BioSci D145 lecture 1 page 1 © copyright Bruce Blumberg 2014. All rights reserved BioSci D145 Lecture #3 Bruce Blumberg –4103 Nat Sci

BioSci D145 lecture 1 page 1 ©copyright Bruce Blumberg 2014. All rights reserved

BioSci D145 Lecture #3

• Bruce Blumberg ([email protected])– 4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment)– phone 824-8573

• TA – Ron Leavitt ([email protected])– 4351 Nat Sci 2, 824-6873 – office hours M 2:30-3:30 4206 Nat Sci

2

• check e-mail and noteboard daily for announcements, etc..– Please use the course noteboard for discussions of the material

• Updated lectures will be posted on web pages after lecture – http://blumberg.bio.uci.edu/biod145-w2016– http://blumberg-lab.bio.uci.edu/biod145-w2016

• Last year’s midterm is posted.

http://blumberg.bio.uci.edu/biod145-w2016

http://blumberg-lab.bio.uci.edu/biod145-w2016


Genome mapping• The problem – genomes are large, workable fragments are small

– How to figure out where everything is?– How to track mutations in families or lineages?

• analogy to roadmaps– The most useful maps do not have too much detail but have

major features and landmarks that everything can be related to• Allows genetic markers to be related to physical markers

• What sorts of maps are useful for genomes?– Restriction maps of various sorts (most often of large insert libraries)

• RFLPs, fingerprints– Recombination maps, how often to traits segregate together– Physical maps – which genes occur on same chunks of DNA


Genome mapping (contd)• How are maps made?

• What do we map these days?

– Restriction digestion and ordering of fragments to build contigs• Fingerprinting

– Location of marker sequences onto larger chunks– Hybridization of markers to larger chunks– Calculation of recombination frequencies between loci

– BACs are most common target for mapping of new genomes– Radiation hybrid panels still in wide use– Goal is always to map markers onto ordered large fragments and

infer location of genes relative to each other.– HAPPY mapping becoming widely used again


Genome mapping (contd) (stopped here)• Useful markers

– STS – sequence tagged sites• Short randomly acquired sequences• PCRing sequences, then prove by

hybridization that only a single sequence is amplified/genome

– VERY tedious and slow• validated ones mapped back

to RH panels• Orders sequences on large chunks of DNA

– STC – sequence tagged connectors• Array BAC libraries to 15x

coverage of genome• Sequence BAC ends• Combine with genomic maps

and fingerprints to link clones– Average about 1 tag/5 kb

• Most useful preparatory to sequencing


Genome mapping (contd)• Useful markers (contd)

– ESTs – expressed sequence tags• randomly acquired cDNA sequences• Lots of value in ESTs

– Info about diversity of genes expressed– Quick way to get expressed genes

• Better than STS because ESTs are expressed genes• Can be mapped to

– chromosomes by FISH– RH panels– BAC contigs

– Polymorphic STS – STS with variable lengths• Often due to microsatellite differences• Useful for determining relationships• Also widely used for forensic analysis

– OJ, Kobe, etc



– SNPs – single nucleotide polymorphisms• Extraordinarily useful - ~1/1000 bp in humans• Much of the differences among us are in SNPs• SNPs that change restriction sites cause RFLPs (restriction

fragment length polymorphisms• Detected in various ways

– Hybridization to high density arrays (Affymetrix)– Sequencing– Denaturing electrophoresis or HPLC– Invasive cleavage

• Tony Long in E&E Biology has method for ligation mediated SNP detection that they use for evolutionary analyses



– RAPDs – randomly amplified polymorphic DNA• Amplify genomic DNA with short, arbitrary primers• Some fraction will amplify fragments that differ among

individuals• These can be mapped like STS• Issues with PCR amplification• Benefit – no sequence information required for target

– AFLPs – amplified fragment length polymorphisms• Cut with enzymes (6 and 4 cutter) that yield a variety of small

fragments ( < 1 kb)• Ligate sequences to ends and amplify by PCR• Generates a fingerprint

– Controlled by how frequently enzymes cut• Often correspond to unique regions of genome

– Can be mapped• Benefit – no sequence required.


Genome mapping (contd)• Fingerprinting

– Array and spot ibraries– Probe with short oligos (10-mers)

• Repeat– Build up a “fingerprint” for each clone– Can tell which ones share sequences

• tedious


Genome mapping (contd)• Mapping by walking/hybridization

– Start with a seed clone then walk along the chromosome– Takes a LOOONNNNGGG time– Benefit – can easily jump repetitive sequences


Genome mapping (contd)• Mapping by hybridization

– Array library – pick a “seed clone” – See where it hybridizes, pick new seed and repeat– Product


Genome mapping (contd) Restriction mapping of large insert clones

• Mapping by restriction digest fingerprinting– Order clones by comparing patterns from restriction enzyme

digestion


Genome mapping (contd)• FISH - Fluorescent in situ hybridization – can detect chromosomes or

genes– Can localize probes to chromosomes and– Relationship of markers to each other– Requires much knowledge of genome being mapped

– Chromosome painting marker detection


Genome mapping (contd)• Radiation hybrid mapping

– Old but very useful technique (Geisler paper)• Lethally irradiate cells with X-rays• Fuse with cells of another species, e.g., blast human cells then

fuse with hamster cells– Chunks of human DNA will remain in mouse cells

• Expand colonies of cells to get a collection of cell lines, each containing a single chunk of human cDNA

• Collection = RH panel– Now map markers onto these RH panels

• Can identify which of any type of markers map together– STS, EST (very commonly used), etc

• Can then map others by linkage to the ones you have mapped– Compare RH panel with other maps

• Utility – great for cloning gaps in other maps

• HAPPY Mapping – – PCR-based method – see Ron’s presentation


Genome mapping (contd)• How should maps be made with current knowledge?

– All methods have strengths and weaknesses – must integrate data for useful map

• e.g, RH panel, BAC maps, STS, ESTs– Size and complexity of genome is important

• More complex genomes require more markers and time mapping

– Breakpoints and markers are mapped relative to each other– Maps need to be defined by markers (cities, lakes, roads in

analogy)– Key part of making a finely detailed map is construction of

genomic libraries and cell lines for common use• Efforts by many groups increase resolution and utility of maps

• Current strategies– BAC end sequencing– Whole genome shotgun sequencing– EST sequencing– HAPPY mapping– Mapping of above to RH panels– Fancier techniques (Dovetail, Chicago reads, Hi-C assemblies)

BioSci D145 lecture 4 page 15 ©copyright Bruce Blumberg 2004-2007. All rights reserved

Nobel Prize in Chemistry 1980Walter Gilbert (Harvard) & Frederick Sanger (MRC Labs)(Sanger also won Nobel in 1958 for protein sequencing)

DNA sequence analysis (first gen sequencing)• DNA sequencing = determining the nucleotide sequence of DNA

– Two main methods– shared Nobel prize in 1980

• Chemical cleavage – Maxam and Gilbert

• Enzymatic sequencing (based on polymerization reaction)


DNA sequence analysis• Maxam and Gilbert

– One of the first reasonable sequencing methods– Very popular in late 70s and early 80s– VERY TEDIOUS!!

• Totally superceded by dideoxy sequencing now


DNA sequence analysis (contd)• Dideoxy sequencing – Sanger

1977– Virtually all sequencing is

done this way now– Requires modified

nucleotide• 2’3’-dideoxy dNTP

– DNA polymerase incorporates the ddNTP and chain elongation terminates

– Original method used 4 separate elongation reactions

– Products separated by denaturing PAGE and visualized by autoradiography


DNA sequence analysis (contd)• Dideoxy sequencing (contd) – Sanger 1977

– Dideoxy NTPs present at ~1% of [dNTP]– Each reaction has identified end– In principle, all possible chain lengths are represented

• varies by [dNTPs], [ddNTPs], [primer] and [template] and ratios


DNA sequence analysis (contd)

A C G T A C G T

A C G T


1. Trace files (dye signals) are analyzed and bases called to create chromatograms.

2. Chromatograms from opposite strands are reconciled with software to create double-stranded sequence data.

Automated DNA sequence analysis• How to improve throughput of sequencing?

– Incorporate fluorescent ddNTPs, separate products by PAGE• Base calling and lane calling issues

– Key advance was capillary sequencers• Separate DNA in a thin capillary instead of gel• Very accurate, no tracking errors, much more automation

friendly


Automated DNA sequence analysis

• Capillaries vs gels– Capillaries much faster – higher field strength possible– Fully automated = higher throughput


PCR – polymerase chain reaction amplification of DNA• PCR is most routinely used method to

amplify DNA– Exponential amplification of DNA by

polymerases – Saiki et al, 1985• 2n fold amplification, n= # cycles

– 35 cycles = 235 = 3.4 x 1010 fold

• Originally used DNA polymerase I– Needed to add fresh enzyme

at every cycle because heat denaturation of template killed the enzyme

– Not widely used – too painful to do manually

– Nobel Prize to Kary Mullis in 1993 for deciding to use Taq DNA polymerase for PCR

• He was middle author on paper!


Hot water bacteria: Thermus aquaticusTaq DNA polymerase

Life at High Temperatures by Thomas D. BrockBiotechnology in Yellowstone© 1994 Yellowstone Association for Natural Sciencehttp://www.bact.wisc.edu/Bact303/b27

PCR – polymerase chain reaction amplification of DNA (contd)


Cycle sequencing – fusion of PCR and fluorescent ddNTP sequencing• http://www.dnalc.org/ddnalc/resources/animations.html• Combine PCR amplification with

dideoxy sequencing – cycle sequencing– Linear amplification of template

in the presence of fluorescent ddNTPs– When nucleotides are used up

reaction is over– Separate on capillary electrophoresis

instrument– Advantages

• Fast, single tube reaction• Works with small amounts of

starting material– Disadvantages

• Still need to prepare highquality template to sequence

• Cost and time– Many sequencing centers spend

time, $$ on template prep– Automation requirements

http://www.dnalc.org/ddnalc/resources/animations.html


Isothermal amplification – the solution to template preparation• How to make template preparation faster, easier and more reliable?

– Eliminate automation requirement, amplify starting material in some other way

– Φ29 DNA polymerase (aka TempliPhi)– http://www.gelifesciences.com/aptrix/upp01077.nsf/content/samp

le_preparation~product_selection_category~rolling_circle_amplification

– Enzyme has high processivity and strand displacement activity• Isothermal reaction produces huge quantities of DNA from tiny

amount of input• More efficient than PCR (no temp change, no machine, no

cleanup)

http://www.gelifesciences.com/aptrix/upp01077.nsf/content/sample_preparation~product_selection_category~rolling_circle_amplification




Modern DNA sequence analysis• Cycle sequencing

– Virtually all commercial DNA sequencing today is done by cycle sequencing with fluorescent ddNTPs

• ABI Big Dye chemistry– Template preparation still tedious for small scale

• TempliPHi used in genome centers (obviated need for most automation)

– Capillary sequencers predominant form of technology in use

• But, next generation sequencing is already coming online and will rapidly displace old technology in genome centers.– 454 sequencing (Roche)– Solexa (Illumina) – SoLID (Applied Biosystems)

• 3rd generation sequencing (individual DNA molecule) now available– e.g., Pacific Biosciences (sequence reads of 1,000-10K bases)


Other sequencing technologies• Sequencing by hybridization

– Construct a high-density microchip with all possible combinations of a short oligonucleotide

• Up to 25-mers • By photolithography

– Synthesized onchip directly

– Label and hybridize fragment to be sequenced

– Wash stringently– Read fluorescent spots– Reconstruct sequence

by computer


Other sequencing technologies (contd)• Sequencing by hybridization rarely used for de novo sequencing

– Extremely fast and useful to sequence something you already know the sequence of but want to identify mutation - resequencing

– Disease causing changes• e.g in mitochondrial DNA

– SNP discovery– Works best for examining sequence of <10 kb


Other sequencing technologies (contd)• http://www.affymetrix.com/products/arrays/index.affx• SNP discovery

– Photo shows mitochondrial chip

– Right panel shows pairs of normal (top) vs disease (bottom) (Leber’s Hereditary Optic Neuropathy)

• Top 3 disease mutations

• Bottom control with no change

http://www.affymetrix.com/products/arrays/index.affx


Other sequencing technologies – Next Generation sequencing• 2nd generation = high throughput, short sequences• 3rd generation = single molecule sequencing

• Small number of sequence templates (thousands) but very long reads (~105 bp)

• What is the immediate implication of this technology for genome assembly?

• Key review is Metzger, M.L. (2010) Sequencing technologies — the next generation, Nature Reviews Genetics 11, 31-46.

We should now be able to completely sequence large insert clones directly and avoid fragmentation by repetitive elements!

3rd generation


Other sequencing technologies (contd)• Pyrosequencing –

– http://www.454.com– Based on synthesis of complementary strand to a template (like

Sanger)– Detection of elongation with chemiluminescence

• Fragment genome to appropriate size (depends on application)

• add adapters to each end• Isolate those with different adapters on each end• PCR to amplify

http://www.454.com/


Other sequencing technologies (contd)• Pyrosequencing (contd)

– PCR – capture template on micro beads such that each bead gets 1 molecule of DNA – how?

– Emulsify in water/oil microreactors– Amplify DNA– Break and recover DNA containing beads

Use a large ratio of beads to DNA



– Sequencing – load beads into picotiter wells• Add enzymes (sulfurylase and luciferase)• Run reaction – flow nucleotide/buffer

solution across wells one at a time• Complementary nucleotide addition

leads to light output– light output is proportional

to # consecutive nucleotides



– What is the point?• Can generate 400,000 reads in parallel (FLX)• Or > 1,000,000 (FLX Titanium)• Each read is 200-400 bp (FLX), or 400-600 (FLX Titanium)• So you can get

– 8 x107 bp per run! (FLX)– 4-6 x 108 bp/run (FLX Titanium)

• What is massively parallel sequencing good for?– Rapid sequencing of genomes, or resequencing of known

sequences– Ancient DNA (even dinosaurs? – Svante Pääbo says ~200K years

is limit)– ChIP-sequencing (week 6)– Sequencing ESTs or other tags– Determining microbial diversity in field samples– Transcriptome sequencing– Identifying variations in

• Viral populations• Gene sequences in mixed populations


Amplicon sequencing

• Idea is to sequence many copies of the same thing– Gene sequence– mRNA transcript


Amplicon sequencing (contd)• What is amplicon sequencing good for?

– Discovery of rare somatic mutations in complex samples (e.g., cancerous tumors - mixed with germline DNA) based on ultra-deep sequencing of amplicons

– Sequencing collections of exons from populations of individuals to identify diversity

– Sequencing collections of human exons from populations of individuals for the identification of rare alleles associated with disease

– Analysis of viral quasispecies present within infected populations in the context of epidemiological studies

– Evolutionary biology in populations

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BioSci D145 lecture 1 page 1 © copyright Bruce Blumberg 2014. All rights reserved BioSci D145 Lecture #3 Bruce Blumberg –4103 Nat Sci