GEGE2x01 Genetics and Genomics LECTURE 15: Writing your prac report Writing Writing your prac report Gene mapping in Bactrocera tryoni GEGE2x01 Genetics and Genomics LECTURE 15 Dr Emily Remnant [email protected] Lecture 15 Writing your prac report • Refresh what we did in Week 3-4 prac Background, experimental setup and results • Report format Each section explained • Assessment criteria How the marking criteria relate to the sections of the report • Question time Opportunity to ask for direction, not answers! Here’s what we’ll cover today: To ask questions throughout, go to www.menti.com and use the code 56 28 67 Practical report assessment task • 25% of final grade. Due October 11th (Canvas) • Report guidelines: Canvas > Assessments > Practical report • Report sections, info & marking criteria Gene mapping with Molecular Markers What’s the purpose of a scientific report? To communicate the results of an experiment clearly and succinctly To ask questions throughout, go to www.menti.com and use the code 56 28 67 What’s the purpose of a scientific report? • To do this you need to: 1. Put the experiment into a biological context • What is the question? Why is it important? What has been done previously? 2. Describe how the experiment was performed • The key details required for someone else to independently repeat the experiment 3. Explain the results obtained • A factual account of your findings, clearly laid out using text, tables and figures 4. Discuss the significance, implications and limitations of the results • Synthesise your results back into the original context and tie in with future directions …in a language that is clear, accurate and scientifically appropriate To communicate the results of an experiment clearly and succinctly Background • The Queensland fruit fly or Q-fly, Bactrocera tryoni Order: Diptera Family: Tephritidae … etc. To ask questions throughout, go to www.menti.com and use the code 56 28 67 Background • https://www.youtube.com/watch?v=qxfdtr1BMxE • *PLEASE NOTE: while tiny sensors on the backs of fruit flies are awesomely cool, the particular strategy in this video is not necessarily relevant to our study • For YOUR report, think about: • Why we need genetic markers, why we need to understand more about the genetics of Bactrocera tryoni • Novel and relevant genetic techniques that could be employed to help control this pest The Experiment • Use molecular and phenotypic markers for linkage mapping in Bactrocera tryoni • Molecular markers • RFLP: restriction fragment length polymorphism • Restriction site in PCR product from white gene • Microsatellites: short repeat sequences, with short and long alleles • Data from multiple microsatellites as shown Table 3, pg 12 • Phenotypic marker : white marks • Determine if the two genes white and white marks are linked, and on which chromosomes they are located To ask questions throughout, go to www.menti.com and use the code 56 28 67 Phenotypic and molecular markers white marks wm+ Ra and Rb See also the Figure 3 schematic, page 4 of lab notes white RFLP 680bp Ra 130bp 360bp 190bp RsaI RsaI Rb 130bp 550bp 680bp RsaI Male and Female Backcross • A test cross is done by crossing a heterozygote with a double-recessive individual: AaBb X aabb • A backcross is when the double-recessive has the same genotype as original parent (cross ‘back’ to parental genotype) • Female Backcross: heterozygous female crossed to double-recessive male AaBb♀ X aabb♂ • Male Backcross: double-recessive female crossed to heterozygous male aabb♀ X AaBb♂ To ask questions throughout, go to www.menti.com and use the code 56 28 67 Male Backcross in B. tryoni • Like Drosophila, B. tryoni males do not have recombination during meiosis • A male backcross* can be used to determine if genes are on the same or different chromosomes • *double-recessive female crossed to heterozygous male Parents wm Rb wm Rb ♂♀ X wm+ Ra wm+ Ra P2- Female: homozygous mutant for white marks and Rb RFLP allele P1- Male: homozygous for wild type and Ra RFLP allele Male Backcross in B. tryoni F1: *G2: ♂ Expected ratios: Phenotype Genotype Unlinked Linked white marks, b b wm Rbwm Rb 1 1 white marks, a b wm Rawm Rb 1 0 wild type, b b wm+ Rbwm Rb 1 0 wild type, a b wm+ Rawm Rb 1 1 P: Xwm Rb wm Rb ♀ wm+ Ra wm+ Ra ♂ wm+ Ra wm Rb * G2: different from F2, (F2 are specifically offspring from an F1 x F1 cross) To ask questions throughout, go to www.menti.com and use the code 56 28 67 Outcomes of B. tryoni Male Backcross Rawm+ wm Rb wm+ Rb wm Ra 1 wm+ Ra 1 wm+ Rb 1 wm Ra 1 wm Rb Rawm+ wm Rb 2 genes; Different chromosomes; Unlinked: Independent assortment Four types of gametes with parental and non- parental phenotypes wm+ Ra wm Rb Rawm+ wm Rb wm+ Rb wm Ra 2 genes; Same chromosome; Linked: No Recombination in males 1 wm+ Ra 0 wm+ Rb 0 wm Ra 1 wm Rb 2 types of gametes with parental phenotypes only Linked Unlinked à Male backcross: Great way for mapping genes to chromosomes with limited offspring What is happening in the F1 (heterozygous) male? ♂ wm+ Ra wm Rb How did we determine if white marks and white are linked? Our strategy: • DNA was provided from a male backcross: • Parent 1 (male, wild-type, Ra Ra) • Parent 2 (female, white marks, Rb Rb) • F1 (male, wild-type, Ra Rb) • Sixteen G2 offspring (F1 male x female P) • Each with known phenotype at white marks but unknown RFLP genotype at white gene • PCR of the white gene (lab notes, p6-7) • Restriction enzyme digest of PCR product with RsaI (p8) • Gel electrophoresis of uncut and cut PCR products (p9) Gel electrophoresis results: • Size standards: known fragment sizes shown in Figure 5, pg 16 • Use size standards to estimate your sample RFLP fragment sizes, based on the expected band sizes- are they similar to expected? • Fill out Table 3 (pg 12) with RFLP ‘phenotypes’ (AB or BB) ♀P♂P F1 G2-1 G2-2 G2-3 G2-4 G2-5 G2-6 G2-7 G2-8 G2-9 1kbpUC19/ HpaII 250bp 500bp 750bp 1000bp 489bp 404bp 501bp 331bp 242bp 190bp 147bp 110bp multiple bands (2000-10000bp) To ask questions throughout, go to www.menti.com and use the code 56 28 67 Table 3: Microsatellite data/chromosomes S = short allele L = long allele Bt1 = a microsatellite marker known to be located on B. tryoni chromosome 2 Bt2 = chromosome 5 Bt5 = chromosome 3 … etc Microsatellite question: Prelab exercises (p3): Each microsatellite (Bt1, Bt2, etc.) will have a similar format, with short and long alleles segregating in the offspring Note the difference between prelab question (F2 offspring from F1xF1 intercross) vs our results (G2 offspring from male backcross) To ask questions throughout, go to www.menti.com and use the code 56 28 67 Same or different chromosomes? Lwm+ wm S wm+ S wm L 1 wm+ L 1 wm+ S 1 wm L 1 wm S Lwm+ wm S 2 genes; Different chromosomes; Unlinked: Independent assortment Four types of gametes with parental and non- parental phenotypes wm+ L wm S Lwm+ wm S wm+ S wm L 2 genes; Same chromosome; Linked: No Recombination in males 1 wm+ L 0 wm+ S 0 wm L 1 wm S Two types of gametes with parental phenotypes only Linked Unlinked à Linkage analysis: Examine segregation of your genes with the microsatellite markers Any questions so far? To ask questions throughout, go to www.menti.com and use the code 56 28 67 Report format • Scientific report similar to a research journal article • 1500 words of written text, plus figures, tables, references, and supplementary information • Contains the sections: • Introduction • Methods • Results • Discussion • Supplementary Read some journal articles • To help you get used to the ‘scientific voice’- how scientists write • Note: you are not required to write an abstract in your report • Journal: Biology letters: short-format, brief articles. Introduction • Include a relevant, informative title for your article • Not just the generic name of the prac • Approximately 2-3 written paragraphs to set the scene of the r
eport • Describe the background and put the experiment into context. Think about the big picture: • Study organism- significance: why do we want to understand this species? • Describe molecular markers, how their use has aided in genetic mapping • Include references to support your statements • State the aims of the experiment. To ask questions throughout, go to www.menti.com and use the code 56 28 67 Introduction: some tips • The introduction moves from the general to the specific in 2-3 paragraphs and should cover the following areas: • Identify the main context and draw on background literature to summarise key points of previous related research • Explain: ‘why this study?’; ‘what are the aims?’ • Think carefully about what to include and what to leave out • Choose relevant background to the topic – no need to go into a history of genetic mapping, or to assure us of the benefits of human genome sequencing • Cover briefly what molecular markers are and their uses, not a complete theoretical background Methods • Describe the experiment • What cross was set up, which samples were examined? • Which molecular techniques were used? Which analytical/statistical techniques? • Write as a paragraph, not a protocol, briefly describing the key information so it can be understood/repeated. • Include necessary details • concentrations of reagents, temperatures, incubation times • Don’t include unnecessary information that is obvious • no need to say that you used PCR tubes, or equipment that is implied. To ask questions throughout, go to www.menti.com and use the code 56 28 67 Methods: some tips DON’T: Write it like a protocol, recipe or set of instructions: • “First, put 10ul of buffer into a PCR tube, then add ….” DO: Write a brief paragraph, essential details only • “The PCR reaction contained…” DON’T: State only the volumes used • “The PCR reaction contained 2µl of PCR buffer” DO: Include reagents used at their final concentration in the reaction • “The PCR reaction contained 67mM Tris (pH 8), 16.6mM[NH4]SO4 … etc” Which one sounds more like a journal article? • Take 1.5ml of bacterial cells and put into an eppendorf tube. • Centrifuge for 5 minutes until a pellet is formed, then pour off the supernatant into the waste bins provided. • Next, add 1ml of sterile minimal media to the pellet and vortex to resuspend. • Repeat the above steps, resuspending in 0.5ml of media the second time • Take four agar plates and streak out bacterial suspensions using a sterile swab… Bacterial cells from four E. coli strains (wild type, mutants 1, 2 and 3) were pelleted by centrifugation from 1.5 ml liquid culture. Cells were rinsed in 1 ml of sterile minimal media and re-centrifuged, followed by resuspension in 0.5 ml of sterile media. Swabs of each strain were plated onto four sterile minimal medium L-agar plates, including three plates that had each been supplemented with a different component of the tryptophan biosynthetic pathway (tryptophan, indole or anthranillic acid). Plates were incubated at 37°C for 24 hours. Option 1: Option 2: Discussion board question about methods: • Q: “In explaining the PCR and gel methods, I was unable to determine a few concentrations such as concentration of template DNA (which was reported as 3 uL in the notes) as well as the concentration of white PCR product used in the restriction digest. Would it be acceptable to report just the volumes used?” • A: Yes- absolutely. We have not provided concentrations of the DNA template used in the PCR, and we did not measure the DNA concentrations of our white PCR product, so it is acceptable to report the volume used in this instance. Discussion board question about methods: • Q: “How do we display the genetic cross? Should we make a diagram similar to the one in the lab notes or just summarise with words?” • A: Up to you! There is often not one single way to represent each element of the report, and this is a good example. Think about the clearest way to represent the information. • Note that using figures taken directly from the lab notes is discouraged- you always need to adapt a diagram in your own way, using your own words. Any other questions so far? To ask questions throughout, go to www.menti.com and use the code 56 28 67 Results • Clear, precise description of your findings that includes: • data presentation such as figures and tables • linked to a verbal summary of your findings • This section describes but does not explain your results. • It provides a factual account of your findings, for example: outcomes and data produced as well as any analysis and tests performed on the data, described clearly (but the implications are not discussed) To ask questions throughout, go to www.menti.com and use the code 56 28 67 Results: some tips • Include a written paragraph detailing the results obtained • Use data from your group where possible. If data is missing, observe from other groups. Check all results against ‘perfect cut gel’. • Exercise 5, questions 1-4* on page 11 of the lab notes provides a good guideline of what should appear in the results • * don’t write the question followed by the answer; incorporate your answers into a written paragraph • Present a summary of the results in a concise table • Refer to all tables/figures that you include somewhere throughout the text • Include the analysis of how you determined linkage • Statistically test your hypothesis and include in results • Non-essential figures/tables can be placed in the Supplementary Discussion board question about Results: • Q: “Processed results for linkage analysis – is there a specific format for this results table or is this up to us? Would this table simply summarise the linkage relationships or also show how we were able to deduce those relationships? ” • A: The format is again up to you. The table can summarise the linkage relationships, though I would recommend explaining somewhere (perhaps in an accompanying paragraph) how you deduced the linkage relationships. If you can find a way to include this information in the table, that is fine too. Discussion board question about Results vs Discussion: • Q: “Should we be interpreting the results in the Results section or is that reserved for the Discussion section? For example, instead of just stating there was an x # of bands in a lane and their respective sizes, can we interpret these results and say which allele they represent in the ‘Results’ section? I’m a bit unclear what extent of analysis should be in the Results and the Discussion sections.” • A: Results include analysis of the data, so in this case you would include the number of bands, AND which allele they represent in results. You would also include your linkage analysis to determine which chromosome the genes are on. Discussion • An informed commentary based on your actual results • Relates back to your introduction and the key context/background literature/ related research • Tells the reader what your results mean in terms of the context, background and aims. • To enable this you must include • Key points of your results and discussion of how they allowed you to achieve your aim • Limitations of your results, e.g. what you would do differently if you revisited the research and/or what you would explore further • Suggestions for future research in this area • A final summary of the main findings and why they are important To ask questions throughout, go to www.menti.com and use the code 56 28 67 Discussion: some tips • Demonstrate a clear understanding and interpretation of the results and their biological significance • Exercise 5, questions 5-6, pg11 provides some important discussion points that should be considered • Consider limitations and future directions • Could you improve the methodology used? • Think about which modern techniques are now available that could complement this research. • What would you
do next to increase our understanding of the genetics of B. tryoni? • Finish with a brief conclusion to the report bringing it back to the aims References • Both the Introduction and Discussion are opportunities to integrate your report with the literature. • You should cite at least 2-3 relevant primary references that support any factual statements that you make about previous work, or provide inspiration for techniques that you propose as future directions • Citing the lab manual: Generally not encouraged unless the information does not appear elsewhere (eg. something specific about our methods) • Author-date referencing system: • Pleska, M. and Guet, C.C. (2017). Effects of mutations in phage restriction sites during escape from restriction-modification. Biol Lett 13(12): 20170646 To ask questions throughout, go to www.menti.com and use the code 56 28 67 Supplementary section • Comes at the end of the report • Contains information that you used to determine the results, but are too detailed to include in the results • Raw data such as • Labelled gel photos • Table recording approximate band sizes for each genotype • Large table where you recorded all the results (eg. a completed Table 3 from lab notes) To ask questions throughout, go to www.menti.com and use the code 56 28 67 Discussion board question about Supplementary: • Q: “In reference to “summary of the banding pattern for each individual” in the Supplementary section, is this referring to including a table like Table 3 from our lab notes?” • A: You can certainly include a summary of the gel banding patterns within a similar table to Table 3, which I would consider to be a record of the raw data, and too large to be included in the Results section. Any more questions? To ask questions throughout, go to www.menti.com and use the code 56 28 67 Assessment criteria • Context (20%): Place the experiment into context in the introduction and throughout the report. Provide relevant background information on the study organism, the techniques used and the aims. • Technical accuracy (25%): Presentation of the methods and results in a technically accurate way, including tables, figures and Supplementary information. • Understanding, interpretation and extension (40%): Demonstrate understanding and interpretation of the results, and discussion of the implications, relevance, and suggestions of future directions/experiments. • Presentation (15%): Overall clarity of presentation, appropriate scientific style, good use of English and appropriate use of references. Context (20%) • Largely represented by the Introduction, also elements of the Discussion • Description of the relevance and importance of the study • Explanation of key points in a clear and concise way • Lack of irrelevant information • Source information is appropriately referenced To ask questions throughout, go to www.menti.com and use the code 56 28 67 Technical accuracy (25%) • Largely represented by the Methods description, also includes presentation of the Results (and Supplementary) • Correct, scientific description of the Methods used • Accurate, concise representation of all necessary results and analysis • Figures and tables presented clearly and labelled correctly, with figure legends and table headings • Inclusion of raw data in the supplementary section • (the point above still applies for supplementary- figures/tables labelled etc…) To ask questions throughout, go to www.menti.com and use the code 56 28 67 Understanding, interpretation, extension (40%) • Largely represented by the analysis, understanding and interpretation of Results and Discussion. • Analysis of results provided the correct biological explanation (Did you get the ‘correct’ result?) • Adequate coverage of questions from Exercise 5, pg 11of the lab notes and include a discussion of these points in your results and discussion section • Consideration of the implications, relevance, and suggestions of future directions/experiments. • Extend from this study to suggest ways of expanding on the results, using modern techniques Presentation (15%) • Clarity of writing throughout the report • Correct use of genetic nomenclature and scientific style – not too wordy, clear and concise, information is easy to interpret from what has been written • Overall layout • Ordering of sections • Information placed in correct sections • Placement of figures/tables in a linear order, integrated with written paragraphs • Spelling, grammar, and correct use of english • Choice of relevant references that have been correctly cited and referred to within the report • Adherence to word count To ask questions throughout, go to www.menti.com and use the code 56 28 67 Questions? • See Canvas for Report Guidelines > Assignments > Practical Report • Discussion Board for any other questions To ask questions throughout, go to www.menti.com and use the code 56 28 67 External report writing tools • Help with structure, scientific language, general guidelines for each section* of the report, with interactive questions and examples: http://learningcentre.usyd.edu.au/wrise/biochemistry3/overall_structure/os _structure.html • work through each screen by clicking ‘next’ • *Note: we don’t require an Abstract/Summary in your report
