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?

Introduction to Biotechnology

Defining Biotechnology.  Biotechnology is the use of biological organisms and processes to produce commercially valuable products.  Many of the earliest biotech products made use of bacteria to produce recombinant proteins.  That is, the bacteria produced human proteins that could be used as therapeutics in humans.  This feat is possible because both humans and bacteria shared a common ancestor.  Remarkably, the genetic information that human cells use to make proteins can be used by bacteria found in human feces. 

Sometimes, the use of bacteria and yeast in the production of foods such as cheese and beer is placed under the umbrella of biotechnology.  However, for our purposes in this class, biotechnology will be limited to the products created using modified genetic information.  For example, the recombinant proteins mentioned above are the result of taking genetic information from human cells and, through the use of numerous molecular biology processes, bringing that information into bacterial cells in the context that these organisms can understand. 

Central Dogma of Biology.  Despite the need to go back in time a couple billion years, humans and bacteria share a common ancestor.  Evolutionary theory as well as the central dogma of biology weave together to allow bacteria to read genetic information derived from human cell to make human proteins.  The central dogma of molecular biology posits that genetic information flows from DNA to RNA to protein. While the “DNA to RNA” step can be reversed, the same is not true for genetic information flowing from the form of protein back to nucleic acids such as DNA and RNA.

Last Universal Common Ancestor.  Of all known living organisms, DNA serves not only as the information that is both passed down from one generation of organisms to the next but also as the source code for proteins using RNA as an intermediate, or messenger, molecule.  This fact implies that the last universal common ancestor (aka LUCA) had a DNA genome.  All organisms since have made use of DNA genomes as well as the genetic code used to decipher the information.  With DNA genomes in all living organisms and a shared used of a genetic code, biotechnology is built on the ability of scientists to use molecular biology tools to edit and modify DNA.  The possible modifications are nearly endless. 

Model organisms. The extent of biodiversity makes it impractical to study all organisms in a laboratory setting.  Instead, representative organisms from the tree of life have been disproportionately studied.  Multiple factors have guided the selection of these “model organisms” including the ease of culturing asexual organisms and breeding sexual organisms.  Biological principles that are determined in model organisms are usually applicable to non-model organisms because of both the central dogma of biology and the concept of LUCA. 

In a positive feedback loop, research of a model organisms incentivizes additional research of that model organism because tools to study the organism are developed in one study that could be used in follow-up studies of that organism.  However, recent improvements in molecular biology and biotechnology methods are reducing the barriers of entry for studying new organisms and, thus, reducing our reliance on any particular model organism.

Transgenic Animals…Other Than Mice

For Biotechnology Course

  1. Drosophila embryos are injected at the syncytium stage. What is syncytium?  What is the advantage to transgene injection at this stage in development?
  2. Why are two P elements often used to create transgenic flies? Why use a partially-deleted inverted repeat?
  3. Can you envision how alcohol dehydrogenase could be used as a selectable marker in fruit flies?
  4. Look at the number of parasites in the various stages of Plasmodium development (Fig. 16.19). What stage of the life cycle does the defensin A strategy “attack”?  How about bee venom phospholipase?  Single-chain antibodies? What would be the practical reason for targeting therapies to these stages of the life cycle?
  5. The generation of sterile female transgenic salmon includes some interesting steps. What is the purpose of these steps?
    • Fertilize with Charr Sperm and Pressure Shock
    • Fertilize with Salmon Sperm and Pressure Shock
    • Methyl Testosterone Sex Reversal
  6. The book states, “Using antisense RNA and ribozymes have largely been discontinued in favor of RNA interference.” Why?  Is it an obsolete statement?  (Stay up-to-date with CRISPR Journal.)
  7. Why does the book use so many words to discuss “Natural Transgenics and DNA Ingestion”?

Transgenic Animals…Mostly Mice

For biotechnology course

  1. The first transgenic mice had a growth hormone, somatotropin, expressed under the regulation of a metallothionein promoter. What supplement was added to the diet to induce somatotropin expression?
  2. In Greek mythology, what does a chimera look like? What does a chimeric mouse (usually) look like?  Would you expect to see a chimeric mouse from a nuclear microinjection-based approach?  How about an ESC/blastocyst injection-based approach?  How about a retrovirus-based approach?
  3. Does rBST or rTPA come from a transgenic farm animal?
  4. How might you know whether a DNA cassette was inserted into a genome?
  5. The LCR is full of HS. What does this tell you about the DNA in that region?  What would you expect for expression levels if a gene was cloned into the beta-globin cluster?
  6. What benefit could come from having insulator sequences on either side of your transgene?
  7. Graff thinks figure 16.9A is drawn wrong. What do you think?
  8. How does the Lac operon work? How is this system used in transgenic animals?  Do you use lactose to induce transgene expression in mice?
  9. Clontech sells “Tet-Off and Tet-On Gene Expression Systems”. How can it be both?
  10. What would you do with a TRIM69-floxed mouse to knock out expression of TRIM69 in monocytic cells?

Genome Editing with CRISPR

For biotechnology

Many questions were inspired by this CRISPR review.

  1. PAM stands for “protospacer adjacent motif”. What does this mean?
  2. Compare CRISPRi and CRISPRa. Which Cas9 variant would be useful for these approaches?  What Cas9 fusion protein would you create for CRISPRi?  CRISPRa?
  3. How would you set up your CRISPR-based experiment to preferentially use NHEJ DNA repair pathway? What type of mutation would you expect?
  4. How would you set up your CRISPR-based experiment to preferentially use HR DNA repair pathway? What kind of mutation could you expect?
  5. Why would you likely use two gRNAs in Cas9n- and RFN-based experiments? What does it mean that this would reduce your chances of causing off-target effects?
  6. The ribozyme-gRNA-ribozyme method of creating a sgRNA sounds complex. Why wouldn’t you just clone sgRNA sequence alone in a normal PolII-based promoter?  How can this RGR system produce sgRNAs in a tissue-specific manner?
  7. Discuss the similarity between processing the CRISPR array in bacteria and processing polycistronic tRNA-gRNA (PTG) sequences.
  8. CRISPR systems are adaptive immune systems in bacteria and archaea that provide resistance to bacteriophage. How has this been adapted to plants?  How has this been adapted to animals?
  9. Compare GMOs and GEs. Why might the latter be more acceptable?
  10. How do gene drives work?

Plant culture and plant transgenics

For Biotechnology course

  1. Describe Gregor Mendel’s experiments. What were some important conclusions from this work?
  2. Compare traditional breeding, mutation breeding, and transgenic approaches to creating new kinds of plants.
  3. Many plant cells are totipotent. In what ways can plant researchers make use of this trait?
  4. What does “Ti” stand for in Ti plasmid? What kind of cells are found in the “T”?  What cells make use of the “opines” generated from the gene products of the T-DNA?
  5. What is a two-component system? What are the roles of VirA and VirG in Agrobacterium?
  6. What DNA elements are removed from Ti plasmids in order to provide “room” for transgenes to fit? What types of transgenes would be introduced into the Ti plasmid?
  7. What is a gene gun?
  8. What reporter genes can be used to identify successful transgene integration into the plant genome?
  9. How can you get rid of the reporter gene? Is this necessary?  Where does the loxP “scar” come from?

Recombinant Protein Technologies (Part 1)

For Biotechnology course

  1. Describe a couple of reasons why cDNA is cloned into plasmids when using bacterial protein expression systems rather than genomic sequence for a particular gene.
  2. How could you remedy a situation where the 5′ end of a cloned gene forms a hairpin structure with the Shine-Delgarno sequence?
  3. Insulin is first created as preproinsulin in the pancreas.  What processing steps occur to arrive at active insulin?  How was the production of insulin modified to avoid these processing steps?
  4. What additional modification(s) can be made to create fast-acting insulin for type I diabetics?
  5. What additional modification(s) can be made to create slow-acting insulin for type II diabetics?
  6. Describe two strategies for addressing inefficient codon usage.

Protein analysis (part 3)

Biotechnology Chapter 9 Day 3 Questions

  1. How does the yeast two-hybrid system work?
  2. Examine figures 6 and 7 from this paper.  Draw each panel and explain the experimental design as well as the interpretation of the co-immunoprecipitation data.
  3. Experiments are designed to answer questions that researchers have.  What type of questions would be answered using an antigen capture immunoassay?  A direct immunoassay array?
  4. In experiments using protein interaction arrays, what types of molecules could “bind to” the spotted proteins?
  5. Discuss the analogy that networks of metabolites can be compared to city streets.