Next Generation Sequencing 1: Overview Questions

For Biotechnology

Questions to answer while watching this video:

  1. What are the four types of sequencing that will be discussed in this video.
  2. What was the cost of the original human genome sequencing effort?  What was the cost (using Sanger sequencing) immediately after the completion in 2001?
  3. What does it mean that DNA is made of anti-parallel strands?
  4. DNA can be denatured by raising the temperature.  What “R” word did Dr. Chow use as a synonym for annealing the matching strands?
  5. How does a polymerase know what new nucleotide to add to the 3′ hydroxyl group of a growing DNA strand?
  6. What does dNTP stand for?  What are the four types of dNTPs?
  7. What are two ways that a fluorescent terminator differs from the dNTPs?
  8. In a Sanger sequencing-based PCR reaction, how many primers are added?
  9. In a Sanger sequencing-based PCR reaction, which molecules are more common: dNTPs or fluorescent terminators?
  10. What is a typical length of sequence that can be acquired using traditional (Sanger) sequencing?
  11. The Human Genome project used genomes from fewer than 10 people.  How many genomes need to be sequenced to better understand the functions of genes?
  12. Advances in sequencing technology from what company was behind the drops in sequencing costs around the years 2007, 2010, and 2015?
  13. The results from sequencing one stretch of DNA is called a “read”.  How many reads can be obtained from a run on a 384-well plate Sanger sequencing instrument vs. a NovaSeq instrument?
  14. How many gigabases of sequences can be obtained on the Sanger vs. NovaSeq instruments?
  15. What is the cost of sequencing a human genome with the latest Illumina technology (at the time this video was made)?
  16. When showing the different sizes of Illumina flow cells, what was the object that was used as a reference to show scale?
  17. Which spelling do you prefer: adapter or adaptor?
  18. The adaptor sequences have two parts: primer binding sites and capture sequences. 
    1. Once the (denatured) DNA has been loaded into the flow cell, which adaptor sequence part is hybridized to other single-stranded DNA first?
    2. How many copies of the original ssDNA are found in a cluster by the time the sequencing primer is added?
  19. In fluorescent reversible terminator chemistry, which carbon of the ribose sugar is the terminator molecule attached to?
  20. Once the terminator molecule is removed, what functional group resides at that carbon of the ribose sugar?
  21. What is the sequence of the first cluster that Dr. Chow walks through?
  22. What is the limit of the read length for this sequencing approach?
  23. What is the solution for the problem created when two cluster are so close together that they partially overlap?
  24. What is an advantage of switching from 4-color to 2-color chemistry?
  25. What is the pore size in the Oxford Nanopore system’s membranes?
  26. Detectors measure a change in what electrical property as a ssDNA flows through the pore?
  27. How many ssDNA molecules can flow through the pore at the same time?
  28. What features are found on the 3′ carbon and 5′ carbon of the ribose sugar in nucleotides used in PacBio sequencing?
  29. How does a PacBio flow cell limit the number of ssDNA molecules being sequenced at a given position?
  30. Why is a movie created to “watch” the PacBio sequencing, rather than a single image per round like in Illumina sequence technologies?
  31. How does data from PacBio get “corrected” to make up for the 10-15% error rate during the reading of a single stretch of ssDNA?
  32. Why are sequencing technologies that produce long reads beneficial for genome assembly? (3 bullet points for this answer)
  33. Which medical application did you find to be the most interesting? Why?

Buster Bear Goes Fishing

For Animal Stories for Kids

Buster Bear was having a lazy day.  But as the sun reached higher into the sky, his fur started to get hot and his stomach was starting to growl. 

“All right” he said to himself.  “Time to get a move on.”

Slowly and methodically, Buster Bear started to head downhill to the Laughing Brook.  The movement felt good to his well-rested muscles and the water ahead looked inviting. 

As he approached the Laughing Brook, Buster noticed that he wasn’t the only one that had thought about having fish as a mid-morning snack.  Once he was close, he realized that it was Little Joe Otter.  He also came to realize that the otter had been successful.  Little Joe Otter had a nice big trout in his mouth!

Buster Bear crept up to the unsuspecting otter and snarled.  As you can imagine, poor Little Joe Otter panicked at the sound, dropped the fish, and dove into the nearby water. 

“Just as I had hoped!” Buster Bear thought as he pounced on the trout before it flip-flopped its way down the stream bank.

Little Joe Otter popped his head out of the water of the water and stared coldly at Buster Bear’s mouth. 

Buster Bear stubbornly stood there and eventually said, “If you would like the fish back, just come get it.”

This did nothing to get Little Joe Otter to stop his unblinking gaze, so Buster Bear repeated his half-hearted offer.  “Come get your fish if you want it.”

Furious, Little Joe Otter dove under the water and slipped downstream until he met Billy Mink.  It didn’t take long to tell Billy about his encounter with Buster Bear.  Billy Mink listened to Little Joe’s tale and nodded sympathetically.  However, being a practical sort of animal, Billy Mink could only say, “There’s not much you can do about it, is there?”

Stewing in his anger, Little Joe Otter thought over his options until a plot for revenge took root in his mind.  Without a word, Little Joe Otter took to the water again and made his way upstream until he spotted Buster Bear.

It was clear that one fish did not satisfy the lazy bear’s appetite, so Buster Bear was progressing from one deep pool to the next.  Little Joe Otter picked up on Buster’s plan and moved quickly into action.  He first went to the pool Buster Bear was going to next.  Once there, he swam at a frantic pace in circles to scare away the fish in the pool and disturb the mud to make the water murky.

Buster Bear fished unsuccessfully there for a while and, discouraged, moved on to the next pool only to find the conditions to be similar.  All day long, Little Joe Otter sabotaged the lazy bear’s fishing.  Buster Bear’s stomach continued to growl and Little Joe Otter’s stomach joined in the chorus. 

Biology connection: Food resources, like fish, are prized by many different types of animals.  The battle for food is called competition.  The population size of one type of animal is often limited not only by the amount of food in an area but also by whether other types of animals eat that food, too.

Re-write of “Thornton Burgess Bedtime Stories” chapter 1.

Central Dogma of Biology

For Conversational Biology series – topic POLS

To learn a topic, it is nice to have a central framework upon which to build your “mind map”.  When it comes to biology, my central framework is called “The Central Dogma of Biology”.  To get us started learning about biology, I think it is appropriate to provide this concept for you to use when building your mind map of biology.

If we go back in time to 1956, we would find that Francis Crick was an important figure in biology, particularly molecular biology.  Just a few years earlier, he had looked at Rosalind Franklin’s data and, along with James Watson, had described the structure of deoxyribonucleic acid (DNA).  This was a big deal because biologist were beginning to come to grips with the idea that DNA is the molecule (a type of macromolecule under the umbrella of “nucleic acids”) that stores genetic information.  Prior to this, protein was the top dog in the minds of most scientists.  DNA was considered “boring” and proteins (another type of macromolecule) were considered to be more interesting, so it stood to reason that proteins would have been suspected to be the storage molecule for molecular biology. 

Anyway, back to the central dogma of biology (aka the central dogma of molecular biology).  This concept was proposed by Francis Crick.  His framework can be overly simplified to “DNA is transcribed to RNA and RNA is translated to protein” or, even more simply, “DNA à RNA à protein”, where the arrows are steps that read information of one molecule to create the next molecule in the progression.

Let’s back up a step again.  What’s RNA?  It, like DNA, belongs to the nucleic acid macromolecule class.  It looks a lot like DNA, but it is one oxygen molecule short, so RNA (ribonucleic acid) has a similar name to DNA (deoxyribonucleic acid).  Notice that the difference in the names is “deoxy”, which is a scientific way of saying “lacks an oxygen”.

Francis Crick had one more arrow in his “DNA à RNA à protein” framework and that was a reverse arrow between DNA and RNA (DNA ß RNA).  This left open the possibility that information in the form of RNA could be used to create a new molecule of DNA.  This turns out to be true, so this process is called “reverse transcription” since the process of DNA à RNA is called transcription.

One final point, Crick wisely avoided drawing an arrow from protein back to RNA (RNA ß protein).  When we get to the topic of the genetic code, we will see why “reverse translation” isn’t a thing.

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Preface for writing this series

For Conversational Biology series

Welcome to Life and Biology’s new blog series called “Conversational Biology”.  There are several reasons that I’m initiating this effort and I wanted to put them up front. 

*Before all that, I have a confession to make.   I am fairly proficient at starting projects and not seeing them through to completion.  This particular project is one that I have considered for quite some time and have finally started.  Time will tell if I stick with it or not.*

My first reason for writing this blog series is that I would like to create a body of work that is approachable for everyone, but goes into college-level depth on all topics in biology.  This is an effort that contrasts with textbooks that are often difficult to wade through or are simply not a pleasure to read.  A collection of books have been written recently that include the F-bomb in their title.  A couple years ago, I had a failed attempt at my “Conversational Biology” goal whereby I started explaining biology with the crudest, most vulgar language I could come up with.  While it was fun to write in such a profane approach, it just didn’t feel authentic.  In another science outreach effort, I created a series of YouTube videos that presented various biological topics in a conversational tone.  That was a far better approach as it felt more authentic to me.  However, the videos (despite their low production value) were time-consuming to create and have thus failed to continue.  I have high hopes that this blog-based approach will be more manageable and yet valuable to the reader.

The second reason for this series is that I turned 40 years old this past year.  If you’re older than 40, you might be able to relate to my experience where I started to do a philosophical examination of my life by reflecting on the “first half” of my life and thinking through what my “second half” of life should look like.  My first 40 years were fairly productive with 20+ years of education, a variety of jobs, and building a family.  At first this made my second half of life seem like it would need to be exhausting to keep up the pace I started. This series of blog posts will be an attempt to conquer one of the biggest stumbling blocks I had up to age 40…writing.  If there is something that grinds me into the ground every time, it’s writing.  Blog posts seem less daunting than writing a book, so I’ve decided that I’m just going to take on blogging as a way of daily-ish writing motivation.  Yesterday was my oldest son’s 13th birthday.  I’m using his milestone as an arbitrary “fork in the road” whereby I take my writing output seriously.  How did I mark the occasion?  I wrote 1415 words!  Very likely this was the highest word count I’ve ever achieved in a single day.  I hope to look back at this and remember how hard writing was until I took it on with full effort.

Enough about me.  My third reason for writing this series is to provide my students with supplementary reading for the courses that I teach at Montana Tech.  Currently, those courses are: Principles of Living Systems (POLS), POLS lab, Discover Biology lab, Introduction to Evolution, Virology, Immunology, and Biotechnology.  I’m always asking students to read the material and then write out the most important concepts as if their parents or non-biology majors were the audience.  The writing professors call this “synthesis”.  I just figured it was a good way of remembering the material.  In fact, when I was a student, I had a habit of daydreaming during my study sessions about how I would teach the topics I was learning about to other people. 

Finally, even though my third reason was based on helping my students in college, I suspect that education is about to go through a major upheaval.  There is so much information available to everyone that can muster a Google search, the high cost of college will soon be viewed as outrageous (assuming the consensus already haven’t put the cost in this category).  Perhaps in the future, advanced degrees will be merit based.  That is, passing a “class” will be based solely on whether the student shows an understanding for that material.  Will college degrees be boiled down to passing tests rather than sitting in lecture halls?  If so, students will still need to learn the material.  Perhaps writing a series of blog posts in a conversational manner to cover all the topics you might come across in current biology courses will be a way of replacing the lecturer of the future.

Next article: Central Dogma of Biology