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

Transcription

1 BIOSC 1285: GENOMICS LABORATORY Department of Biological Sciences University of Pittsburgh Faculty Zuzana Swigonova Ph.D. Office: A356 Langley Hall Phone: Office hours Office hours: by appointment You are encouraged to contact your instructor when you need help, and you are expected to do so to review your project. Many questions may be answered in class, your instructor to set up an appointment when you need more time to discuss your project, course material, or course related issues. Classes: meeting times & places Course overview Textbook CourseWeb Course policies Grading Bioinformatics classes: Hughes Computer Lab, 120 Clapp Hall Wet lab classes: 102 Clapp Hall This course is designed to guide students through the design and evaluation of a hypothesis driven research project in the area of comparative and functional genomics of bacteriophages. In the first half you will learn methods of sequence analysis and comparative genomics while working with genomes of recently sequenced bacteriophages. Based on the comparison of closely related bacteriophages you will propose hypotheses about which genes are or are not essential for phage survival and growth. In the second half you will perform a series of experiments in which you will test these hypotheses using novel recombineering techniques that generate phage gene knockouts and assess their survival and ability to propagate. 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. Optional reading is Introduction to Bioinformatics by AM. Lesk (2005, Second edition. Oxford University Press). Class materials and updates will be posted on course web. You are expected to check regularly for lecture notes, 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), a parent (family emergency), or a University official (University business)). A written excuse with acceptable documentation has to be submitted to the instructor no latter than one week after the missed class. If you miss more than 20% of the classes you should discuss possible options available to you with your advisor or the CAS Dean s Office. Final grade will be based on scores obtained from homework and assignemnts (20% each), two midterm exams (30%), evaluation of laboratory notebook (10%), final class project (30%), and a short (10 mins) presentation of the final project (10%). 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 Biosc 1285, p. 1 of 5

2 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 There will be two midterm examinations. Please check the class schedule to avoid any scheduling conflicts. First exam will cover basic concepts introduced in the comparative genomics part of the course and the background biology of phages, second midterm will focus on data interpretation and recombineering. 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. Homework There will be three homework assignments throughout the course. These will be due on the week specified in the class schedule. Assignments there will be several assignments during the term that will assess you overall understanding of the course objectives. Some may be given in class; some may be given as take home assignments. These are not planned in the course schedule and can be given at any time during the term. Laboratory notebook - 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. Class project Each student will be working on a class project throughout the semester. This will consist of (i) a hypothesis statement generated from comparative analyses and (ii) its experimental testing using phage recombineering techniques. Students are expected to write a research project summary consisting of an abstract, introduction, materials and methods, results, and discussion sections. Each project will be presented to the class in a brief (~10 mins) presentation. 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. G grade Academic Integrity Special Accommodations 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. 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. Violation of the Academic Integrity Code requires the instructor to submit an Academic Integrity Violation Report to the Dean. If you have a disability that requires special testing accommodations or other classroom modifications, you need to notify both the instructor and the Disability Resources and Services Biosc 1285, p. 2 of 5

3 no later than the 2nd 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 / (TTY) to schedule an appointment. The Office is located in 216 William Pitt Union. E- mail Communication Policy: Statement on classroom recording Each student is issued a University e- mail address (username@pitt.edu) upon admittance. This e- mail address may be used by the University for official communication with students. Students are expected to read e- mail 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 e- mail via other service providers (e.g., Hotmail, AOL, Yahoo). Students that choose to forward their e- mail from their pitt.edu address to another address do so at their own risk. If e- mail is lost as a result of forwarding, it does not absolve the student from responding to official communications sent to their University e- mail address. To forward e- mail 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 E- mail Communication Policy, go to 01.html.) 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. Biosc 1285, p. 3 of 5

4 COURSE SCHEDULE, SPRING 2012 W Date Activity / Assignments * 1 1/04, /09,10 1/11,12 Lecture 1: Phage biology, including dirt and phagehunting program Com. lab: NCBI databases, phagesdb.org; Introduction to lab notebook 1 (Reading: DNA Master annotation guide section 1, 3, 4) Lecture 2: Next generation sequencing Comp. lab: auto- annotation, glimmer/genemark intro to DNA Master (Reading: Pedulla et al 2003; DNA Master annotation guide sections 2, 6) Lecture 3: Genome architecture; keeping electronic notes Comp. lab: BLAST, Phamerator basic (Reading: DNA Master Annotation Guide section 5,7,8 through 8.4.2, 9 through 9.3) 1/16,17 No class Dr. Martin Luther King (University closed); 1/17 add/drop period ends 1/18,19 1/23,24 1/25,26 1/30,31 2/01,02 2/06,07 2/08,09 Comp. lab: Sequence alignments local vs global/ continue annotation refinements (Reading for week 4: DNA Master annotation Guide section 10) Comp. lab: Continue annotation Lecture 5: Functional assignments/ BLAST, Conserved Domains, HHPred Comp. lab: Continue annotation (Reading: DNA Master Annotation Guide, rest of section 8, 9) Lecture 6: trnas, frameshifts, parasitic elements; Com. Lab: continue gene assignments Lecture 7: Promoters, terminators, AttP sites, Comp. lab: Wrap up gene assignments for merge (homework 1 due: Genome Profile) Lecture 8: Analyze discrepancies as a class; (Reading: Hatfull et al. 2010) (homework 2 due: Merged genome discrepancies) Lecture 9: Comparative genomics and Clusters/DotPlots, Phamerator advanced functions, identifying genes of interest for research project (Reading: Pope et al PLoS One 2011) Lecture 10: Phylogeny Com. Lab: Compare genomes in DNA Master, Gene vs genome phylogenies, ANIs, gene content, Splitstree (Reading: Pace Microbiol Mol Biol Rev, 2009) 2/13,14 Comp. lab: Comparative Genomics computer time finish analyses 2/15,16 Lecture 12: Polymerase chain reaction; Intro to lab notebook 2 (wet lab) Comp. lab: Design PCR primers & deletion oligos (Homework 3 due: Comparative Genome Map; Research Project Hypothesis with supporting bioinformatics) (Reading: van Kessel & Hatfull, Methods Mol Biol. 2008) 2/20,21 MIDTERM EXAM 1 (full class) 2/22,23 Lecture 13: Recombineering Wet lab: Practice infection and plating (Reading: Marinelli et al, PLoS One 2008; van Kessel at al, Nat Rev Microbiol. 2008) 9 2/27,28 Wet lab: Check plates for plaques; PCR of deletion substrate Biosc 1285, p. 4 of 5

5 2/29,01 Lecture 14: Writing a scientific paper Wet Lab: PCR/visualization of deletion substrate End of Monitored Withdrawal (3/2) 10 3/4-11 Spring Recess (no classes) /12,13 Lecture 15: Transformations Wet lab: Deletion substrate clean up / PCR/visualization of deletion substrate 3/14,15 Wet Lab: Transformations / recovery / plating 3/19,20 Wet Lab: Transformations / recovery / plating / Plague picking 3/21,22 Wet lab: Transformations / Plaque picking / PCR screening for mutants (Homework 4 due: Recombineering) 3/26,27 Wet lab: Transformations / Plaque picking / PCR screening for mutants 3/28,29 Wet lab: Plaque picking / PCR screening for mutants 4/02,03 Wet lab: Plaque picking / PCR screening for mutants 4/04,05 Wet lab: Plaque picking / PCR screening for mutants / purification of mutants MIDTERM EXAM 2 (1 hour) 4/09,10 Wet lab: Transformations / Plaque picking / PCR screening for mutants 4/11,12 Wet lab: Finish experiments / phage mutant lysates due / lab cleanup 4/16,17 Finish presentations / review / discussion 4/18,19 Final Presentation during last week of classes *The class schedule is subject to revision as the term progresses. Any significant changes will be announced in class, posted on courseweb or an will be sent to the students. If you do not use pitt account, make sure that your pitt are forwarded to your account you use on a regular basis. PAPERS TO READ: 1. Pedulla Ml et al. Origins of highly mosaic mycobacteriophage genomes. Cell 2003, 113(2): Pope WH et al. Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4 PLoS ONE (10): e Hatfull GF et al. Comparative genomic analysis of 60 Mycobacteriophage genomes: genome clustering, gene acquisition, and gene size. JOURNAL OF MOLECULAR BIOLOGY Vol 397, Issue 1, Pages Pace NR. Mapping the tree of life: progress and prospects. Microbiol Mol Biol Rev Dec; 73(4): Marinelli LJ, Piuri M, Swigonová Z, Balachandran A, Oldfield LM, van Kessel JC, Hatfull GF. BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One. 2008;3(12):e van Kessel JC, Marinelli LJ, Hatfull GF. Recombineering mycobacteria and their phages. Nat Rev Microbiol Nov; 6(11): Review. 7. van Kessel JC, Hatfull GF. Mycobacterial recombineering. Methods Mol Biol. 2008; 435: Biosc 1285, p. 5 of 5