Sorefan Flashcards
(54 cards)
1
Q
What is whole genome sequencing?
A
- complete genome seq of organism at single time
- inc seq of chromosomal DNA and mito/chloro etc DNA
2
Q
What are the challenges for genome sequencing?
A
- NA extraction from cells –> needs high quality and conc
- fragmentation
- sub-fractionation size selection –> to isolate fragments of correct size
- separating indiv molecules
- amplification of signal
- reading signal
- data analysis
3
Q
What were the 3 phases of human genome project?
A
- genetic and physical maps of human and mouse, seq yeast and worm
- -> technology dev
- draft seq –> inc many gaps and errors
- finished seq –> fill in gaps and correcting errors
4
Q
How are genetic maps made?
A
- analyse genetic distance between genes by measuring recombination freq
- markers rely on variation of seq between parents and individuals
- distance measured in centimorgans
- mostly PCR based, eg. polymorphisms in genes and DNA markers
- linkage map by looking at relative distances of 2 or more polymorphic genes and measuring RFs
- DNA markers superseded phenotypic markers
- DNA based mol markers could be RFLPs
- -> methods to analyse are slow so moved onto using SSLPs as easy to analyse w/ PCR
5
Q
What are SSLPs?
A
- simple seq length polymophisms
- repeat regions in genome that vary in length between pops
- usually mini and microsatellite seqs
6
Q
What are minisatellites?
A
- repeat units up to 25bp
- not spread evenly around genome, mostly at telomeric regions
- several kb long
- difficult to PCR
7
Q
What are microsatellites?
A
- usually di or trinucleotide repeats
- few 100 bases long
- easy to PCR
- 650,000 in genome
8
Q
Why are genetic maps in humans limited?
A
- large pops of siblings don’t exist, so limited no. recombination events to study
- recombination events not at random genome positions –. recombination hotspots
9
Q
How are physical maps created?
A
- restriction mapping locates relative positions on DNA molecule of recognition seqs for for REs
- FISH = map marker locations by hybridising probe containing marker to intact chromosomes
- STS = map positions of short seqs by PCR
10
Q
What are the advantages of creating BAC libraries from indiv chromosomes?
A
- BAC clone library can be used to seq genome
- BACs w/ inserts from each chromosome could be shared across consortium
11
Q
How is genome sequencing carried out clone by clone?
A
- extract DNA
- fragment DNA
- -> ideally completely random so no parts missed out
- -> by physical methods = sonication, hydrodynamic shearing, restriction enzymes and transposase
- -> by chemical methods (mostly used to fragment RNA) = heat and divalent cation (Zn and Mg)
- size selection –> gel electrophoesis
- clone 100-200kbp fragments into BAC plasmids to create library
- transformation of bacteria for BACs
- pick indiv colonies and extract vector (each tube has many copies of indiv DNA insert)
12
Q
How are clones positioned on genetic and physical maps?
A
- test clones for PCR markers w/ known locations
- BAC end sequencing using Sanger
- -> known seq so can design primer
- -> denature vector and Sanger seq
- -> design primer to reverse strand to seq other direction
- -> end seqs from same insert, so are paired end read
13
Q
Why are paired end read useful?
A
- can physically link 1 end of seq w/ another, so can be used to resolve seq gaps
14
Q
How is it decided which BAC has insert next to insert of interest?
A
- gen contiguous set of clones
- if any of BACs inc end seq, then insert they contain must be next to it
- test BAC library for end seq from desired vector by PCR
- repeated over and over again until all BACs placed in order on each chromosome
- created contig
15
Q
Why was shotgun seq of BAC clones needed?
A
- as BAC end seq leaves most of middle of genome insert to seq
16
Q
How was shotgun seq BAC clones carried out?
A
- each BAC clone broken up into 5-10kb fragments
- cloned into diff vector that accepts smaller inserts
- if seq lots of paired end seqs can assemble large fragment (=consensus seq)
17
Q
How did Celera seq human genome?
A
- fragmented genome into 2-50kbp fragments
- cloned 2, 10 and 50kbp fragments into plasmids to create library
- assemble reads to create consensus seq and seq contigs
- draft genome had 98% bases
18
Q
Why did the IHGP use clone by clone instead of whole genome shotgun seq?
A
- to prove feasible for complex repeat rich genome
- assembly easier and could be performed confidently
- could target gaps for finishing
- better suited to diverse international consortium
19
Q
What needed to be done to finish the human genome?
A
- fill in sequencing gaps and physical gaps
20
Q
Why were gaps present in human genome, and how could these problems be solved?
A
- cloning bias
- no restriction sites –> use diff RE, use physical or chem fragmentation method
- insert unstable –> use diff vector
21
Q
How were seq gaps closed?
A
- paired end seqs align to either side of gap
- if gap < 1kbp = PCR across gap
- if gap > 1kbp = sequential seq along insert
22
Q
How were physical gaps closed if know order of scaffolds?
A
- if gap region absent from all gene libraries
- PCR used to amplify genomic DNA spanning gaps and amplified DNA seq directly w/ or w/o cloning into vector
- PCR products over 3kbp hard to amplify
23
Q
How were physical gaps closed if don’t know order of scaffolds?
A
How do we know which pairs of primers are adj and will give product?
- try every poss and look for PCR reaction products using gDNA as template
- in singleplex PCR reaction each combo of primers tested w/ genomic DNA as template
- process sped up w/ multiplex PCR, as multiple pairs of primers tested in single PCR tube, so fewer reactions need to be performed
- use algorithm to decide min no. primer combos
24
Q
Where are repetitive seqs found in genome?
A
- approx 45% of genome
- mini and microsatellites
- centromeres
- telomeres
- transposons
- duplicated genes
25
What are the problems w/ repetitive seqs?
- hard to assemble and usually resolved last
- many poor quality never get these regions resolved
- better if had tech to seq v long fragments that span repetitive seq
26
How can repetitive seqs cause rearrangements?
- if break up seq and reassemble can get shorter as don't know original order
27
How can repetitive seqs confuse computer, and how can this be solved?
- tandem repeats can cause truncations --> computer can't be sure where to map reads to
- worse if repetitive seqs on diff chromosomes
- seq across whole repetitive region to identify flanking seqs that are unique (IGHL couldn't do this as not poss at time)
- anchor flanking seq regions to known positions of genome --> need genetic and physical maps
28
What factors influence the species chosen for genome projects?
- genetic model
- commercial/medical relevance
- genome size --> small easy, so bacteria most common
29
Why was NGS needed?
- Sanger and Celera slow, expensive, cloning bias, low coverage and 1 seq at time
30
How was NGS initially dev?
- 1st was 454 sequencing dev by Roche
- Applied biosystems dev competing tech called SOLiD
- both replaced by Solexa
31
How does NGS compare to Sanger seq?
- NGS involves fragmenting genome and seq all fragments or in parallel
- no plasmid cloning req and cheap
- NGS still has similar challenges --> 1 key challenge is how to separate indiv seqs as amplify insert so machine can measure signal
32
How is Illumina library prep carried out?
- need to create library of gDNA inserts flanked by adaptors of known seq
- fragment gDNA using physical/enzymic/chemical methods
- size selected on gel
- end repair DNA so has blunt ends
- -> DNA pol I fills in 5' overhang
- -> exonuclease removes 3' overhang
- add A tail so DNA ends not compatible and DNA cant concatermerise
- adaptors ligated to ends of fragments using DNA ligase (adaptors are ds oligonucleotides w/ T overhang)
- adaptor seqs elongated by PCR using extended oligonucleotide primers that inc unique 6 base index seq
- allows amplification and quantification of library
33
What are the functions of adaptor seqs?
- provide priming sites for PCR amplification
- allow index seqs to be added
- priming sites for bridge amplification
- priming sites for seq
34
What are indexes in Illumina indexed libraries, and why are these included?
- unique 6 base codes that identify each sample
| - can distinguish diff samples, so multiple samples can run at same time
35
Why is Illumina bridge amplification necessary?
- Illumina sequencer not sensitive enough to measure signal from 1 DNA molecule
- so bridge amplification used to prod localised clusters of ≈1000 identical molecules on glass dide
36
How is Illumina bridge amplification carried out?
- after library prep, library diluted and denatured, ss library washed onto flow cell w/ lawn of oligonucleotides complementary to adaptor seqs
- indiv molecules hybridise to oligos on flow cell
- fragment strand then bends over, hybridising to complementary oligo on flow cell forming bridge
- pol used to create complementary copy of fragment strand
- original strand then washed away
- repeated to create cluster of identical molecules at discrete location
- next stage = seq fragments using fluorescently labelled nucleotides
37
Why is there an optimum length of fragments for bridge amplification?
- want 2 initial molecules to be far apart so don't coalesce, so can be spread effectively
- done by washing over at low conc
- long arms mean could reach quicker and coalesce
38
How is Illumina sequencing by synthesis carried out?
- sequencing primer hybridised
- pol and 4 nts added
- fluorophores at each cluster read by lasers
- cleave fluorophore and unblock nt
- wash
- repeat (until achieve desired read length)
- index seq primer hybridised
39
Why are Illumina sequencers so expensive?
- optics, lasers and cameras req
40
What are the adv and disadv of Illumina seq by synthesis?
- enormous output
- accurate but not as accurate as Sanger
- all nts can be added simultaneously
- relatively slow
- short read lengths
- sample needs to be amplified --> PCR bias as doesn't amplify GC rich seq efficiently
- ligation of adaptors biased --> ligases prefer certain seqs
41
What would the features of a better sequencing machine be?
- single molecule seq w/o amplification
- continuous reads (no stop starting)
- v long reads
- solid state e-s
- cheap
- small
42
What are some examples of 3rd gen seq?
- Helicos (now obsolete)
- Pacific Biosciences (PacBio)
- Oxford Nanopore
- NABsys
43
What are the advantages of PacBio?
- v long reads
- 1 mil reads
- v high accuracy
- shortest run time
- least GC bias (can easily seq through v high/low GC content)
- no amplification bias
- discover broad spectrum of DNA base mods
44
How is PacBio library prep carried out?
- prod genomic library of fragments
- fragment DNA
- repair DNA damage and ends A tailed
- ligate SMRTBell adaptors
- anneal seq primer to SMRTBell templates
- -> complementary, so adaptor partially ds and partially ss
- -> t overhand, complementary to A tail
- streptavidin tagged pol and primer bound to SMRTBell adaptor cloned insert
- sequence
45
Is PacBio library prep diff to other library preps?
- similar
| - but genomic fragments ≈10kb and SMRTBell adaptors used
46
How is SMRT (single molecule real time) sequencing carried out?
- add diff fluorescent label to each type of nucleobase but attach it to terminal phosphate released during polymerisation
- measure fluorescence each time new base added, decays away when fluorescent tag released
- use zero mode waveguide chambers to improve detection from tiny signals
- read lengths prod of ≈500-3200 bases
47
How is Nanopore library gen?
- rapid
- DNA isolated using beads that bind DNA
- transposase complex used to simultaneously cut and ligate 1st adaptor
- then 2nd 1D adaptors and motor proteins added
- amplification nt req as device can read single molecules
- 2 diff types adaptor can be added
- 1D adaptors allow 1 strand to be seq
- 2D adaptors allow both strands to be seq
48
What does nanopore tech involve?
- protein nanopore
- -> heptameric protein α-hemolysin
- -> separated from bacteria allowing low cost and robust nanopores
- -> pore embedded into synthetic membrane w/ high electrical resistance
- -> 512 pores per minion cell
- synthetic polymer membrane
49
What occurs during 1D sequencing?
- DNA attached to pore and motor protein controls DNA translocation speed through pore
- 1 strand seq and other strand discarded
50
What occurs during 2D sequencing w/ hairpin adaptor?
- hairpin adaptor allows seq of both strands
- 1st strand seq then adaptor unwound and seq
- opp strand seq
- opp strand seq complementary to 1st strand, used to correct errors in seq to create '2 direction read'
- not same as paired end seq
51
What are the adv of Nanopore seq?
- no amplification --> decreases artifacts from PCR
- rapid
- long reads --> simplifies assembly
- solid state electronics --> cheaper and more reliable machines
- portable
- versatile --> can be changed to measure RNA, proteins or other compounds
52
What are the applications of NGS?
Research tools:
- de novo genome seq
- re seq genome and comparing to reference genome
- seq transcript
- methylation of DNA
- seq small RNAs
- protein binding sites
Clinical apps:
- diagnosis
- biomarkers
- prenatal testing
53
How can NGS be used as a research tool?
- seq genome of species
- cataloguing variation between individuals in species
- characterising differences between cells w/in individuals
- describing underlying cellular mechanisms
54
What is NGS now used in clinic for?
- personalised medicine
