FSC350 Flashcards
(25 cards)
What is a read (1)
A small sequence of nucleotides that are a particular sequence from a fragment
What is the difference between read length and read depth (2)
Read depth is the amount of coverage a nucleotide has (how many times that nucleotide was sequenced)
Read length is how many nucleotides the fragment read is
What are the main differences between SBS and semi-conductor sequencing (4)
SBS uses terminators to block further polymerization (going one base at a time)
SBS terminators are fluorescent and indicate which nucleotide was added
SCS is pH mediated relying on hydrogen ions released during polymerization of DNA
Can record multiple nucleotides added at once as the value of pH change is theoretically proportional to the number of nucleotides added
What is coverage (1)
Read depth - average number of times a nucleotide was sequenced
What is the typical average threshold for read coverage (1)
40x
How does whole genome sequencing differ from targeted sequencing (4)
WGS covers the whole genome and typically has coverage of around 40x
Can seen large variants
Targeted targets specific regions within the genome and can have great coverage such as 500x
Cost effective
What is the difference between small and large whole genome sequencing (4)
Small used for sequencing genome of microbes <5mb
They mutate a lot so they can be very different from references which is why the whole genome is sequenced
Large uses for diseases and population studies >5mb
Provides high rez base by base view of genome
What are some applications for DNA sequencing (3)
D
What are some applications for RNA sequencing (4)
mRNA - analyze transcriptomes of disease states or biological processes
Total RNA - whole transcriptome analysis
Targeted RNA - sequence transcripts of interest
Small RNA - sequence small RNAs like microRNAs
What are some differences and similarities between single vs paired end sequencing
SES makes one pass at replicating the template dna
Can capture longer reads but likelihood of error increases
PES makes two passes at replicating the template, in either direction
Will capture shorter reads but if your target sequence is smaller you can have more confidence in your sequence
What are some examples where the entire genome could be sequenced in one read (2)
Bacteria or viruses
Advantages of whole genome sequencing (4)
Comprehensive view of whole genome
Can detect all types of mutations including structural variants
Standardized processing and analysis for all patients and all tumour types
Does not require any prior knowledge of the disease
Disadvantages of whole genome sequencing (5)
More expensive
Large dataset presents a challenge of data management, analysis and interpretation
Findings might not be actionable
Risk of accidental findings
Shallow sequencing less sensitive than targeted approaches
Advantages of whole exome sequencing (6)
About half the cost of whole genome sequencing
Small data set is easier to manage, analyze, and interpret
Standardized processing and analysis for all patients and all tumor types
Will detect indels, SNPs, and CNVs
Does not require any prior knowledge of disease
Provides deep sequencing with good sensitivity for rare clones
Disadvantages of whole exome sequencing (4)
Only 1.5% of genome is sequenced
May miss fusion genes and oncogenes
Findings may not be actionable
Risk of incidental findings
Advantages of targeted sequencing (4)
Cost effective
Results easy to interpret
Findings actionable for cancer-relevant genes
Very deep sequencing with very high sensitivity for rare clones
Disadvantages of targeted sequencing (3)
Will miss many mutations
Requires prior knowledge of genes of interest
Delays diagnosis of patients with rare diseases that are not represented on the panel
How can sequencing be used in commercial agriculture (3)
Genotyping for trait screening
Back crossing to move a single trait of interest from a donor parent to progeny
Animal traceability for GMO characterization
How can MPS be used for microbial genomics (3)
WGS - map novel or unfinished organisms
Detect strain specific mutations
Denovo sequencing for novel organisms
How can sequencing be used for reproductive health (4)
Figure out why miscarriages or failed IVF implantations
Embryo screening for correct number of chromosomes
Worries about inherited conditions
Non invasive prenatal tests
How can sequencing be used in translational research (5)
Understand diseases by identification, pathology, prognosis, treatment, and prevention
How can sequencing be used in oncology (9)
Cancer whole genome sequencing
Identify cancer specific variants in the tumour sample that are not in the normal sample
Targeted cancer sequencing
Solid tumors, hematology, germline, pan-cancer, immuno-oncology
Non-invasive biomarkers
Cancer exome sequencing
Coding mutations that contribute to tumour progression
Cancer rna sequencing
Gene expression changes in transcriptomes
Explain the steps to cluster generation in the ion torrent technology (2)
Fragments of the template dna and beads fall into a well
The template dna binds to the bead and amplifies until it covers the bead vja emulsion PCR
What is the point of adapters in MPS (1)
They are attached to the ends of the dna strands so that you can identify each sample from all the others in the reaction
