12.1DNA sequencing and Genotyping Applications Flashcards
1
Q
What sequencing methods does shotgun genotyping use?
A
next generation and some help from 3rd generation
2
Q
How does shotgun sequencing work?
A
- sheer genomic DNA into short pieces
- sequence by next generation
- assembler software looks for sequence overlaps to assemble them into larger fragments
- problems with repetitive DNA sequences solved by nanopore sequencing, which is longer and helps with alignment
3
Q
What are the problems with shotgun sequencing? what is used to address these issues?
A
- smaller pieces of DNA have repetitive DNA sequences
- 3rd generation (eg; nanopore) used to overcome this problem
4
Q
What are the advantages of long reads in shotgun sequencing ?
A
- longer are more specific
- helps with the assembly and alignment of the shorter fragments
5
Q
when a genome is first sequenced, how many times is it sequenced? Why are there so many copies?
A
- on average, hundreds-thousands of times
- greater number of copies ensures accuracy
6
Q
what is sequencing depth?
A
- the number of times a base is represented within in all reads
- greater read depth gives greater confidence a base is accurately read: known as base calling
7
Q
What is the read depth of the first sequencing event? How about a repeat event?
A
- first sequencing event has depth several hundreds to thousands
- repeats: usually lower is satisfactory
8
Q
What are the steps to transcriptonomics?
A
- isolate mRNA
- convert to cDNA
- sequence by next gen
- bioinformatic softward sequences into different genes
- the number of times the gene appears revealed how expressed the gene was in in that organism
9
Q
what sequencing methods does shotgun sequencing and transcriptonomics use?
A
- shotgun: next generation, and some 3rd gen
- transcroptonomics: next gen
10
Q
Which DNA gene is used to identify animals?
A
- mtDNA COI gene most widely used for identifying animal species (other genes used for plants/fungi)
- uses sanger sequencing (since its a small amount DNA)
11
Q
how was the evolutionary relationship of humans discovered?
A
- using mtDNA and sanger dideoxy sequencing
- used estimates of how fast mtDNA was evolving to date key events = molecular clock
- count the number of mutations that separate individuals or groups
found that all living humans have common ancestor: mitochondrial eve
12
Q
how is next gen sequencing good for mixed up and degraded DNA?
A
- its good for detecting traces of ancient hybridization too
- because of the massively parallel genome sequencing
13
Q
Describe studying microbiomes, which sequencing method is used?
A
- uses next gen
- microbiomes account for most of diversity on earth and a huge amount of biomass
- isolate DNA from environmental sample
- amplify microbial sequences using primers that amplify 16srDNA gene
- sequence using next gen (illumina)
- run data though data base to seed what species are present and abundance
14
Q
Describe the use of genome sequencing in eDNA?
A
- uses next gen and qPCR
- DNA can be isolated from environmental samples
- use appropriate primers DNA sequences amplified, then sequenced
- species present can be identified using speciesDNA databases
OR - using taxon specific primers and then qPCR to see if present
15
Q
what are the 5 applications of DNA sequencing and which methods do they use?
A
- shotgun: uses next gen + 3rd gen
- transcriptonomics: uses next gen
- species identification: sanger
- evolutionary history of humans: sanger, later next gen.
- eDNA: next gen and qPCR
16
Q
why is it useful to study genetic variation at the molecular level?
A
- determine the genetic bases of disease/phenotypic traits
- identifying relatedness/ population mixture
- identifying species
- identifying criminals
17
Q
Who invented DNA fingerprinting?
A
Alec Jeffries, 1980s
- invented DNA fingerprinting using minisatellites
18
Q
What are minisatellites?
A
- consist of ~10-1000 bp sequences that are repeated many times in tandem arrays
- mini satellite arrays have extremely high allelic variation due to frequent mutations (replication slippage/unequal crossing over)
19
Q
Why was minisatellites tedious?
A
- it relied on tedious method of southern blotting, replaced by microsatellites
20
Q
What are microsatellites?
A
- similar to minisatellites but have shorter arrays (<10, ~2-5 bp)
- shows much allelic variation due to slippage and unequal crossing over
- can be amplified using qPCR
21
Q
Describe micro satellite genotyping?
A
- primers fluorescently labeled
- amplify products of different size
- separate products by electrophoresis
- genotypes of products identified by size of products
22
Q
What do the bands of DNA fingerprint look like when there is co-dominance?
A
- co-dominance = 2 bands
- dominant/recessive = 1 band
23
Q
Do micro satellite arrays differ between all people? what is the exception?
A
Twins have the same (because they share DNA copies)
24
Q
What electrophoresis machines are used for micro satellite genotyping?
A
- capillary electrophoresis machines are used for dideoxy sequencing
- multiple microsatellites amplified at same time using primers labelled different colours (multiplex analysis)
25
What is multiplex analysis?
- when multiple micro satellites are amplified at the same time, and identified using different fluorescence primers
26
How many standard microsatellites are used in criminal forensics?
- 13 standard microsatellites are used: detect enough variability to distinguish individual humans (Except twins)
27
How much DNA does micro satellite require? aer there and problems associated?
PCR based micro satellite genotyping requires a small amount of DNA (perfect for criminal forensics)
- methods are very precise; contamination may be a problem
28
what forms of DNA can be used to establish identities in forensic analysis?
- microsatellites and mtDNA can be used to identify remains
- microsatellites can enable identification via kinship analysis
- materanal mtDNA can be used to establish close relationships via maternal lineages
29
Do any microsatellites cause genetic disorders?
- most microsatellites have no impact on health: selectively neutral
- occur outside of exon
- in humans however a few cause disease: trinucleotide repeats within genes/important DNA sequences
30
Describe the difference between healthy and affected individuals regarding micro satellite mutations
- all humans have these mutations, but in healthy: allele means repeats are small
- genetic disorder people allele with genes with too many repeats : = abnormal proteins
31
What are some examples of disease called by dysfunctional microsatellites?
- Huntington's disease, myotonic dystrophy, fragile X syndrome
32
describe the impacts of mutations on restriction endonuclease sites?
- mutations can either destroy or create restriction endonuclease sites
- gain or loss of these sites can be detected using gel electrophoresis
- restriction site polymorphisms are most commonly caused by single nucleotide polymorphisms (SNPS)
33
What are SNPs?
- caused by single base mutations are the most common genetic variations
- SNPS occurs every 800-1000 bp in human DNA
- average human differs from the reference genome at 4-5 M sites
- usually di-allelic (A or G at particular position)
- SNPS close others on a chromosome are normally inherited together forming a haplotype
34
What is a haplotype?
- when SNPs are close together on a chromosome they are normally inherited together forming a haplotpye
- an arbitrarily long stretch of DNA characterized by particular alleles at the SNP positions in that sequence
35
what are SNP chips?
- AKA microarrays, designed to allow many SNPs to be genotyped at once
- used DNA hybridization baed assay to determine genotypes at known snps
- general method of choice for rapid screening (will be supplanted by super cheap genome sequencing)
36
What are Genome wide associations?
- GWAs
- aim to find genetic links to disease/traits
- looks for SNPS that have alleles correlated with presence of disease/trait
- many gene influences by environment / many genes: must surgery many genes and many individuals to find a link
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