Sequencing and Identification

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Indiana University, Purdue University, Indianapolis *

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Biology

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Feb 20, 2024

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Task: 1. Which of the following sequencing technologies utilizes labeled nucleotides with the fluorescent molecule on the phosphate terminal? (2 points) a. Sanger sequencing b. Illumina sequencing c. PacBio SMRT sequencing d. Nanopore sequencing e. None of the Above 2. Which sequencing technology produces very short sequence reads, usually less than 300 bp? a. Sanger sequencing b. Illumina sequencing c. PacBio SMRT sequencing d. Nanopore sequencing e. None of the Above 3. Illumina sequencing utilizes which of the following to allow massively parallel sequencing? a. Microfluidic channels b. Nanopores c. Flow cells with bound template DNA d. Electrophoresis gels e. None of the Above 4. What is a key advantage of PacBio SMRT sequencing over Illumina sequencing? a. Higher throughput b. Longer read lengths c. Lower cost d. Higher accuracy

e. None of the Above 5. Nanopore sequencing involves threading DNA through a tiny pore in order to directly sequence the molecules. What type of sensor is used to detect the bases? a. Fluorescent sensor b. Chemically treated sensor c. Thermal sensor d. Optically labeled sensor e. None of the Above 6. RNA sequencing provides all but which of the following advantages over microarrays? a. Higher sensitivity b. Higher dynamic range c. Higher resolution d. No prior sequence knowledge required e. None of the Above 7. Lower abundance transcripts are better detected by: a. Microarrays b. RNA sequencing c. Mass spectrometry d. All techniques give relatively equal results e. None of the Above 8. In mass spectrometry protein identification, ionized peptides are separated based on: a. Size b. Hydrophobicity c. Mass/charge ratio d. Fluorescent activity e. None of the Above 9. Fragmentation of proteins prior to mass spectrometry is typically done by: a. Trypsin digestion

b. Gel electrophoresis c. Thermal decomposition d. Restriction enzymes e. None of the Above 10. Gene expression data can be used to help in all but which of the following endeavors? (2 points) a. Disease diagnosis b. Determining gene function c. Studying metabolic pathways Vaccine development d. None of the Above 11. Match each of the following terms with its corresponding technology. Each term should match only a single technology: Protein Identification, Microarray, Illumina Sequencing, Sanger Sequencing, SMRT sequencing, and Nanorpore sequencing. (30 points) a. Mass spectrometry Protein Identification b. Reverse transcription Micro Array c. Bridge amplification illumina sequencing d. Modified polymerase Sanger sequencing e. Motor protein SMRT Sequencing f. Trypsin digestion Protein Identification g. Squiggles Nanopore sequencing h. Sequencing by synthesis illumina sequencing i. Gel electrophoresis sanger sequencing j. In/del errors nanopore sequencing 12. Despite the goals being largely the same, identifying the sequence of molecules in a macromolecule, the technologies used for nucleotide identification are called nucleotide sequencing, and the technologies used for protein identification is not called protein sequencing. Write a few sentences explaining why there is this distinction between the two technologies. (Hint: consider how the technologies mechanically work) (20 points)

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I would argue that this distinction comes from the fundamental aspects of how the nucleotides and proteins are sequenced/identified. For nucleotides, there are several methods of sequencing that are based upon the nucleotides that compose the DNA or RNA molecule being examined. This is different than proteins which are based upon amino acids and not nucleotides. That is why there is such variance in the technologies used to analyze them. In slide 17 of lecture 22, we discussed that RNA-sequencing and microarray showed high levels of agreement only when genes have moderate levels of expression. What are the contributing factors for why they don’t agree at both low and high expression levels? Based upon your knowledge of each technique, which results do you think are more accurate? (30 points) Based off what we learned in class as well as some brief research it seems that RNA sequencing is more accurate. This is primarily because it has a broader dynamic range, as well as having a higher sensitivity which allows it to identify lower abundance transcripts. It also seems that it is more common for microarray to experience illegitimate due to batch effects as well as bad labeling practices that vary based on the experimenter. I also found that at high expression levels, microarrays may hit a saturation threshold in which higher signals then become less proportional to the actual abundance of the transcripts. For lower expression levels there tends to be some error due to the background noise that can be cause by things like cross hybridization or artifacting.

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