BIOSC 1285: GENOMICS LABORATORY Department of Biological Sciences University of Pittsburgh

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1 BIOSC 1285: GENOMICS LABORATORY Department of Biological Sciences University of Pittsburgh Faculty Office hours Classes: meeting times & places Course overview Textbook CourseWeb Course policies Zuzana Swigonova, Ph.D. Office: A356 Langley Hall Phone: Office hours: by appointment. For general course-related issues I will be available before and after each lab class. You are encouraged to contact me when you need help, and you are expected to do so to review your project. Many questions will be answered in class. me to set up an appointment when you need more time to discuss your project, course material, or course related issues. Mo/We: 8:30-10:20AM Location: G5 Clapp Hall First half of the term will be heavier on wet laboratory experimentation to collect data while the second half of the class has an increased time spent on bioinformatic analysis of your data and writing research reports. You have to have a computer to do bioinformatics and analyze your data in class. This course is designed to guide students through the basics of metagenomic approach to study association of a host and its microorganisms. Using an insect model, students examine the diversity of microbes associated with the insect gut and study the potential roles of microbiota in biology of the host (parasitism, commensalism, symbiosis, etc). Based on the preliminary data students formulate a hypothesis about metabolic interaction between the host and its microbiome and test their hypothesis using experimental or bioinformatics approaches. In this course, students learn methods of insect dissection, molecular barcoding, PCR, cloning, sequencing, and bioinformatics approaches to sequence comparison, phylogenetic analyses, and comparative genomics. Emphasis is given on reading scientific literature, formulation of scientific questions and hypotheses, experimental and bioinformatics methods of data analyses. No textbook is required. The required readings are recently published papers on the covered subject. They are listed in the course schedule and will be available through the courseweb. You will be provided with pages of written protocols for computational and experimental procedures and lecture notes. To keep all material organized; bring a three-hole binder to the class. Optional reading is Introduction to Bioinformatics by AM. Lesk (2008, Third edition. Oxford University Press). Class materials and updates will be posted on course web. You are expected to check regularly for lecture notes, protocols, assignments, announcements, and other material. Main communication with the class will be via CourseWeb announcements. Make sure you know how to get on CourseWeb: After log in on the main page you will have access to your My CourseWeb page where you find the link to the class. If you need help, contact computer help desk at HELP. Attendance is mandatory and there will be no make-up labs. Absence has to be properly excused and documented (a note from a doctor (illness) or a University official (University business)). A written excuse with acceptable documentation has to be submitted to the instructor before a planned absence or no latter than one week after the missed class. If the excuse is not submitted on time, you will obtain zero points for missed work and/or 5-10 points deduction from your grade. If you attend an interview, you need to arrange a make-up work with your instructor. It is your responsibility to communicate your absence with your instructor. Point penalties will be automatically used for unexcused absence. If you miss more than 20% of Dr. Swigonova Biosc 1285, p. 1 of 6

2 the classes you should discuss possible options available to you with your advisor or the CAS Dean s Office. Grading G grade Final grade will be based on scores obtained from assignments (20%), two midterm exams (30%), evaluation of laboratory notebook (10%), research reports (24%), and a panel discussion (or oral presentation) of the final project (16%). All coursework has to be completed to obtain a grade in this course. Final grade will be determined by the percentage of the total points you earn during the course. During the term you can estimate your grade by applying the following scale: % = A; 80-89% = B; 70-79% = C; 60-69% = D; less than 59% = F. There is no curve in this class, thus your grade is determined based on the percentage as specified above. Examination (30%) There will be two midterm examinations. Please check the class schedule to avoid any scheduling conflicts. First exam will be a practical exam covering experimental approaches introduced in the wet lab of the course. Second exam will cover methods used in bioinformatics. There will be no make-up exam except in special cases such as illness, severe personal trauma, and (rarely) University business. If you miss the exam and you do not have an acceptable excuse, you will receive score of 0 for that exam. Assignments (20%) there will be several assignments during the term that will assess your overall understanding of the experimental and analytical approaches covered in class. Some may be given in class (short quizzes); some may be given as take home assignments (reading assignments). Additional assignments may be introduced based on the class progress and needs. Laboratory notebook (10%) Each student has to maintain a laboratory notebook, where information about the bioinformatics data analyses, performed experiments, or any additional work has to be recoded in detail. Brief introduction to writing a laboratory notebook will be provided. Laboratory notebooks will be evaluated based on the quality of laboratory note taking. Research reports (24%) Each student is required to write three research reports. Some may be given as collaborative assignments while others may be result of individual work. The emphasis of the evaluation will be given on the understanding of applied concepts and data analyses and a detailed description of the research topic and background. Detailed grading rubric will be available on courseweb. Panel discussion/oral presentations (16%) Pannel discussion of final reports or Final Oral presentation will conclude the term, the choice depends on the overall class progress and timing at the end of the term. If you wish to petition for a G grade, you must submit a request for this change in writing and you must document your reason(s). You will be required to make arrangements, in person, for the specific tasks you must complete in order to remove the G grade. You will be expected to sign documentation describing the work that has to be completed and the due date. All required work must be completed by the specified date otherwise a zero will be assigned for the work and final grade will be determined using this score. Remember that G grades, according to CAS guidelines, are to be given only when students who have been attending a course and have been making regular progress are prevented by circumstances beyond their control from completing the course after it is too late to withdraw. If you miss the final exam, you may receive a G grade if the above conditions are met. Academic Integrity Students in this course will be expected to comply with the University of Pittsburgh s Policy on Academic Integrity Code ( Any student suspected of violating this obligation for any reason during the semester will be required to participate in the procedural process, initiated at the instructor level, as outlined in the University Guidelines on Academic Integrity. This may include, but is not limited to, the confiscation of the examination of any individual suspected of violating the code. Furthermore, no student may use any unauthorized materials during an exam, including notes, dictionaries, pagers, telephones, PDAs, programmable calculators, any devices that can connect to the internet. Dr. Swigonova Biosc 1285, p. 2 of 6

3 Special Accommodations Accessibility Communication Policy: Copyright notice Statement on classroom recording If you have a disability that requires special testing accommodations or other classroom modifications, you need to notify both the instructor and Disability Resources and Services ( no later than the second week of the term. You may be asked to provide documentation of your disability to determine the appropriateness of accommodations. To notify Disability Resources and Services, call (412) (Voice or TTD) to schedule an appointment. The Disability Resources and Services office is located in 140 William Pitt Union on the Oakland campus. Blackboard is ADA Compliant and has fully implemented the final accessibility standards for electronic and information technology covered by Section 508 of the Rehabilitation Act Amendments of Please note that, due to the flexibility provided in this product, it is possible for some material to inadvertently fall outside of these guidelines. Each student is issued a University address (username@pitt.edu) upon admittance. This address may be used by the University for official communication with students. Students are expected to read sent to this account on a regular basis. Failure to read and react to University communications in a timely manner does not absolve the student from knowing and complying with the content of the communications. The University provides an e- mail forwarding service that allows students to read their via other service providers (e.g., Hotmail, AOL, Yahoo). Students that choose to forward their from their pitt.edu address to another address do so at their own risk. If is lost as a result of forwarding, it does not absolve the student from responding to official communications sent to their University address. To forward sent to your University account, go to log into your account, click on Edit Forwarding Addresses, and follow the instructions on the page. Be sure to log out of your account when you have finished. (For the full Communication Policy, go to Blackboard is ADA Compliant and has fully implemented the final accessibility standards for electronic and information technology covered by Section 508 of the Rehabilitation Act Amendments of Please note that, due to the flexibility provided in this product, it is possible for some material to inadvertently fall outside of these guidelines. To ensure the free and open discussion of ideas, students may not record classroom lectures, discussion and/or activities without the advance written permission of the instructor, and any such recording properly approved in advance can be used solely for the student s own private use. Looking forward to a productive and successful semester, Dr. Swigonova Dr. Swigonova Biosc 1285, p. 3 of 6

4 BIOSC 1285(2164): Lab schedule All protocols will be available in Protocols folder on CourseWeb. Be sure to read any introductory and background material associated with the protocols (specified in class and posted on courseweb). You will be responsible for it! All reading assignments are due as hard copies at the beginning of class (check the dues dates specified for each assignment). WK Date Protocols & associated readings scheduled (Schedule will be modified based on class progress) 1 1/04 Course business. Course organization & expectations. Introduction to laboratory space. Lecture: Introduction to Insects. 2 1/09 1/11 Host basic anatomy and biology. Anatomical dissections & identification of body parts. Class discussion: Homology vs. homoplasy; Ants vs. termites Reading assignment: Life cycle, biology, and taxonomy of termites and ants (Borror D. J. et al. (1989), An introduction to the study of Insects, 6 th edition; pg OR pg ; handout) DNA isolation - Host & its microbiome (Protocol 1 & 5) DNA purity and quantification (Protocol 2) Assessment #1 (Quiz): Insect host anatomy & biology 1/16 No class Martin Luther King Jr. Day (add/drop ends on Wednesday, 1/18) /18 1/23 1/25 1/30 2/1 PCR amplification of microbial mtg 16S rdna (Protocol 3) Paper discussion: Rubin et al. (2014), DNA extraction protocols cause differences in 16S rrna amplicon sequencing efficiency but not in community profile composition or structure. MicrobiologyOpen, 3(6): Reading Assessment #1 (due): Rubin et al. (2014) answer 2 questions posted on courseweb. Gel visualization of microbial mtg 16S rdna PCR product (Protocol 4) Molecular barcoding: PCR amplification of host COI gene (Protocol 3) Paper discussion: Yun et al. (2014), Insect gut bacterial diversity determined by environmental Habitat, diet, developmental stage, and phylogeny of host. Appl. Environ. Microbiology 80(17): In this discussion we will focus on the main findings and interpretations in respect to microbial diversity within the host body. Reading Assessment #2 (due): Yun et al. (2014) answer 4 questions posted on coursweb. PCR amplification of microbial mtg 16S rdna (Protocol 3) set up samples with no dilution; 1/10 and 1/20 dilution Gel visualization of insect COI amplification (Protocol 4) PCR & Gel electrophoresis of PCR products (Protocol 4) Assessment #2 (Quiz): on laboratory experimentation (background & principles of DNA isolation, PCR, and gel electrophoresis) Paper discussion: Park DS, Suh SJ, Oh HW, and Herbert PD Recovery of the mitochondrial COI barcode region in diverse Hexapoda through trna-based primers. BMC Genomics. 11:423 PCR as needed: Molecular barcoding: PCR amplification of host COI gene (Protocol 3) Gel purification of mtg 16S rdna (Protocol 6, step 1-8); DNA purity & quantification (Protocol 7) Reading Assessment #3 (due): Park et al. (2010) answer 3 questions posted on courseweb due online by the start of the class 6 2/6 TOPO cloning and transformations of microbial 16S rdna PCR products (Protocol 7) Dr. Swigonova Biosc 1285, p. 4 of 6

5 /8 2/13 2/15 2/20 2/22 2/27 3/01 3/06 3/08 3/13 3/15 TOPO: Streak-purify transformants (Protocol 7.5) each group follows up on 18 transformants and one negative control (half-plate streak LB/Kan) Gel purification of COI gene PCR product (Protocol 6 you will use this protocol, since samples contain non-specific band) Reading: read TOPO cloning background (Protocol 7 the TOPO cloning manual full) Update your Laboratory notebook: All entries are dated, pages are numbered, results discussed and conclusions stated. All troubleshooting notes have to be included. Include the reasoning behind class switch to one host (termites). TOPO: Plasmid DNA isolation from transformants (Protocol 9) part 1: set up overnight cultures If applicable - Gel purification of COI gene PCR product (Protocol 6 part2) Assessment #4: LAB PRACTICAL part 1 (1 hour): written & wet lab: molecular barcoding, PCR & background on DNA isolation, PCR, gel electrophoresis, and TOPO cloning (be able to interpret results and suggest troubleshooting) Lab notebook for feedback (part1) TOPO: Plasmid DNA isolation from transformants (Protocol 9) part 2: isolate plasmid DNA (Protocol 9) Submission for sequencing: COI (Protocol 11) Assessment #4: LAB PRACTICAL part 2: gel electrophoresis Lab notebook for feedback (par2) TOPO: Screening of TOPO clones by restriction digest (Protocol 10) ± TOPO: part 1 set up overnight cultures (Protocol 9 part 1) Paper discussion: Husseneder, C. (2010). Symbiosis in subterranean termites: A review and insight from molecular studies. Envi. Ent. 39(2): Assessment #5: Husseneder (2010) answer 3 questions posted on courseweb; due by 8:30am Submission for sequencing: 16S rdnas (Protocol 11) submit 5 of your samples ± TOPO: part 1 plasmid isolation (Protocol 9 part 2) Paper discussion: Su et al. (2016), Comparative gut microbiomes of four species representing the higher and lower termites. J. Insect. Sci 16(1): 97 Assessment #6: Su et al. (2016) answer 3 questions posted on courseweb; due by 8:30am TOPO: Screening of TOPO clones by restriction digest (Protocol 10) Submission for sequencing: 16S rdnas (Protocol 11) submit another 5 of your samples Bioinformatics: How to proof read trace files (Protocol 12) Submission for sequencing: 16S rdnas (Protocol 11) submit last set of 5 of your samples Bioinformatics: Proof reading trace files and construction of consensus sequence (Protocol 12) Homework: Establish RDPII account (Protocol 13, pg.1) you need your account ready after spring break! Spring Break reenergize! Bioinformatics: Introduction to sequence alignment Analysis of termite microbiome RDPII database and taxon sampling (Protocol 13) Bioinformatics: Sequence alignment (NW & Smith-Waterman) Analysis of termite microbiome data analysis; RDPII database/blast Assessment #7: REPORT #1 Host life cycle and biology (guidelines posted on courseweb); due online today by 8:30am Dr. Swigonova Biosc 1285, p. 5 of 6

6 12 3/20 Bioinformatics: Multiple sequence alignment and molecular evolution Project: Analysis of termite microbiome Assessment #8 (Quiz): Sequence alignment 3/22 Project: Analysis of termite microbime 13 3/27 3/29 Complete analysis of termite microbiome Discussion of independent project Independent project Assessment #9: REPORT #2 Termite microbiome (guidelines on courseweb); due by 8:30am /03 Independent project 4/05 Independent project 4/10 Independent project 4/12 4/17 4/19 Independent project Assessment #10: Submit your lab notebook for grading. Assessment #11: FINAL EXAMINATION (take home exam) Free lab on bioinformatics: Independent project finish bioinformatics analyses FINAL EXAMINATION (take home exam due at the beginning of 8:30am) Clean up 20 mins Assessment #12: REPORT #3: Summary of independent project due as a hard copy in class Assessment: Oral presentations independent project Dr. Swigonova Biosc 1285, p. 6 of 6