Microsoft Word - BIOL2499_BIOL XXXXXXXXXXPractical Report 1 Assessment details.docx
BIOL2499/BIOL XXXXXXXXXXAssessment: Practical Report 1
(Comparative genomics using Oxford nanopore next-generation DNA sequencing)
Assessment details – Course: Systems Biology
Assessment
task and
purpose
To provide an opportunity for students to demonstrate technical ability in the critical analysis and
interpretation of omics data.
Course
learning
outcomes
(CLO)
This assessment relates to the following CLOs
1. Demonstrate theoretical and technical understanding of Systems Biology (Omics) approaches
to the study of biological systems from the level of molecules to cells, tissues, organisms and
populations.
2. Critically analyse, interpret and explain omics data.
3. Summarise and synthesize and evaluate primary scientific literature.
4. Communicate clearly and effectively using co
ect scientific language and conventions.
Assessment
description /
equirements
Working as an individual follow the demonstration in Collaborate Ultra and use the instructions in the
practical manual to complete the practical activities in weeks 2-5. Include the results of the activities and
complete the questions relating to the assessment criteria in the report.
This assessment will demonstrate a capacity to evaluate complex ideas and concepts in research of
omics datasets.
Assessment
type &
weighting
This is a summative assessment. This assessment is worth 25% of the total mark for this course.
Assessment
submission
details
Deadline: Midnight Sunday the 8h of May
Method: Submit the assessment onto CANVAS by the due date. The file format should be Word or pdf.
Maximum 4000 words (not including references and figure legends).
Grading /
performance
standards
A ru
ic is available on CANVAS. It outlines the grading and performance standards. Students are
encouraged to read the ru
ic in conjunction with the assessment description. The Discussion Board
provides opportunities for Q&A about grading and performance throughout the assessment period.
Assessment
preparation/
Resources
https:
usegalaxy.org.au/
https:
last.ncbi.nlm.nih.gov/Blast.cgi
http:
www.softbe
y.com
e
y.phtml?topic=fgenesh&group=programs&subgroup=gfind
https:
web.expasy.org/translate/
https:
web.expasy.org/sim/
https:
fungidb.org/fungid
Assessment
feedback
A ru
ic will be used to grade individual performance. Assessment feedback will be provided to
individuals on CANVAS at the conclusion of the marking period. Students can ask for input (feedback)
throughout the assessment period using the Discussion Board’s Q&A. In addition, at the conclusion of
the marking period, video feedback addressing the strengths and weaknesses of all students’
performance (collective feedback) will be provided.
Indicators of
authentic and
lended
assessment
Authentic in this assessment
• Relates to world-wide trends e.g. commonly used techniques in systems
iology
• Requires students to use technology in a practical context
Blended in this assessment
• Digital technology
Microsoft Word - BIOL2499:BIOL XXXXXXXXXXPractical Report 1 Assessment Guidelines.docx
BIOL2499/BIOL2512 Systems Biology – Practical Report 1
1
BIOL2499/BIOL2512 Practical Report 1
Use your results from Practical Activities from weeks 2-5 to complete this assessment using a
standard practical report structure: Introduction (
ief), Aim, Results, Methods (just refer to
the practical manual in this section) and Discussion. The focus should be on the results and
discussion sections as these are worth the most in the marking ru
ic.
Maximum 4000 words (not including references and figure legends)
Include in the report:
Introduction (
ief) and aim
Include the information relevant to understanding the context of the practical activities and
the aim. Use primary references when information is supplied from an external source (this
can be the practical manual)
Results
Wet lab practical session/ Bioinformatics practical activity 1: The fluconazole MIC (in
µg/mL) of the C. neoformans wildtype and the mutant determined by E test.
Bioinformatics practical activity 1: The FastQC analysis that you generated in Galaxy. Using
these results, describe the quality of your next generation sequencing data.
Bioinformatics practical activity 2: Include the results of the variants that you have identified
in Galaxy. Describe how many sequence variants you identified after sorting and discuss how
confident are you on these results.
Bioinformatics practical activity 3:
• Include the results of your nucleotide BLAST and blastx. Describe what the results
mean.
• The ID and name of the gene identified (e.g. CNAG_00682 kinesin)
• Include an image of your gene structure generated using Softbe
y FGENESH in your
eport. Describe how many exons the gene has.
• Include an image (screenshot) of your protein alignment in your report. Describe the
molecular consequences of the mutation that you have identified.
Methods
Refer to the practical manual in this section and just state any changes that were made.
Discussion: Use your results and the information in fungiDB from omic experimental
approaches to provide evidence for a role of this gene in antifungal drug resistance.
Bioinformatics Practical
Activity 1
Nanopore genome sequencing
Introduction
This practical activity is designed to introduce the tools, data types and workflow of
nanopore next-generation sequencing of genomes. In this practical activity you will
e interpreting the various file formats used in storing reads, assessing the
experimental output and performing quality control of the sequencing reads.
Learning objectives
At the end of this activity you will be able to:
• explain the two main steps of rapid li
ary preparation and the theory of nanopore
next-generation genome sequencing
• identify and interpret the file formats used in storing read data
• calculate the overall coverage of a sequencing experiment using the key figures
from nanopore sequencing raw data
• interpret the read length histogram from a nanopore sequencing experiment
• perform quality control on sequencing reads (adapter trimming)
• interpret FastQC read quality reports
Cryptococcus neoformans
India Ink stain of polysaccharide
capsule of C. neoformans
C. neoformans grown on Sabaroud medium
• C. neoformans is a fungus with a worldwide distribution and is associated with
soil and avian habitats
• C. neoformans infection (Cryptococcosis) occurs in severely
immunocompromised individuals (eg. advanced HIV/AIDS)
• Soft, creamy colonies in 3-5 days culture on SAB at 37oC and then colonies
ecome mucoid and creamy to tan.
Phenotype of the C. neoformans mutant
Fluconazole
susceptibility
using E-test
Wild type Mutant
• The aim of the practical is to investigate the genetic determinants of antifungal
drug resistance in Cryptococcus neoformans. This will be achieved using Oxford
Nanopore next generation DNA sequencing and comparative genomics to
identify the mutation/s causing the phenotype of a C. neoformans mutant.
Aim
Isolate C. neoformans genomic DNA
Next Generation Sequencing li
ary
Whole genome sequencing
Genome assembly and variant calling
Molecular consequences and biological significance
Laboratory Practical Activity 1
Bioinformatics
Practical Activities
1-3
Nanopore Sequencing
Introduction to Nanopore Sequencing:
https:
www.youtube.com/watch?v=qzusVw4Dp8w
Q1. How is the sequence of the DNA determined using nanopore technology?
a) Single bases are detected as they are incorporated into growing DNA strands
) DNA moving through the nanopores causes disruption of the electrical cu
ent
c) The release of a H+ ion as a base is incorporated will decrease the pH
d) A fluorescent label on the terminal phosphate of the dinucleotides can be detected
Nanopore Sequencing
Nanopore Sequencing
Q2. During sequencing the nanopore analyzes the entire fragment of
DNA or RNA that is presented to it. Therefore the read length is:
a) always the same
) the same as other next-generation sequencing techniques such as Illumina
c) directly related to the length of the DNA or RNA in the sample
d) determined by the amount of cu
ent passing through the nanopore.
Nanopore Sequencing
Q3. Oxford Nanopore next generation genome sequencing generates reads
which are:
a) Short reads of 150 bp
) Short reads of 1 k
c) Long reads of 10-15 k
d) Long reads where all reads are greater than 10 k
e) Reads of varying size depending on the size of the genomic DNA fragment
Nanopore Sequencing
Q4. In Nanopore sequencing DNA and RNA are sequenced directly rather
than through a copy or synthetic strand. What is an advantage of this?
Nanopore Sequencing
Nanopore Sequencing:
https:
www.youtube.com/watch?v=sv9fFeSd3kE
Q5. A motor protein is added to the end of DNA molecules during the li
ary
preparation prior to sequencing. What is the function of the motor protein?
a) To control the speed of the DNA or RNA molecule passing through the nanopore
) To speed up the DNA passing through the nanopore
c) To read the changes in electrical signals as the DNA passes through the nanopore
d) To keep the double stranded DNA molecule intact as it passes through the nanopore
Oxford Nanopore Li
ary preparation
Rapid Adapter
(RAP)
Fragmentation
mix
Q6. What is the role of the barcodes in nanopore li
ary preparation?
a) They allow the DNA to pass through the nanopore
) They allow the sequencing reads to be assigned to a specific chromosome
c) Barcodes cause disruption of the electrical cu
ent
d) To allow multiplexing, where multiple samples can be sequenced simultaneously
Oxford Nanopore Li
ary preparation
Q7. Why do sequencing adapters have to be attached to the DNA?
a) They allow single bases to be detected as they are incorporated into
growing DNA strands
) They are loaded with motor protein to allow the DNA to pass through the
nanopore
c) The adapters are used to determine the origin of the genomic DNA
sample
d) They allow DNA Polymerase to attach to the DNA
Oxford Nanopore Li
ary preparation
The MinION device and flow cell
Priming and loading the MinION Flow Cell
The sequencing results from your experiment
The MinKNOW software is used to start the sequencing experiment, terminate the
experiment after completion and create a base-called sequence file.
Q8. Why does Nanopore result in
less sequencing reads than Illumina
sequencing?
a) Simultaneous parallel sequencing
cannot occur in Nanopore
) Only one DNA strand is sequenced
at a time in Nanopore sequencing
c) The Nanopore sequencing reads are
longer than Illumina reads
d) Illumina is newer technology
Summary
of raw data:
The MinKNOW software is used to start the sequencing experiment, terminate the
experiment after completion and create a base-called sequence file.
Q9. What does coverage mean in
elation to genome sequencing?
a) The number of sequences which cover
a position in the genome
) How the heterochromatic DNA is
covered in histone proteins
c) How big the genome sequence is
d) How many sequencing reads were
performed in an experiment
Summary
of raw data:
The sequencing results from your experiment
The MinKNOW software is used to start the sequencing experiment, terminate the
experiment after completion and create a base-called sequence file.
The C. neoformans genome is 19 Mbases
Q10. Using the Total Yield, work out
the overall genome coverage of your