12.1DNA sequencing and Genotyping Applications Flashcards
What sequencing methods does shotgun genotyping use?
next generation and some help from 3rd generation
How does shotgun sequencing work?
- 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
What are the problems with shotgun sequencing? what is used to address these issues?
- smaller pieces of DNA have repetitive DNA sequences
- 3rd generation (eg; nanopore) used to overcome this problem
What are the advantages of long reads in shotgun sequencing ?
- longer are more specific
- helps with the assembly and alignment of the shorter fragments
when a genome is first sequenced, how many times is it sequenced? Why are there so many copies?
- on average, hundreds-thousands of times
- greater number of copies ensures accuracy
what is sequencing depth?
- 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
What is the read depth of the first sequencing event? How about a repeat event?
- first sequencing event has depth several hundreds to thousands
- repeats: usually lower is satisfactory
What are the steps to transcriptonomics?
- 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
what sequencing methods does shotgun sequencing and transcriptonomics use?
- shotgun: next generation, and some 3rd gen
- transcroptonomics: next gen
Which DNA gene is used to identify animals?
- 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)
how was the evolutionary relationship of humans discovered?
- 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
how is next gen sequencing good for mixed up and degraded DNA?
- its good for detecting traces of ancient hybridization too
- because of the massively parallel genome sequencing
Describe studying microbiomes, which sequencing method is used?
- 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
Describe the use of genome sequencing in eDNA?
- 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
what are the 5 applications of DNA sequencing and which methods do they use?
- 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
why is it useful to study genetic variation at the molecular level?
- determine the genetic bases of disease/phenotypic traits
- identifying relatedness/ population mixture
- identifying species
- identifying criminals
Who invented DNA fingerprinting?
Alec Jeffries, 1980s
- invented DNA fingerprinting using minisatellites
What are minisatellites?
- 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)
Why was minisatellites tedious?
- it relied on tedious method of southern blotting, replaced by microsatellites
What are microsatellites?
- 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
Describe micro satellite genotyping?
- primers fluorescently labeled
- amplify products of different size
- separate products by electrophoresis
- genotypes of products identified by size of products
What do the bands of DNA fingerprint look like when there is co-dominance?
- co-dominance = 2 bands
- dominant/recessive = 1 band
Do micro satellite arrays differ between all people? what is the exception?
Twins have the same (because they share DNA copies)
What electrophoresis machines are used for micro satellite genotyping?
- capillary electrophoresis machines are used for dideoxy sequencing
- multiple microsatellites amplified at same time using primers labelled different colours (multiplex analysis)
What is multiplex analysis?
- when multiple micro satellites are amplified at the same time, and identified using different fluorescence primers
How many standard microsatellites are used in criminal forensics?
- 13 standard microsatellites are used: detect enough variability to distinguish individual humans (Except twins)
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
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
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
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
What are some examples of disease called by dysfunctional microsatellites?
- Huntington’s disease, myotonic dystrophy, fragile X syndrome
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)
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
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
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)
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
