Genomic DNA Libraries

for Shotgun Sequencing

Projects

William C. Nierman

TIGR
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Whole Genome Shotgun Sequencing
Library construction Random Sequencing Phase Closure Phase

a. sequence DNA
a. assemble sequences
(15,000 sequences/ Mb)
a. isolate DNA
b. close gaps

b. fragment DNA
c. edit
c. clone DNA GGG ACTGTTC ...

d. annotation

237 239

COMPLETE 238
GENOME SEQUENCE

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 Genomic Sequencing Overview
Marker1 Marker2
Genomic DNA

Large Insert Library (20 - 500 Kb)

Physical
Map

Shotgun Library (2-3 Kb)

Sequencing
(6-8 X)

Assembly

Gap Closure
Analysis
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 Genomic Sequencing Overview
Marker1 Marker2
Genomic DNA

Shotgun Library (2,10, 50 Kb)

Sequencing
(6-8 X)

Assembly

Gap Closure
Analysis
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Shotgun Sequencing
Phase
Library Construction

Clone Picking

Template Preparation
Sample
Tracking
Sequencing Reactions

Electrophoresis and
Base Calling

Sequence Files

Genome Assembly

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Consensus
quality values

graphical representation of phred quality values

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 3 Tier Whole Genome Shotgun
Library Strategy

1. Moderate copy number plasmids containing ~2-

kb inserts

2. Moderate copy number plasmids containing

~10-kb inserts

3. Fosmid or other clones containing 40 - 200-kb

inserts

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 Repetitive
sequences

TIGR Assembly Viewer. Green arrows represent F and R sequences from the same clone.
Red arrows represent sequences with a sequence mate in a different contig. 5’ end of the
assembly points to a telomeric repeat and is linked to a clone containing telomeric sequence

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 Repetitive Regions

Large-insert
spanning clone,
DMGRG22

Output from the TIGR software tool repeat Display showing a section of an assembly. The
black boxes represent a 700 bp repeat (7V, 24 copies/genome) and a 3100 bp repeat (9D, 9
copies/genome). Both repeats are spanned by clone DMGRG22. To confirm the sequence
of these repeats, this clone was transposed.

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 TIGR
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 4737A
TIGR
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4738A
 Library Requirements
1. Free vector should be at low or undetectable level.

2. None of the clones should contain chimeras derived by

insertion of two or more random fragments from separate
parts of the genome.

3. The inserts should be of relatively uniform size.

4. Libraries of different insert sizes for linking should be used.

5. Libraries should be representative of genome.

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 BstXI adaptor cloning system
DNA insert
BstXI adaptor
CTTTCCAGCACA
Ligate GAAAGGTC

Ligate Complementary to BstXI adaptor

CTGGAAAG
GTGTGACCTTTC

Vector

Vector
plus insert

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 Library Requirements
1. Free vector should be at low or undetectable level.

2. None of the clones should contain chimeras derived by

insertion of two or more random fragments from separate
parts of the genome.

3. The inserts should be of relatively uniform size.

4. Libraries of different insert sizes for linking should be used.

5. Libraries should be representative of genome.

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 Genome representation in
shotgun libraries is limited by
the need for faithful
propagation in Escherichia coli

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 TIGR
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 TIGR
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 TIGR
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 What is “Unclonable” DNA ?
Difficult cloning targets include several
different types of sequences, such as:
– Toxic coding sequences
– Promoters
– A/T Rich DNA
– Modified bases
– Repetitive regions

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Library Coverage and Randomness

• Tolerance of cloned DNA by E.

coli host

• Vector copy number

• Insert size

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 Vector Design Issues
• Vector driven transcription and translation into the
insert induce expression of the cloned sequence.
• Fortuitous transcription out of the insert can interfere
with vector maintenance.
• False positives and false negatives arise from
inappropriate transcription.
• High copy number can cause plasmid instability.

P lac

Cloned fragment

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 Sequencing Project Vector Features
1. The sequencing primer sites immediately flank the cloning
site to avoid excessive re-sequencing of vector DNA.

2. PCR primer sites are located immediately outside of the

sequencing primer sites to allow PCR amplification for
template preparation.

3. The entire cloning region including the primer sites is isolated

from RNA transcription.

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 Design Features of a BstXI Adaptor
Cloning System
Forward sequencing primer Reverse sequencing primer
BstXI site BstXI site
Forward PCR primer Reverse PCR primer
rrnBT1 rrnBT2

ter2

pHOS vector plus insert

Ori, copy
Pr number

AmpR ter1

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 Construction of Linking Library in pHOS2Kan
genomic DNA with BstXI adaptors
~50 kb Ligation
CTGGAAAG CTTTCCAGCACA
ACACGACCTTTC GAAAGGTC pHOS2
Amp
Restriction
Digest
Double Amp/Kan
Selection
pHOS2
Kan
Amp
pHOS2

Amp Phosphatase
Ligate Kan Cassette

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 TIGR
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 Fosmid Library Construction

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Copy Number Induced (+) vs. Uninduced (-) Fosmid DNA preps

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 TIGR
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 TIGR
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 Library Mix
Wolbachia (endosymbiont of B. malayi)
Sequenced to 20X
True genome size: 1,080,471 bases.
At 7.6X coverage:
0% small, 100% large gave 1 scaffold
of 1,076,660 bp and 10 contigs
5% small, 95% large gave 1 scaffold of
1,077,210 bp and 12 contigs
60% small, 40% large gave 14 scaffolds
(largest=160 kb), 79 contigs

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 Redundancy Analysis from Completed Projects

Genome & Coverage Coverage Scaffolds Contigs

Insert Size Est. Actual
GBS Small 9.0X 8.9X 75 95
GBS Large 2.1X 4.7X 94 279
GMX Small 2.2X 3.4X 996 1321
GMX Large 3.2X 4.1X 38 1238
GFS Small 10.8X 10.4X 123 210
GFS Large 2.5X 5.1X 104 422

TPG Small 6.5X 6.5X 91 321

TPG Large 3.2X 4.2X 128 1620

gbs = Streptococcus agalactiae

gmx = Myxococcus xanthus
gfs = Fibrobacter succinogenes
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tpg = Theileria parva
 GBS of
Comparison EFFORT
Library Strategies
PHYS GAPS
23%
2 DIFFICULT REPEATS
27%

SEQ GAPS
12%
5 RNA'S
8%

MATT'S HELP
3%
COVERAGE
3%
REPEATS
EDITING 15%
FAILED MATES
8%
Genome BSP GBS 1%
GSA GSE
S. pneumoniae S. agalactiae S.aureus S. epidermidis
Size MB 2.1 2.1 2.8 2.7
Groups 160 58 134 12
Seq Gaps 290 46 198 24
Start Date Nov 95 Dec 00 Mar 99 Feb 01
In Closure 49 months 10 months 26 months 7 months

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 Myxococcus xanthus Sequencing Statistics
• Total shotgun sequences _ 130,436
– TIGR Library insert sizes 2-3 kb, 10-12 kb
– Sequence coverage of 9X
• Assembled into single scaffold of 103 contigs
• Two rounds of autoprimer sequencing
reduced contig number to 36
• 9,131,959 bases, 3500 Ns in gaps

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 Aspergillus fumigatus karyotype

1,789 Kb

3,779 Kb

2,021 Kb

3,992 Kb

4,018 Kb

4,834 Kb

4,891 Kb

3,933* Kb
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 Optical Analysis

• Molecule maps generated from images of

single DNA molecule digested with NheI
• Resolution (avg fragment size) 8.28kb
• Total coverage: 8,987 Mbase, or 300x
• Total of 8 chromosomes
• Total size: 29.189 Megabases

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 A. fumigatus chr5-7 contig placement

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 Aspergillus fumigatus
Chromosomes

1 2.2 2.7 4.9 Mb

2 1.8 3.0 4.8 Mb

3 1.3 2.8 4.0 Mb

rRNA
4 0.4 0.7 2.5 3.9 Mb
0.3

5 1.2 2.6 3.9 Mb

6 1.3 2.5 3.6 Mb

Mitochondrion
7 0.7 1.3 2.0 Mb
32 Kb
8 0.8 1.0 1.8 Mb

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Telomere
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 TIGR
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 TIGR
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