Transcript An Integrative Study of Metabolic Control and Complexity

Genome Sequencing
Gibson and Muse (on reserve) Ch 2
The first objective of most genome projects is the
determination of the DNA sequence either of the genome or
of a large # of transcripts
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Sanger (di-Deoxy) DNA sequencing
Most current genome projects rely on chain termination
sequencing developed in 1974 by Frederick Sanger
The basic idea behind Sanger Sequencing is to
generate all possible single-stranded DNA molecules
complementary to a template
The template starts at a common 5’ base and extends
up to 1.5 kb
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Sanger (di-Deoxy) DNA sequencing
ssDNA fragments are labeled to allow identification of
the 3’-most base
Fragments are separated by size by gel electrophoresis
Resulting ladder of fragments is then read
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Sanger (di-Deoxy) DNA sequencing
Template can be cloned
DNA, genomic DNA or
PCR product
DNA polymerase, primer
(3’OH) and dNTPs are
used to synthesize DNA
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Sanger (di-Deoxy) DNA sequencing
Dideoxy method uses DNA
bases containing modified
deoxyribose sugars =
dideoxyribose which
contain H at the 3’ position
of the ribose sugar rather
than OH
Modified sugars cause
chain termination
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Sanger (di-Deoxy) DNA sequencing
ddNTPs can be labeled w/ 35S and run as 4 different rxs (~500bp)
ddNTPs can be labeled w/ fluorescent dyes and run as 1 rx (~1kb)
Fragments are separated by differential retardation of migration
of molecules of different size
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Improvements on Sanger Sequencing
•Four-color fluorescent dyes, dye-terminators, also dyelabeled primers (req.s 4 lanes)
•Seq. rx products are “laser” scanned just before running off
an electrophoresis medium - reading is automatic
•Improvements in chemistry and bioengineered DNA
polymerase - better handling of secondary structure, longer
(~1.2kb) reads
•Replacement of slab gels w/ capillary electrophoresis longer reads, no gel pouring - the art of pouring gels is dead
In total, ~20x increase in sequencing productivity
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New Sequencing Methods - SBH
Emerging technologies will reduce the cost and time associated
with genome sequencing
Sequencing by hybridization (SBH) - uses complementarity
between 2 strands to detect whether an exact match to an oligo
is present in a sample of DNA. This can be used in “resequencing” looking for SNPs, e.g. a variant detector array
(VDA) contains all possible oligionucleotides in a molecule
several kb in length as well as mismatches at each sites - look
for binding of sample to different spots in the array
This technique is being used to resequence primate genomes
and scan for 1.5 million SNPs in human genome
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New Sequencing Methods - Mass Spec.
Mass Spec. techniques have been used to seq.
fragments up to 50 bp, identity assigned by time
of flight through a vacuum chamber. In principle,
it should be possible to determine seq. of
molecule that is devided into all possible
oligonucleotides
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New Sequencing Methods - nanopore
technology
Nanopore sequencing strategies - one approach is
to use monitor electrical current as a single strand
of DNA or RNA passes through a pore in a
membrane, another approach channels nucleic
acid through a single-molecule fluorescence
reader
These methods have the potential of reading 100s
of kb of sequence in minutes
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New Sequencing Methods - singlemolecule Sanger
Some single-molecule approaches using the Sanger
method focus on detecting fluorophore incorporation
into single molecules on an array
454 Corporation machines amplify single molecules
using massively parallel picoliter scale amplification
and detection
“Pyrosequencing” platform GS20 - DNA sequence is
determined by analyzing flashes of light released by
release of pyrophosphate with chain extension using
a predetermined sequence of nucleotide addition in
picoliter-sized reactions
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Typing HPV by Pyrosequencing
Pyrosequencing, a bioluminometric DNA sequencing technique, is replacing Sanger
Sequencing in some applications
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Typing HPV by Pyrosequencing
Pyrogram showing
genetic variation
between strains
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New Sequencing Methods pyrosequencing
A single run GS 20 generates up to 25 million high-quality bases
in hundreds of thousands of short sequences(~100bp)
2 sequencing runs produced 99.75% of 2 chloroplast genomes
(~150kb each) to 25x and 17x coverage w/ error rates tested
against conventional sequencing of ~0.04%
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New Sequencing Methods pyrosequencing
Comparison of Sanger,
454, Illumina, and ABI
SOLiD
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Reading Sequence Traces
Base-calling is routinely done by automated software that
reads bases, aligns similar sequences and allows easy
editing of sequences
phred - software that converts traces into sequence data
that can be deposited into database, including probability
scores for each base call
•4 fluorescent spectra are merged, maintaining peak register
•Mean peak distance is used to determine where peaks
should be - insert “n”s for missing peaks, call doublets as
single bases (heterozygotes)
•Adjust peak distance for changes as run proceeds and for
variation in GC content
<1 second required to call ~1kb
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Reading Sequence Traces
1st ~50 bases “messy”
SNPs and indel between two
sequences
Traces also become difficult to
read at end of seq. - diffusion
and sm. relative difference in
size of fragments
Depending on end use, seq.s
can be manually edited
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Reading Sequence Traces - Errors
All automated base-calling algorithms make errors, so we
assign probabilities to each call
phred uses 4 parameters
1. The variance in peak spacing over a 7-peak window
centered on called base
2. The ratio of the largest uncalled to the smallest called
peak in that window
3. The same ratio over a 3-peak window
4. The # of bases between the current base and the
nearest unresolved one
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Reading Sequence Traces - Errors
The error probability, p, is converted to a phred score, q =
negative logarithm of p,
q < 13 means that greater than 0.05 probability that the
base is miscalled
q>30 means an error probability of 0.001
q > 20 is regarded as high-confidence
Bases much further from the start get progressively
smaller phred scores
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Contig Assembly
Sequences of DNA fragments longer than ~1kb have to be
assembled - aligned and corrected - again often done w/
software, e.g. phrap assembler and consed graphic editor,
general features:
•Color highlighting of features such as different bases, quality
scores, regions of seq. conservation, manual vs automated
calls
•Ability to view and work between actual traces and edited seq.
•Display of complementary strand
•Manual editing tools - insertion/deletion etc, linked to output
•Alignment algorithm
•Computation of probability scores for consensus
•Ability to id potentially polymorphic sites
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Contig Assembly
Editing starts w/ chromatogram files which are generally left
untouched
Base call files (phred) include quality scores, base calls, peak
positions
Assembly files (phrap) include alignment, consensus contig
seq. and score
Additional flies can be saved after editing, etc
Averaging across multiple reads provides the high confidence
in automated DNA sequence determination
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Genome Sequencing
2 traditional approaches to genome sequencing
Hierarchical - ordered, start w/ low res. physical alignment
Shotgun - break the genome into small manageable pieces
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Hierarchical Sequencing
Earlier form, aka - Top-down, map-based, clone-byclone
Efficient use of sequencing and computational
resources, fosters high-res. physical and genetic maps
and allows sharing of work across consortia
First step is cloning fragments into manageable units
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Cloning Vectors
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DNA Libraries
Digested or sheared genomic or chromosomal DNA is ligated
into the MCS of a vector
Aim for 5-10x redundancy - every region represented 5-10 times
Select clones by matching end seq. to reconstruct entire
chromosome = tiling path
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DNA Libraries - hybridization
Chromosome
walking uses
restriction fragment
lengths - restriction
profiling - and
hybridization using
labeled probes to
order clones
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DNA Libraries - Fingerprinting
Clones can be ordered by comparing restriction digest profiles
using alignment software
All cones
that share
the labeled
probe seq.
Band profile
– shared
bands,
vector band,
unique
bands
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Restriction
digest of
clones =
finger print
1,3,4 form a
tiling path
DNA Libraries - End-sequencing
Gaps remaining after fingerprinting can be filled by sequencing
both ends of a collection of BAC clones and comparing w/
already the sequenced genome - finding a match to one end
implies that the clone covers a gap
Once a tiling-path is chosen, BAC clones are sheared into small
fragments that are subcloned for automated sequencing, often
into phagemid vectors (hold 1kb of DNA) or plasmid (2-3kb) and
seq. both ends
Enough seq. reactions are conducted to ensure ~10x coverage
Remaining (small) gaps are filled in the sequence “finishing”
phase
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Shotgun Sequencing
Computer algorithms are used to assemble contigs derived
from 100,000s of overlapping seq.s
Construct plasmid library from whole genome and seq. to
achieve 5-10 fold redundancy
Multiple sequences are aligned using algorithms that screen out
repetitive sequences
This assembly can resolve ~90% of the genome - filling in gaps
can take as long as initial phase
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Shotgun Sequencing - software
Screener - marks and hides (masks) seq. containing repetitive
DNA (e.g. microsatelites, LINEs, ribosomal DNA)
Seq. are not removed, but screened from alignment algorithm
so they do not contribute to overlap - length and location is
taken into account in genome assembly
Overlapper - compares all the reads searching for overlaps of
predetermined length and identity, e.g. 40bp overlaps w/ no
more than 6 errors - 1 in 1017 by chance
“errors” account for seq. errors, polymorphisms, heterozygotes
Parallel processing on 40 supercomputers, each w/ 4Gb of RAM
overlapped 27 million human seq. reads in <5 days
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Shotgun Sequencing - unitig
Unitigger - resolves repeat-induced overlaps due to low copy
number repeats (tandem or dispersed duplicates)
A unitig is a contig formed w/ a series of overlapping,
unambiguously unique sequences
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Shotgun Sequencing - overcollapsed
unitig
Overcollapsed unitigs are repetitive elements that have been
incorrectly collapsed into a single unitig
Overcollapsed unitigs can be identified b/c they appear to have
a much higher level of coverage (b/c of repeats through
genome) than the rest of the seq.
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Shotgun Sequencing - coverage
Highly likely unitigs (U-unitigs) formed from 6x coverage can
cover up to 98% of nonrepetitive euchromatin in stretches of
unique DNA greater than 2kb in length = 75% of the human
genome
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Shotgun Sequencing - software
Scaffolder - uses mate-pair information to link U-unitigs into
scaffold contigs
HGP and D. melanogaster project primarily seq.ed from both
ends of 2kb or 10 kb clones - these were correctly paired ~98%
of the time
Contiguity of assembled scaffold was verified using 50kb matepairs
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Shotgun Sequencing - gaps
Many remaining gaps are from repeats - and many can be
resolved by computationally aggressive, but more error-prone,
techniques
The level of coverage must balance genome size, funds and
needs of project
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Shotgun Sequencing - gaps
3 approaches to filling gaps
1. If gap from removed repeat, seq. can be reinserted as a
generic sequence - no polymorphisms etc
2. It may be possible to find a cloned seq. that bridges gap and
seq. that (e.g. 50 kb clone instead of 2 or 10kb clones)
3. Gaps may be bridged using seq. from different projects e.g.
cDNA seq., comparison w. other species in search of loci
that may bridge gap (design PCR primers to direct seq.ing)
Finally scaffolds are assembled into chromosomal locations,
compare with genetic map or place on cytological maps w/
fluorescent in situ hybridization (FISH)
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Phred
scoring
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Sequence Verification
Whole genome seq. must be assessed at 3 levels:
1. Completeness. Microbial genomes seq.ed for single isolates
in entirety, may contain ~kb gaps
Most higher eukaryotic genomes contain heterochromatin that
may never be seq.ed, except in small islands
2. Accuracy. Assessed by probability scores, can be increased
w/ greater coverage (overall or in spots)
3. Validity of assembly. Assessed by internal consistency or
contrasting w/ pre-existing genetic or physical maps. Can
compare w/ predicted restriction profiles w/ observed
fingerprints, and spacing of paired end seq.s
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Sequence Verification
At time of publication IHGSC and Celera human genomes, draft genomes,
contained hundreds of inconsistencies and gaps
Only chromsomes 21 and 22 were “finished” and these contained far fewer
problems = differences likely due to assembly
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