell is a bit tricky, as the wrong action can completely destroy the whole cell. By sucking up the primer mixture carefully with a pipette through a disposal vent, a kind of suction is created over the actual pore-membrane area which lets you (with some quick manouvering skills) drop by drop load your sample into that chamber with the help of a pipette which sucks it all down.<br><br>
 
   
 
   
 
Being to slow in this process can result in bubbles forming across the membrane which effectively kills all of the pores.
 
Being to slow in this process can result in bubbles forming across the membrane which effectively kills all of the pores.
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<h2>Result</h2>
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<h2 id="Res">Result</h2>
 
<p>Down below are some example pictures from one of our sequencing runs, giving a clue as of what kind of information that can be gathered.</p>
 
<p>Down below are some example pictures from one of our sequencing runs, giving a clue as of what kind of information that can be gathered.</p>
  
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                                 <p><b>Figure 2:</b> State of the sequencing pores after a full sequencing. Notice the amount of sequencing pores (light green) dropping of as issues like saturation (black, orange) starts to increase.</p>
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                                 <p><b>Figure 2.</b> State of the sequencing pores after a full sequencing. Notice the amount of sequencing pores (light green) dropping off as issues like saturation (black, orange) start to increase.</p>
 
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                                 <p><b>Figure 3:</b> Passed versus failed reads.</p>  
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                                 <p><b>Figure 3.</b> Passed versus failed reads.</p>  
 
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                                 <p><b>Figure 4:</b> Distribution of read lengths.</p>
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                                 <p><b>Figure 4.</b> Distribution of read lengths.</p>
 
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                                 <p><b>Figure 5:</b> Quality score distribution for the different read lengths, were a quality score of at least 7 is acceptable.</p>  
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                                 <p><b>Figure 5.</b> Quality score distribution for the different read lengths, were a quality score of at least 7 is acceptable.</p>  
 
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<p>An ideal sequencing run would mean that a lot of the material (depending on run time) has been read and converted into large amounts of ”passed reads” data files containing lines and lines of base sequences. However during our multiple sequencing runs a couple of re-emergin issues consistently showed up, therefore a lot of time was dedicated to troubleshooting this. It was later hypothesized that the source of poor sequencing was because of mRNA left in the samples due to inadequate reverse transcription. See cDNA synthesis </p>
 
<p>An ideal sequencing run would mean that a lot of the material (depending on run time) has been read and converted into large amounts of ”passed reads” data files containing lines and lines of base sequences. However during our multiple sequencing runs a couple of re-emergin issues consistently showed up, therefore a lot of time was dedicated to troubleshooting this. It was later hypothesized that the source of poor sequencing was because of mRNA left in the samples due to inadequate reverse transcription. See cDNA synthesis </p>
 
   
 
   
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<h3>Low throughput</h3>
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<h3>Low Throughput</h3>
  
 
<p>Because it is impossible to have a 100% purely adaptor ligated mRNA sample we initially decided on that it would be acceptable to run the sequencing for a longer time with a below average throughput. However it was noted during repeated sequencing runs that the throughput was actually sub-par, showing that something in the sample(s) was consistently clogging the pores. This meant that only a fraction of our total material was actually being sequenced, eventually leading to near-zero throughputs.</p>
 
<p>Because it is impossible to have a 100% purely adaptor ligated mRNA sample we initially decided on that it would be acceptable to run the sequencing for a longer time with a below average throughput. However it was noted during repeated sequencing runs that the throughput was actually sub-par, showing that something in the sample(s) was consistently clogging the pores. This meant that only a fraction of our total material was actually being sequenced, eventually leading to near-zero throughputs.</p>
 
   
 
   
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<h3>Failed reads</h3>
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<h3>Failed Reads</h3>
  
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  <p>Another reoccuring theme was the issues with ”failed” reads. A failed read consitutes a failed characterization of the base passing through the pores, effectively discarding it. What this means is that whatever came through the pore was actually not cDNA but something foreign.<p><br><br>
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  <p>Another reoccuring theme was the issues with ”failed” reads. A failed read consitutes a failed characterization of the base passing through the pores, effectively discarding it. What this means is that whatever came through the pore was actually not cDNA but something foreign.<p><br>
 
   
 
   
  
 
<p>Even though some progress was made creating read-data that passed, the amount of data as well as the issue with consistency in form of duplicate or triplicate sequencing runs made it deemed as not significant enough to carry over to the bioinformatics.</p>
 
<p>Even though some progress was made creating read-data that passed, the amount of data as well as the issue with consistency in form of duplicate or triplicate sequencing runs made it deemed as not significant enough to carry over to the bioinformatics.</p>
  
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<h1>References</h1>
 
  
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<p><b>[1]</b> Oxford Nanopore, 2018. How does Nanopore DNA/RNA sequencing work? https://nanoporetech.com/how-it-works Date of visit 2018-10-15<p><br>
 
  
  
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<h2 id="References">References</h2>
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<p><b>[1]</b> Oxford Nanopore, 2018. How does Nanopore DNA/RNA sequencing work? <a href="https://nanoporetech.com/how-it-works">https://nanoporetech.com/how-it-works</a> Date of visit 2018-10-15</p><br>
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Latest revision as of 16:19, 3 December 2018