DNA Sequencing. CS262 Lecture 9, Win07, Batzoglou Method to sequence longer regions cut many times at random (Shotgun) genomic segment Get one or two.

DNA Sequencing

CS262 Lecture 9, Win07, Batzoglou

Method to sequence longer regions

cut many times at random (Shotgun)

genomic segment

Get one or two reads from each segment

~500 bp ~500 bp


Reconstructing the Sequence (Fragment Assembly)

Cover region with ~7-fold redundancy (7X)

Overlap reads and extend to reconstruct the original genomic region

reads


Definition of Coverage

Length of genomic segment: LNumber of reads: nLength of each read: l

Definition: Coverage C = n l / L

How much coverage is enough?

Lander-Waterman model:Assuming uniform distribution of reads, C=10 results in 1 gapped region /1,000,000 nucleotides

C


Repeats

Bacterial genomes: 5%Mammals: 50%

Repeat types:

• Low-Complexity DNA (e.g. ATATATATACATA…)

• Microsatellite repeats (a1…ak)N where k ~ 3-6(e.g. CAGCAGTAGCAGCACCAG)

• Transposons SINE (Short Interspersed Nuclear Elements)

e.g., ALU: ~300-long, 106 copies LINE (Long Interspersed Nuclear Elements)

~4000-long, 200,000 copies LTR retroposons (Long Terminal Repeats (~700 bp) at each end)

cousins of HIV

• Gene Families genes duplicate & then diverge (paralogs)

• Recent duplications ~100,000-long, very similar copies


Sequencing and Fragment Assembly

AGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCT

3x109 nucleotides

50% of human DNA is composed of repeats

Error!Glued together two distant regions


What can we do about repeats?

Two main approaches:• Cluster the reads

• Link the reads




• Link the reads




• Link the reads




3x109 nucleotides

C R D

ARB, CRD

or

ARD, CRB ?

A R B




3x109 nucleotides


Strategies for whole-genome sequencing

1. Hierarchical – Clone-by-clonei. Break genome into many long piecesii. Map each long piece onto the genomeiii. Sequence each piece with shotgun

Example: Yeast, Worm, Human, Rat

2. Online version of (1) – Walkingi. Break genome into many long piecesii. Start sequencing each piece with shotguniii. Construct map as you go

Example: Rice genome

3. Whole genome shotgun

One large shotgun pass on the whole genome

Example: Drosophila, Human (Celera), Neurospora, Mouse, Rat, Dog


Hierarchical Sequencing


Hierarchical Sequencing Strategy

1. Obtain a large collection of BAC clones2. Map them onto the genome (Physical Mapping)3. Select a minimum tiling path4. Sequence each clone in the path with shotgun5. Assemble6. Put everything together

a BAC clone

mapgenome


Methods of physical mapping

Goal:

Make a map of the locations of each clone relative to one another

Use the map to select a minimal set of clones to sequence

Methods:

• Hybridization

• Digestion


1. Hybridization

Short words, the probes, attach to complementary words

1. Construct many probes

2. Treat each BAC with all probes

3. Record which ones attach to it

4. Same words attaching to BACS X, Y overlap

p1 pn


2. Digestion

Restriction enzymes cut DNA where specific words appear

1. Cut each clone separately with an enzyme2. Run fragments on a gel and measure length3. Clones Ca, Cb have fragments of length { li, lj, lk }

overlap

Double digestion:Cut with enzyme A, enzyme B, then enzymes A + B


Some Terminologyinsert a fragment that was incorporated in a circular genome, and can be copied (cloned)

vector the circular genome (host) that incorporated the fragment

BAC Bacterial Artificial Chromosome, a type of insert–vector combination, typically of length 100-200 kb

read a 500-900 long word that comes out of a sequencing machine

coverage the average number of reads (or inserts) that cover a position in the target DNA piece

shotgun the process of obtaining many reads sequencing from random locations in DNA, to

detect overlaps and assemble


Whole Genome Shotgun Sequencing

cut many times at random

genome

forward-reverse paired reads

plasmids (2 – 10 Kbp)

cosmids (40 Kbp) known dist

~500 bp~500 bp


Fragment Assembly(in whole-genome shotgun sequencing)


Fragment Assembly

Given N reads…Given N reads…Where N ~ 30 Where N ~ 30

million…million…

We need to use a We need to use a linear-time linear-time algorithmalgorithm


Steps to Assemble a Genome

1. Find overlapping reads

4. Derive consensus sequence ..ACGATTACAATAGGTT..

2. Merge some “good” pairs of reads into longer contigs

3. Link contigs to form supercontigs

Some Terminology

read a 500-900 long word that comes out of sequencer

mate pair a pair of reads from two endsof the same insert fragment

contig a contiguous sequence formed by several overlapping readswith no gaps

supercontig an ordered and oriented set(scaffold) of contigs, usually by mate

pairs

consensus sequence derived from thesequene multiple alignment of reads

in a contig


1. Find Overlapping Reads

aaactgcagtacggatctaaactgcag aactgcagt… gtacggatct tacggatctgggcccaaactgcagtacgggcccaaa ggcccaaac… actgcagta ctgcagtacgtacggatctactacacagtacggatc tacggatct… ctactacac tactacaca

(read, pos., word, orient.)

aaactgcagaactgcagtactgcagta… gtacggatctacggatctgggcccaaaggcccaaacgcccaaact…actgcagtactgcagtacgtacggatctacggatctacggatcta…ctactacactactacaca

(word, read, orient., pos.)

aaactgcagaactgcagtacggatcta actgcagta actgcagtacccaaactgcggatctacctactacacctgcagtacctgcagtacgcccaaactggcccaaacgggcccaaagtacggatcgtacggatctacggatcttacggatcttactacaca



• Find pairs of reads sharing a k-mer, k ~ 24• Extend to full alignment – throw away if not >98% similar

TAGATTACACAGATTAC

TAGATTACACAGATTAC|||||||||||||||||

T GA

TAGA| ||

TACA

TAGT||

• Caveat: repeats A k-mer that occurs N times, causes O(N2) read/read comparisons ALU k-mers could cause up to 1,000,0002 comparisons

• Solution: Discard all k-mers that occur “too often”

• Set cutoff to balance sensitivity/speed tradeoff, according to genome at hand and computing resources available



Create local multiple alignments from the overlapping reads

TAGATTACACAGATTACTGATAGATTACACAGATTACTGATAG TTACACAGATTATTGATAGATTACACAGATTACTGATAGATTACACAGATTACTGATAGATTACACAGATTACTGATAG TTACACAGATTATTGATAGATTACACAGATTACTGA



• Correct errors using multiple alignment

TAGATTACACAGATTACTGATAGATTACACAGATTACTGATAGATTACACAGATTATTGATAGATTACACAGATTACTGATAG-TTACACAGATTACTGA

TAGATTACACAGATTACTGATAGATTACACAGATTACTGATAG-TTACACAGATTATTGATAGATTACACAGATTACTGATAG-TTACACAGATTATTGA

insert A

replace T with Ccorrelated errors—probably caused by repeats disentangle overlaps

TAGATTACACAGATTACTGATAGATTACACAGATTACTGA

TAG-TTACACAGATTATTGA

TAGATTACACAGATTACTGA

TAG-TTACACAGATTATTGA

In practice, error correction removes up to 98% of the errors


2. Merge Reads into Contigs

• Overlap graph: Nodes: reads r1…..rn

Edges: overlaps (ri, rj, shift, orientation, score)

Note:of course, we don’tknow the “color” ofthese nodes

Reads that comefrom two regions ofthe genome (blueand red) that containthe same repeat



We want to merge reads up to potential repeat boundaries

repeat region

Unique Contig

Overcollapsed Contig



• Ignore non-maximal reads• Merge only maximal reads into contigs

repeat region



• Remove transitively inferable overlaps If read r overlaps to the right reads r1, r2,

and r1 overlaps r2, then (r, r2) can be inferred by (r, r1) and (r1, r2)

r r1 r2 r3





• Ignore “hanging” reads, when detecting repeat boundaries

sequencing error

repeat boundary???

ba

a

b

…


Overlap graph after forming contigs

Unitigs:Gene Myers, 95


Repeats, errors, and contig lengths

• Repeats shorter than read length are easily resolved Read that spans across a repeat disambiguates order of flanking regions

• Repeats with more base pair diffs than sequencing error rate are OK We throw overlaps between two reads in different copies of the repeat

• To make the genome appear less repetitive, try to:

Increase read length Decrease sequencing error rate

Role of error correction:Discards up to 98% of single-letter sequencing errors

decreases error rate decreases effective repeat content increases contig length


• Insert non-maximal reads whenever unambiguous



3. Link Contigs into Supercontigs

Too dense Overcollapsed

Inconsistent links Overcollapsed?

Normal density


Find all links between unique contigs


Connect contigs incrementally, if 2 links

supercontig(aka scaffold)


Fill gaps in supercontigs with paths of repeat contigs



4. Derive Consensus Sequence

Derive multiple alignment from pairwise read alignments

TAGATTACACAGATTACTGA TTGATGGCGTAA CTATAGATTACACAGATTACTGACTTGATGGCGTAAACTATAG TTACACAGATTATTGACTTCATGGCGTAA CTATAGATTACACAGATTACTGACTTGATGGCGTAA CTATAGATTACACAGATTACTGACTTGATGGGGTAA CTA

TAGATTACACAGATTACTGACTTGATGGCGTAA CTA

Derive each consensus base by weighted voting

(Alternative: take maximum-quality letter)


Some Assemblers

• PHRAP• Early assembler, widely used, good model of read errors

• Overlap O(n2) layout (no mate pairs) consensus

• Celera• First assembler to handle large genomes (fly, human, mouse)

• Overlap layout consensus

• Arachne• Public assembler (mouse, several fungi)

• Overlap layout consensus

• Phusion• Overlap clustering PHRAP assemblage consensus

• Euler• Indexing Euler graph layout by picking paths consensus


Quality of assemblies

Celera’s assemblies of human and mouse


Quality of assemblies—mouse


Quality of assemblies—mouse

Terminology: N50 contig lengthN50 contig lengthIf we sort contigs from largest to smallest, and startCovering the genome in that order, N50 is the lengthOf the contig that just covers the 50th percentile.


Quality of assemblies—rat


History of WGA

• 1982: -virus, 48,502 bp

• 1995: h-influenzae, 1 Mbp

• 2000: fly, 100 Mbp

• 2001 – present human (3Gbp), mouse (2.5Gbp), rat*, chicken, dog, chimpanzee,

several fungal genomes

Gene Myers

Let’s sequence the human

genome with the shotgun

strategy

That is impossible, and

a bad idea anyway

Phil Green

1997


Genomes Sequenced

• http://www.genome.gov/10002154

DNA Sequencing. CS262 Lecture 9, Win07, Batzoglou Method to sequence longer regions cut many times at random (Shotgun) genomic segment Get one or two.

Documents

cs262 lecture

batzoglou sequencing

dna sequencing slide

genome sequencing

bp slide

arb slide

dog slide

nucleotides c slide