1. genomic dna libraries for shotgun sequencing projects

8/8/2019 1. Genomic DNA Libraries for Shotgun Sequencing Projects

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TIGRTIGRTIGRTHE INSTITUTE FOR GENOMIC RESEARCHTHE INSTITUTE FOR GENOMIC RESEARCH

Genomic DNA Librariesfor Shotgun SequencingProjects

William C. Nierman


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Whole Genome Shotgun SequencingWhole Genome Shotgun Sequencing

Random Sequencing Phase

a. sequence DNA(15,000 sequences/ Mb)

GGG ACTGTTC ...

a. isolate DNA

b. fragment DNA

c. clone DNA

Closure Phase

a. assemble sequences

b. close gaps

d. annotation

c. edit

237 239

238COMPLETEGENOME SEQUENCE

Library construction


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Genomic Sequencing Overview

Large Insert Library (20 - 500 Kb)

PhysicalMap

Genomic DNAMarker1 Marker2

Shotgun Library (2-3 Kb)Sequencing

(6-8 X)

Assembly

Gap Closure

Analysis


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Genomic Sequencing Overview

Genomic DNAMarker1 Marker2

Shotgun Library (2,10, 50 Kb)

Sequencing(6-8 X)

Assembly

Gap Closure

Analysis


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Library Construction

Clone Picking

Template Preparation

SampleTracking

Sequencing Reactions

Electrophoresis andBase Calling

Sequence Files

Genome Assembly

Shotgun SequencingPhase


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graphical representation of phred quality values

Consensusquality values


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3 Tier Whole Genome Shotgun3 Tier Whole Genome ShotgunLibrary StrategyLibrary Strategy

1. Moderate copy number plasmidsplasmids containing ~2-kb inserts

2. Moderate copy number plasmids containing~10-kb inserts

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


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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 theassembly points to a telomeric repeat and is linked to a clone containing telomeric sequence

Repetitivesequences


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Repetitive Regions

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

Large-insertspanning clone,DMGRG22


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TIGRTIGRTIGRTHE INSTITUTE FOR GENOMIC RESEARCHTHE INSTITUTE FOR GENOMIC RESEARCH 4738A4737A


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Library Requirements1. Free vector should be at low or undetectable level.

2. None of the clones should contain chimeras derived byinsertion of two or more random fragments from separateparts 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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Vector

Vectorplus insert

DNA insertBstXI adaptor

CTTTCCAGCACA

GTGTGACCTTTC

GAAAGGTC

CTGGAAAG

Complementary to BstXI adaptor

Ligate

Ligat e

BstXI adaptor cloning system


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Library Requirements1. Free vector should be at low or undetectable level.

2. None of the clones should contain chimeras derived byinsertion 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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What is Unclonable DNA ?Difficult cloning targets include severaldifferent 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 theinsert induce expression of the cloned sequence. Fortuitous transcription out of the insert can interfere

with vector maintenance.

False positives and false negatives arise frominappropriate transcription. High copy number can cause plasmid instability.

lacP

Cloned fragment


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Sequencing Project Vector Features1. The sequencing primer sites immediately flank the cloningsite to avoid excessive re-sequencing of vector DNA.

2. PCR primer sites are located immediately outside of thesequencing primer sites to allow PCR amplification fortemplate preparation.

3. The entire cloning region including the primer sites is isolatedfrom RNA transcription.


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Design Features of a BstXI AdaptorCloning System

pHOS vector plus insert

AmpR

Ori, copynumber

ter1

ter2

Pr

BstXI site BstXI site Reverse sequencing primer

Reverse PCR primer

Forward sequencing primer

Forward PCR primer

rrnBT1 rrnBT2


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Construction of Linking Library in pHOS2Kan

genomic DNA ~50 kb with Bst XI adaptors

CTTTCCAGCACA

GAAAGGTC

CTGGAAAG

ACACGACCTTTC

pHOS2

RestrictionDigest

Amp

Amp

PhosphataseLigate Kan Cassette

Kan

Amp

Double Amp/KanSelection

pHOS2

pHOS2

Ligation


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Fosmid Library Construction


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


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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

16201284.2X3.2XTPG Large321

916.5X6.5X

TPG Small

4221045.1X2.5XGFS Large

21012310.4X10.8XGFS Small

1238384.1X3.2XGMX Large

13219963.4X2.2XGMX Small

279944.7X2.1XGBS Large

95758.9X9.0XGBS Small

ContigsScaffoldsCoverageActual

CoverageEst.

Genome &Insert Size

gbs = Streptococcus agalactiaegmx = Myxococcus xanthusgfs = Fibrobacter succinogenestpg = Theileria parva


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5 RNA'S8%

REPEATS15%FAILED MATES

1%

EDITING8%

COVERAGE3%

MATT'S HELP3%

SEQ GAPS12%

PHYS GAPS23%

2 DIFFICULT REPEATS27%

Comparison of Library Strategies

Genome BSP GBS GSA GSE S. pneumoniae S. agalactiae S.aureus S. epidermidis

Size MB 2.1 2.1 2.8 2.7 Groups 160 58 134 12Seq Gaps 290 46 198 24Start Date Nov 95 Dec 00 Mar 99 Feb 01In Closure 49 months 10 months 26 mon ths 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 fumigatusChromosomes

Presumed centromeric area

Telomere

3

2

6

5

8

7

1

4

Mitochondrion

rRNA

32 Kb

4.9 Mb

4.8 Mb

4.0 Mb

3.9 Mb

3.9 Mb

3.6 Mb

2.0 Mb

1.8 Mb

2.2 2.7

1.8 3.0

1.3 2.8

2.50.4 0.70.3

1.2 2.6

1.3 2.5

0.7 1.3

0.8 1.0


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1. genomic dna libraries for shotgun sequencing projects

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