Title: Analysis of Sea Lamprey Transcriptomes
Steven Chang, University of Detroit Mercy.
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Abstract Page
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The sea lamprey genome has recently been sequenced, however, annotation and validation of
genes is ongoing. The project described in this manuscript is a novel pipeline of gene annotation
in a basal vertebrate and includes undergraduate involvement from obtaining of biological
samples to library preparation to final annotation of genes in the transcriptome.
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expand the usability of the Lesson will be addressed later in the text.
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Course
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Biochemistry
Cell Biology
Developmental Biology
Genetics
Microbiology
Molecular Biology
Introductory Biology
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Course Level
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o Upper Level
o Graduate
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Class Type
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Audience
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Class Size
o 1 – 50
o 51 – 100
o 101+
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Lesson Length
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One class period
Multiple class periods
One term (semester or quarter)
One year
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Key Scientific Process Skills
o Reading research papers
o Reviewing prior research
o Asking a question
o Formulating hypotheses
o Designing/conducting experiments
o Predicting outcomes
o Gathering data/making observations
o Analyzing data
o Interpreting results/data
o Displaying/modeling results/data
o Communicating results
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Pedagogical Approaches
o Think-Pair-Share
o Brainstorming
o Case Study
o Clicker Question
o Collaborative Work
o One Minute Paper
o Reflective Writing
o Concept Maps
o Strip Sequence
o Computer Model
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Principles of how people learn
o Motivates student to learn material
o Focuses student on the material to be learned
o Develops supportive community of learners
o Leverages differences among learners
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o Requires student to do the bulk of the work
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Vision and Change Core Concepts
o Evolution
o Structure and Function
o Information flow, exchange and storage
o Pathways and transformations of energy and matter
o Systems
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Vision and Change Core Competencies
o Ability to apply the process of science
o Ability to use quantitative reasoning
o Ability to use modeling and simulation
o Ability to tap into the interdisciplinary nature of science
o Ability to communicate and collaborate with other disciplines
o Ability to understand the relationship between science and society
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Key Words: List 3 – 10 key words that are relevant for the Lesson (e.g. mitosis; meiosis;
reproduction; egg; etc.)
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Scientific Teaching Context Page
Learning Goal(s): Provide clearly stated learning goals, which are broad statements of what the students
will know once they have completed the Lesson. Learning goals are typically rather abstract and
use words like “know,” “understand”, “appreciate,” or “demonstrate”.
For example:
 Students will understand the steps in mitosis.
 Students will appreciate the importance of mitosis in the process of reproduction.
Students will understand the principles behind RNA extraction and the advantages and disadvantages of
column-based vs. organic extraction methods.
Students will understand the evolutionary position of sea lamprey in the vertebrate lineage.
Students will understand how genes and gene families evolve over evolutionary time.
Students will demonstrate changes in expression levels of select genes or gene families in different tissues
or treatment groups
Learning Objective(s): Define what students who have successfully accomplished the learning goal can
actually do. Learning objectives describe student behaviors that are observable, measurable, and
testable. Learning objectives should test students’ mastery of the material and use words like
“define”, “predict”, “design” and “evaluate.”
For example:
 Compare and contrast mitosis and meiosis.
 Predict consequences of abnormal meiosis.
Students will be able to:
Extract and isolate high-quality RNA using the Trizol/organic method.
Prepare RNA for library/transcriptome creation
Assemble reads generated by the transcriptome
Statistically discern true and false positive matches of reads to reference genome
Align reads to reference genome.
Discern global pattern differences between transcriptome libraries (i.e. treatment levels, conditions or
tissues)
Create heat-maps of differential expression.
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Main Text
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1. Introduction: The introduction should provide the origin and rationale for the design of the Lesson and
provide enough background information to allow the reader to evaluate the Lesson without
referring to extensive outside material. For complex topics, a Science Behind the Lesson article
may be simultaneously submitted with the Lesson, so that potential instructors will have
sufficient information to implement the Lesson.
The genome of the sea lamprey has recently been sequenced to 70% coverage and 9x depth of coverage
(Li et al. 2013). Annotation efforts of the genome are ongoing to validate the genes contained in the
genome. Moreover, creation of transcriptome databases is a powerful method of confirming expression
of genes and gene families with the added benefit of helping to fill in the missing 30% of the sea lamprey
genome. Additionally, comparison of transcriptome databases allows for global comparison of gene
expression levels of different tissues, developmental stages, treatment levels, etc.
As next-generation sequencing (NGS) technologies mature, price has become less prohibitive and so is
able to be brought into the classroom. A potential barrier has been complexity of the statistics needed for
analysis as well as programming skills. This module is designed as a senior-level undergraduate course
that will take students through the entire transcriptome database process, from extraction of RNA to
annotation of genes and whole transcriptome analysis. This module is laboratory-based and is based off a
4 hour per week meeting time. Pre-requisites for this course are: genetics and evolution.
The introduction should also explain:
 Intended Audience: Describe the intended student population for the Lesson, including level
and major affiliation. For example: first-year students, biology majors, non-majors, advanced
biology students, etc.
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background content knowledge.
2. Scientific Teaching Themes: Explain how the Lesson relates to the Scientific Teaching Themes of:
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Active Learning: How will students actively engage in learning the concepts? List and/or
explain the active learning strategies that are used in the Lesson. For example, activities could
include think-pair-share, clicker questions, group discussion, debate, etc. Include both inclass and out-of-class activities.
Assessment: How will teachers measure learning? How will students self-evaluate their
learning? List and/or explain the kinds of assessment tools used to measure how well students
achieved the learning objectives. For example, assessments might be clicker questions, forced
choice questions, exams, posters, etc.
Inclusive Teaching: How is the Lesson designed to include all participants and acknowledge
the value of diversity in science? List and/or explain how the Lesson is inclusive and how it
leverages diversity in the classroom and beyond. For example, the lesson may use multiple
senses and provide examples of scientists from different backgrounds.
Active Learning
Students will research RNA extraction via columns vs. Trizol and debate the advantages and disadvantages of each
method in a class discussion or paper.
Students will extract RNA via both methods and compare yields and quality of RNA and the topic will be revisited
for a follow-up class discussion.
Students will prepare the RNA for library creation and will discuss the advantages and disadvantages of splitting a
lane to save costs at the expense of depth of coverage. (Robles paper?)
Students will assemble reads and align them to the sea lamprey genome. In this, students will learn the principles
behind sequence alignment and as a group, will decide the e-value cut-off criteria for annotating a gene.
Students will perform gene ontology (GO) categorization to get a big picture/whole transcriptome picture of
differences between transcriptome databases.
Students will perform differential expression analysis across the entire transcriptome to identify patterns for followup studies.
Assessment
Students will orally debate or write a short opinion piece on the two RNA extraction methods. After performing
both methods and comparing yields, students will write a reflection piece as a follow up to their original
paper/discussion.
For a final project, students will present posters at the college-wide celebration of research. Content will be of
differential expression analysis (heat maps, GO categories) and identification of patterns in transcriptome databases
for future analysis.
Inclusive Teaching
Materials will be posted on Blackboard and will endeavor to use Universal Design to enhance accessibility.
Students will be called on for opinions/discussion by choosing ping pong balls from a bag.
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3. Lesson Plan: Provide a detailed description of the Lesson that is sufficiently complete and detailed to
enable another teacher to replicate it. You may need/want to include subsections such as: preclass preparation and in-class script. A Table containing a recommended timeline for the class
should be included. As needed, expand upon aspects of scientific teaching that are particularly
highlighted in the Lesson. As appropriate, provide examples of formative and/or summative
assessments and related rubrics. List materials that are necessary or useful for teaching the
Lesson, whether they are provided as supplementary materials or as links to other websites.
Lesson Plan
Week 1 – Intro to Nucleic Acids – 1 hour pre-lab talk on what DNA and RNA are and why we extract
samples. Intro on trizol vs. column-based method. Students will be divided into 2 groups; trizol/organic
vs column based. Students will review protocols for both methods and read supporting papers on the
method. At the end of class, students will debate the merits and disadvantages of each method.
Week 2 – Introduction to Sea Lamprey
Pre-lab, students will be given papers on sea lamprey phylogenetics and evolution (Potter and Gill,
Hess). Class content will build on the papers, giving evolutionary history of the sea lamprey in the
vertebrate lineage. Control methods will be discussed and alternative control methods will be
brainstormed in a group discussion/report back format, using the biology and physiology of the sea
lamprey to identify potential targets. History of the sequencing of the sea lamprey genome will be given
and compared to the Human Genome Project. Utility of next-generation sequencing will be discussed.
Week 3 – Alignment of sequences to a reference genome, phylogenetic trees and intro to bioinformatics.
Student will be introduced to using BLAST to search the sea lamprey genome for select genes. Data sets
will be given to students to create phylogenetic trees using MEGA. Neighbor-joining (NJ) trees and
bootstrapping methods will be demonstrated and students will apply this to their data sets.
Week 4 – Experimental Design. Students will be refreshed on the scientific method and will have the
opportunity to design their own experiment, using sea lamprey as a model. Focus will be on looking at
differential gene expression between stages of development OR between treatment groups/levels.
Students will be expected to write a proposal for funding, and so sound scientific methods will be crucial
for success and will be part of the assessment of their progress. Proposals will be presented to the class
for peer review in the next week. Before students leave, instructor will approve their question for further
inquiry (i.e. assess feasibility in time frame – 1 week or so).
Week 5 – Students/groups will present their research proposal. Emphasis will be on hypothesis testing,
expected results and backup plans if results are unexpected. Students will be expected to explicitly state
the gap in knowledge and relate their expected findings to developmental or evolutionary biology as a
whole. Students will grade each other, using a developed rubric, on the quality of each others’ proposals
and whether they would fund this project or not.
Week 6 – Treatment/RNA Extraction. Students will obtain animals for RNA extraction. Treatments, if
necessary, will be performed
4. Teaching Discussion: Share your observations about the Lesson’s effectiveness in achieving the stated
learning goals and objectives, student reactions to the Lesson, and your suggestions for possible
improvements or adaptations to different courses or student populations.
 Subheadings: can be included within the sections above to increase readability and clarity.
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Acknowledgments
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References
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number in parentheses after the volume number.
Examples of reference style:
1. Knight JK, Wood WB. 2005. Teaching more by lecturing less. Cell Biol Educ. 4(4):298-310.
2. Samford University. How to get the most out of studying: A video series.
www.samford.edu/how-to-study/. Accessed August 20, 2013.
3. Handelsman J, Miller S, Pfund C. 2006. Scientific Teaching. New York, NY:W.H. Freeman.
3. Please add notes to the end of the reference list; do not mix in references with explanatory notes.
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