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?