Thomas J. Keller, PhD (1985, U.C.S.F, Medicinal Chemistry)

http://www.ohsu.edu/research/core/about.html

MMI Shared Resource Facility

MMI Research Core Facility

Overview

scienceblogs.com—basics_how_do_you_sequence_a_g_1.php

Break the genome into lots of small pieces at random positions.

Clone (or PCR amplify) and purify

Determine the sequence of each small piece of DNA.

Use an assembly program to figure out which pieces fit together.

A bit more detail

Feb. '07: omicsomics.blogspot.com—new-fangled-dna-sequencing.html

Prepare a sample containing many copies of the same DNA molecule

Generate a set of extension products, using a short, sequence specific oligonucleotide (primer), DNA Polymerase, dNTPs, and ddNTPs.

all the fragments have to begin at a specific position (the end of the primer)

and end in a sequence specific nucleotide "base".

Separate the fragments by length using electrophoresis,

either the primer or the ddNTPs must be labeled so the fragments can be easily detected

analysis of the signal as a function of time yields a "read"

characterized by length

and accuracy (1 error in 1,000 for a single read is common)

multiple reads can reduce this to less than 1 error in 10,000

Quality Scores and read length

phred values = ( -10)*Log(error rate)
Phred - Quality Base Calling
Discovering Biology in a Digital World

Current State-of-the-Art Sanger Sequencing

DNA Sequencing & Fragment Analysis

cycle sequencing

Cycle sequencing primer

the types of approaches being used or developed for DNA sequence determination

a set of parameters for evaluating sequencing methods

an overview of the cost in time, effort and money to sequence a small genome with the different approaches

where to go for more information

nrg1325_box1

ref: Shendure, et al., 2004 www.nature.com—nrg1325.html

length

accuracy

quality scores; "Phred values"

coverage

depth

Sample Preparation Approaches

Fragment, Isolate, Amplify, Isolate - then sequence

"traditional" approach

cyclic array approach

mb_fig1

Fragment, Modify, Isolate, Amplify - then sequence

Fragment, Modify, Isolate - then sequence

"single molecule sequencing"

Sequence Determination Approaches

annotated links: arep.med.harvard.edu—Polonator

A. Sequencing by synthesis - direct

new developments in Sanger sequencing

multiplexing and miniturization

microelectrophoretic sequencing

nanopore sequencing

Reversible terminators

flowchart image

nmeth1155-I2

notes from web presentation:
fragment DNA
add adapters (linkers)
covalently bind single-stranded fragments to inside surface of flow cell channels
attached fragments hybridize to primers (pre-attached by Solexa)
perform "bridge amplification": solid-phase PCR (in-situ)

fragments become double stranded

bridge_amplif
cluster

denature double-stranded molecules
cluster amplification

sequence by synthesis

cycle_1
image_1
iterations

fluorescent moiety and "terminator group" are "labile"
after imaging the fluorescence - locate all four colors for that step
remove the fluorescence and blocking groups so strand is ready for next round
continue with next synthesis addition with reversible-fluorescent terminators

Solexa/Illumina

PCR amplification to generate clusters of template prior to std Sanger sequencing (with reversible dye-terminators)
webpage: Solexa technology
pdf: SS_DNAsequencing
ref using 1G: 2. Johnson DS, Mortazavi A, Myers RM, Wold B. (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316(5830):1441-2.

randomly attached fragmented genomic DNA to a planar, optically transparent surface and solid phase amplification to create millions of clusters, each containing ~1,000 copies per template per sq. cm.

add primer that binds to one strand, and 4 different dye-labeled reversible terminators, image each cluster to determine which base was incorporated (like Sanger sequencing)

must cap any ends after adding the reversible fluorescent terminators or you'll get a frame shift (deletion) ???

how do you account for homopolymers?
how do you account for incomplete reactions?

remove fluorophores and 3'-terminal blocker prior to next synthesis with dye-terminators

bind adapter/single stranded templates randomly to surface of flow cell

denature, then perform repeated bridge amplification on flowcell surface to generate sufficient S/N;

n.b. therefore subject to any errors introduced by the polymerase

hybridization to dense lawn of single stranded linkers covalently attached to surface of flow cell

uses linkers ligated to sheared/digested genomic DNA

the unattached end will hybridize to it complementary adaptor to form a bridge structure

but only one end of each strand is covelently attached to the surface

polymerase with unlabeled nts generate a double stranded, bridged sequence

data analysis example

Chaison, et al www.genome.org—324

We have also assembled a human BAC using ap1 million 27-nucleotide Solexa reads generated by the Joe Ecker laboratory at the Salk Institute (150x coverage). The Solexa technology is currently being optimized, and the error model of the Solexa platform remains poorly understood. In particular, the error rates of Solexa reads are position-dependent and highly variable across different machines and even across different runs of the same machine (Alla Lapidus, pers. comm.). As a result, while assembly of Solexa-sized simulated reads was analyzed in Warren et al. (2007), the assembly of real Solexa reads proved to be more challenging.

July '07 opinion piece

biotechnicalcurrency.blogspot.com—helicos-part-i.html

Helicos

sequencing by synthesis, microfluidics, solid phase synthesis
biotechnicalcurrency.blogspot.com—helicos-part-i.html
www.helicosbio.com—Default.aspx

enzymatically tail and add reversible dye to 3' end of single strands from sheared/digested genomic DNA

Helicos BioSciences Corporation

next addition and image is taken

single dye labeled terminater is added by polymerase and image is taken

dye removed, freeing the 3-OH for next addition

B. Sequencing by synthesis - indirect

e.g., pyrosequencing

nmeth1155-I1

nrg1325_fig3a

454 Life Science / Roche

Roche_KBantle

Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Research, 11(1), 3–11.

add duplex adaptors to both ends of sheared/digested gDNA

dilute the non-biotinylated strands and hybridize to xs of beads

presumably you can control the stringency so that the full length complement is the only molecule that binds

one end has biotin, the other does not

the biotinylated strands are bound to Streptavidin coated beads

does it matter whether more than one molecule binds to each bead?

I think it does, you must have to use an excess of beads here to so that you hava a statistical likelihood of getting just one biotin-strand per bead. Also, presumably there are numerous B-adapter sequences elsewhere on the bead to anneal to newly generated pcr products as they are produced in the emulsion pcr.

detection by pyrophosphatase generated luminescence after incorporation of regular nucleotides

problem with homopolymer sections

Quality and read length

References

www.nature.com—449628a.html
genomebiology.com—R143

The work showed that the 454 system was 98% accurate if no culling was used to remove bad bases or reads6.

However, by using a very simple set of rules, which caused somewhere between 10% and 20% of the data to be discarded, the accuracy could be pushed up to 99.75%. And discarding up to 20% of the data for this level of accuracy is a trade-off that is fine with Sogin because the latest 454 system — the Genome Sequencer FLX — can produce up to 400,000 reads per run.

Assembling Short Reads

Chaison, et al www.genome.org—324

de Bruijn Graph: A graph whose nodes are sequences of symbols from some alphabet and whose edges indicate the sequences which might overlap.

mathworld.wolfram.com—deBruijnGraph.html

C. Sequencing by ligation

biotechnicalcurrency.blogspot.com—helicos-part-i.html

Applied BIosystems

June '07 17 minutes: Applied Biosystems presents Michael Rhodes

prepare DNA: shear/enzymes

generate library

or mate pair tags

single fragment

emulsion PCR: 2 poisson dilution to get < 1 bead per drop

enrichment for beads with amplified product

ends in P2 so can be bound to solid support -> from 30 to 80% beads with product

attach beads to array surface

ABI uses smaller beads than Roche/454
ABI: (>20 million beads, up to 40,000 molecules per bead)

more details (35 base read length or 2x25 for same-strand pairs)

4exp5 (1024) octomer probes, only 4th and 5th bases are specific: 2 base encoding with 4 dyes

uses concept of color space

ball and stick model

each of 4 colors represents a set of dinucleotides

eases discrimination between system errors and true polymorphisms

note, each base is read twice, you need to read consecutive color to identify bases

Picture 1

dephosphorylation step to prevent "dephasing", from incomplete ligation reactions

chemical cleavage between bases 5 and 6 of octomer

??

next step -> 9,10 data; then 14,15, then 19,20

reset phase again, etc

reset phase: redo whole process with primer that binds at n-1, so first cycle give data for pos'ns 3 and 4,

then 8,9, etc

2-base encoding

June '07 (14 minutes): Applied Biosystems presents Michael Rhodes

essentially, each base gets read twice, this increased accuracy

Quality and read length

omics omics post

D. Sequencing by hybridization

ShendureEtAl_sFig7

alternative approach: build an "array" of probe sequences

probes cover the the whole genome

Hybridize the unknown sequence to the array in a way that allows you to identify the matches and put them in order

NimbleGen example: ~30-mer 'tiled' oligos spaced every ~7 bp of the genome, both strands

probes are designed to have isothermal hyb characteristics

1st pass: compare signal intensities to the control

Regions of interest from the first pass are reinterigated on a second array having single base resolution

2nd pass: 8 probes per position (all 4 nts for both strands)
the control will clearly differ from the test if a SNP is present

indels or > single SNPs are less conclusive with this method

False negatives can result in erroneous interpretation

i.e., failure to detect a mutation that is actually there

Since you don't know what you don't know these are just missed

Herring and Palsson (2007) compared E. coli strains W3110 (test) and MG1655 (reference)

(could have done it the other way round)

Expected to find 7 SNPs, a single 2 bp insertion, 12 IS element insertions, a single deletion of 6.6 kb, an inversion of 783.1 kb

Found the NimbleGen instrument found 25 SNPs, 4 of which matched the expected sites.

www.pubmedcentral.nih.gov—articlerender.fcgi

Followup sequencing showed that only 2 of the remaining putative SNPs were confirmed

19 were refuted, i.e. false positives
so they had 3 (of 7) false negatives (43%).

it's interesting that the 2 new mutations were confirmed

the problem seems to be in normalizing the hybridization signal

variability of perfect matches is greater than the difference between a perfect match and match with a SNP.

the study revealed that at least 10 mutations had accumulated in strain W3110 from 1987 to 2007

see references to get dates: Kohara, et al (1987) to this work (2007) = 20 years.
2 SNPs, 7 IS insertions (!!), and 1 deletion.

details of sample storage and handling were not known but could have been a "stab" at room temp.

?? but where would the insertions come from??

The single SNP not detected is a substitution in the gene rrlE, which is duplicated 6 other times in the E. coli genome. In previous work, we noted the failure of CGS to detect a 9 bp duplication, a 1 bp deletion and a 28 bp deletion near a transcriptional terminator [8]. It appears that CGS has trouble in regions of high local secondary structure and with sequences repeated multiple times in the genome [15][17].

In addition, detection of SNPs may be less accurate in areas with low overall intensity. Fortunately, such regions are relatively uncommon, and CGS appears to detect most mutations with a reasonable degree of confidence. Resequencing technology allows a sequence to be determined at less than one tenth the cost of traditional Sanger sequencing, but it is less accurate with some types of sequences and requires a closely related reference sequence. Other methods of resequencing [9,12] are likely to differ from CGS in cost and accuracy. The control experiment presented here comparing E. coli strains W3110 and MG1655 can now be performed with these other technologies to allow cross-comparison.

False positives should be found

they get checked by Sanger sequencing and/or PCR

the reference and the unknown must be very similar or the method won't work

Resequencing is very useful:

www.pubmedcentral.nih.gov—articlerender.fcgi

relating phenotype to genotype (SNPs and other polymorphisms)

analyzing natural variation (synteny, copy number, control elements)

commercial

Polonator

DanaherMotion

Affymetrix

slides: Affynetrix_CaseyGates
www.affymetrix.com—foundation.affx
www.affymetrix.com—gseq.affx
www.affymetrix.com—sequence_analysis.affx

NimbleGen

sequencing by cyclic-arrays

E. Mass spectrometry

we won't pursue this one

Detection Approaches: improve S/N and speed

we won't cover this

fluorescence

direct

FRET

luminescence

direct electrochemical

Jan. 08 Review article

mb0701

instead of a homogenous pop (single amplified template) you have multiple amplified templates, each one needing to be read simultaneously

instead of detection after separation, you have detection by incorporation (synthesis methods), so homopolymers are a problem

Illumina/Solexa use cleavable fluorescent dyes at the 5 and 7-positions (pyrimdines and purines respectively) AND a small removable blocking groupt at the 3'-OH postion. The latter prevents multiple incorporations from homopolymers.

www.pubmedcentral.nih.gov—articlerender.fcgi

assembly of short reads

improve methods for "paired-end" reads

ratio of good/bad reads

parameters