EVE 161:Microbial Phylogenomics

Class #10: Genome Sequencing

UC Davis, Winter 2018 Instructor: Jonathan Eisen

Teaching Assistant: Cassie Ettinger

!1

RESEARCH ARTICLE

Whole-Genome RandomSequencing and Assembly ofHaemophilus influenzae Rd

Robert D. Fleischmann, Mark D. Adams, Owen White, Rebecca A. Clayton,Ewen F. Kirkness, Anthony R. Kerlavage, Carol J. Bult, Jean-Francois Tomb,Brian A. Dougherty, Joseph M. Merrick, Keith McKenney, Granger Sutton,

Will FitzHugh, Chris Fields,* Jeannine D. Gocayne, John Scott, Robert Shirley,Li-lng Liu, Anna Glodek, Jenny M. Kelley, Janice F. Weidman, Cheryl A. Phillips,

Tracy Spriggs, Eva Hedblom, Matthew D. Cotton, Teresa R. Utterback,Michael C. Hanna, David T. Nguyen, Deborah M. Saudek, Rhonda C. Brandon,Leah D. Fine, Janice L. Fritchman, Joyce L. Fuhrmann, N. S. M. Geoghagen,

Cheryl L. Gnehm, Lisa A. McDonald, Keith V. Small, Claire M. Fraser,Hamilton O. Smith, J. Craig Ventert

An approach for genome analysis based on sequencing and assembly of unselectedpieces of DNA from the whole chromosome has been applied to obtain the completenucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Hae-mophilus influenzae Rd. This approach eliminates the need for initial mapping efforts andis therefore applicable to the vast array of microbial species for which genome maps areunavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase ac-cession number L42023) represents the only complete genome sequence from a free-living organism.

A prerequisite to understanding the com-plete biology of an organism is the deter-mination of its entire genome sequence.Several viral and organellar genomes havebeen completely sequenced. Bacterio-phage 0X174 [5386 base pairs (bp)] wasthe first to be sequenced, by Fred Sangerand colleagues in 1977 (1). Sanger et al.were also the first to use strategy based onrandom (unselected) pieces of DNA, com-pleting the genome sequence of bacterio-phage X (48,502 bp) with cloned restric-tion enzyme fragments (1). Subsequently,the 229-kb genome of cytomegalovirus(CMV) (2), the 192-kb genome of vaccin-ia (3), and the 187-kb mitochondrial and121-kb chloroplast genomes of Marchantiapolymorpha (4) have been sequenced. The186-kb genome of variola (smallpox) wasthe first to be completely sequenced withautomated technology (5).

At the present time, there are active ge-nome projects for many organisms, includingDrosophila melanogaster (6), Escherichia coli(7), Saccharomyces cerevisiae (8), Bcillus sub-tilis (9), Caenorhabditis elegans (10), andJ.-F. Tomb, B. A. Dougherty, and H. O. Smith are with theJohns Hopkins University School of Medicine, Baltimore,MD 21205, USA. J. M. Merrick is with the State Universityof New York, Department of Microbiology, Buffalo, NY,14214, USA. K. McKenney is with the National Institutefor Standards and Technology, Gaithersburg, MD 20878,USA. All other authors are with The Institute for GenomicResearch (TIGR), Gaithersburg, MD, 20878, USA. Theaddress for TIGR as of 9 September 1995 is 9712 Med-ical Center Drive, Rockville, MD 20850, USA.*Present address: The National Center for Genome Re-sources, Santa Fe, NM, 87505, USA.tTo whom correspondence should be addressed.

496

Homo sapiens (11). These projects, as well as

viral genome sequencing, have been basedprimarily on the sequencing of clones usuallyderived from extensively mapped restrictionfragments, or or cosmid clones. Despiteadvances in DNA sequencing technology(12) the sequencing of genomes has not

progressed beyond clones on the order of thesize of (-40 kb). This has been primarilybecause of the lack of sufficient computa-tional approaches that would enable the ef-ficient assembly of a large number (tens ofthousands) of independent, random se-

quences into a single assembly.The computational methods developed

to create assemblies from hundreds of thou-sands of 300- to 500-bp complementaryDNA (cDNA) sequences (13) led us to testthe hypothesis that segments of DNA sev-

eral megabases in size, including entire mi-crobial chromosomes, could be sequencedrapidly, accurately, and cost-effectively byapplying a shotgun sequencing strategy towhole genomes. With this strategy, a singlerandom DNA fragment library may be pre-pared, and the ends of a sufficient numberof randomly selected fragments may be se-

quenced and assembled to produce thecomplete genome. We chose the free-livingorganism Haemophilus influenzae Rd as a

pilot project because its genome size (1.8Mb) is typical among bacteria, its G+Cbase composition (38 percent) is close tothat of human, and a physical clone mapdid not exist.

Haemophilus influenzae is a small, nonmo-tile, Gram-negative bacterium whose onlySCIENCE * VOL. 269 * 28 JULY 1995

natural host is human. Six H. influenzaeserotype strains (a through f) have beenidentified on the basis of immunologicallydistinct capsular polysaccharide antigens.Non-typeable strains also exist and are dis-tinguished by their lack of detectable capsu-lar polysaccharide. They are commensal res-idents of the upper respiratory mucosa ofchildren and adults and cause otitis mediaand respiratory tract infections, mostly inchildren. More serious invasive infection iscaused almost exclusively by type b strains,with meningitis producing neurological se-quelae in up to 50 percent of affected chil-dren. A vaccine based on the type b capsularantigen is now available and has dramatical-ly reduced the incidence of the disease inEurope and North America.

Genome sequencing. The strategy for ashotgun approach to whole genome se-quencing is outlined in Table 1. The theoryfollows from the Lander and Waterman(14) application of the equation for thePoisson distribution. The probability that abase is not sequenced is P0 = e-", where mis the sequence coverage. Thus after 1.83Mb of sequence has been randomly gener-ated for the H. influenzae genome (m = 1, 1X coverage), P0 = e-1 = 0.37 and approx-imately 37 percent of the genome is unse-quenced. Fivefold coverage (approximately9500 clones sequenced from both insertends and an average sequence read lengthof 460 bp) yields P, = e-5 = 0.0067, or 0.67percent unsequenced. If L is genome lengthand n is the number of random sequencesegments done, the total gap length is Le-",and the average gap size is L/n. Fivefoldcoverage would leave about 128 gaps aver-aging about 100 bp in size.

To approximate the random model dur-ing actual sequencing, procedures for libraryconstruction (15) and cloning (16) weredeveloped. Genomic DNA from H. influen-zae Rd strain KW20 (17) was mechanicallysheared, digested with BAL 31 nuclease toproduce blunt ends, and size-fractionated byagarose gel electrophoresis. Mechanicalshearing maximizes the randomness of theDNA fragments. Fragments between 1.6and 2.0 kb in size were excised and recov-ered. This narrow range was chosen to min-imize variation in growth of clones. In ad-dition, we chose this maximum size to min-imize the number of complete genes thatmight be present in a single fragment, andthus might be lost as a result of expressionof deleterious gene products. These frag-ments were ligated to Sma I-cut, phos-phatase-treated pUC18 vector, and the li-gated products were fractionated on an aga-rose gel. The linear vector plus insert bandwas excised and recovered. The ends of thelinear recombinant molecules were repairedwith T4 polymerase, and the moleculeswere then ligated into circles. This two-

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• Presenters?

• Questions / To Cover?

RESEARCH ARTICLE

Whole-Genome RandomSequencing and Assembly ofHaemophilus influenzae Rd

Robert D. Fleischmann, Mark D. Adams, Owen White, Rebecca A. Clayton,Ewen F. Kirkness, Anthony R. Kerlavage, Carol J. Bult, Jean-Francois Tomb,Brian A. Dougherty, Joseph M. Merrick, Keith McKenney, Granger Sutton,

Will FitzHugh, Chris Fields,* Jeannine D. Gocayne, John Scott, Robert Shirley,Li-lng Liu, Anna Glodek, Jenny M. Kelley, Janice F. Weidman, Cheryl A. Phillips,

Tracy Spriggs, Eva Hedblom, Matthew D. Cotton, Teresa R. Utterback,Michael C. Hanna, David T. Nguyen, Deborah M. Saudek, Rhonda C. Brandon,Leah D. Fine, Janice L. Fritchman, Joyce L. Fuhrmann, N. S. M. Geoghagen,

Cheryl L. Gnehm, Lisa A. McDonald, Keith V. Small, Claire M. Fraser,Hamilton O. Smith, J. Craig Ventert

An approach for genome analysis based on sequencing and assembly of unselectedpieces of DNA from the whole chromosome has been applied to obtain the completenucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Hae-mophilus influenzae Rd. This approach eliminates the need for initial mapping efforts andis therefore applicable to the vast array of microbial species for which genome maps areunavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase ac-cession number L42023) represents the only complete genome sequence from a free-living organism.

A prerequisite to understanding the com-plete biology of an organism is the deter-mination of its entire genome sequence.Several viral and organellar genomes havebeen completely sequenced. Bacterio-phage 0X174 [5386 base pairs (bp)] wasthe first to be sequenced, by Fred Sangerand colleagues in 1977 (1). Sanger et al.were also the first to use strategy based onrandom (unselected) pieces of DNA, com-pleting the genome sequence of bacterio-phage X (48,502 bp) with cloned restric-tion enzyme fragments (1). Subsequently,the 229-kb genome of cytomegalovirus(CMV) (2), the 192-kb genome of vaccin-ia (3), and the 187-kb mitochondrial and121-kb chloroplast genomes of Marchantiapolymorpha (4) have been sequenced. The186-kb genome of variola (smallpox) wasthe first to be completely sequenced withautomated technology (5).

At the present time, there are active ge-nome projects for many organisms, includingDrosophila melanogaster (6), Escherichia coli(7), Saccharomyces cerevisiae (8), Bcillus sub-tilis (9), Caenorhabditis elegans (10), andJ.-F. Tomb, B. A. Dougherty, and H. O. Smith are with theJohns Hopkins University School of Medicine, Baltimore,MD 21205, USA. J. M. Merrick is with the State Universityof New York, Department of Microbiology, Buffalo, NY,14214, USA. K. McKenney is with the National Institutefor Standards and Technology, Gaithersburg, MD 20878,USA. All other authors are with The Institute for GenomicResearch (TIGR), Gaithersburg, MD, 20878, USA. Theaddress for TIGR as of 9 September 1995 is 9712 Med-ical Center Drive, Rockville, MD 20850, USA.*Present address: The National Center for Genome Re-sources, Santa Fe, NM, 87505, USA.tTo whom correspondence should be addressed.

496

Homo sapiens (11). These projects, as well as

viral genome sequencing, have been basedprimarily on the sequencing of clones usuallyderived from extensively mapped restrictionfragments, or or cosmid clones. Despiteadvances in DNA sequencing technology(12) the sequencing of genomes has not

progressed beyond clones on the order of thesize of (-40 kb). This has been primarilybecause of the lack of sufficient computa-tional approaches that would enable the ef-ficient assembly of a large number (tens ofthousands) of independent, random se-

quences into a single assembly.The computational methods developed

to create assemblies from hundreds of thou-sands of 300- to 500-bp complementaryDNA (cDNA) sequences (13) led us to testthe hypothesis that segments of DNA sev-

eral megabases in size, including entire mi-crobial chromosomes, could be sequencedrapidly, accurately, and cost-effectively byapplying a shotgun sequencing strategy towhole genomes. With this strategy, a singlerandom DNA fragment library may be pre-pared, and the ends of a sufficient numberof randomly selected fragments may be se-

quenced and assembled to produce thecomplete genome. We chose the free-livingorganism Haemophilus influenzae Rd as a

pilot project because its genome size (1.8Mb) is typical among bacteria, its G+Cbase composition (38 percent) is close tothat of human, and a physical clone mapdid not exist.

Haemophilus influenzae is a small, nonmo-tile, Gram-negative bacterium whose onlySCIENCE * VOL. 269 * 28 JULY 1995

natural host is human. Six H. influenzaeserotype strains (a through f) have beenidentified on the basis of immunologicallydistinct capsular polysaccharide antigens.Non-typeable strains also exist and are dis-tinguished by their lack of detectable capsu-lar polysaccharide. They are commensal res-idents of the upper respiratory mucosa ofchildren and adults and cause otitis mediaand respiratory tract infections, mostly inchildren. More serious invasive infection iscaused almost exclusively by type b strains,with meningitis producing neurological se-quelae in up to 50 percent of affected chil-dren. A vaccine based on the type b capsularantigen is now available and has dramatical-ly reduced the incidence of the disease inEurope and North America.

Genome sequencing. The strategy for ashotgun approach to whole genome se-quencing is outlined in Table 1. The theoryfollows from the Lander and Waterman(14) application of the equation for thePoisson distribution. The probability that abase is not sequenced is P0 = e-", where mis the sequence coverage. Thus after 1.83Mb of sequence has been randomly gener-ated for the H. influenzae genome (m = 1, 1X coverage), P0 = e-1 = 0.37 and approx-imately 37 percent of the genome is unse-quenced. Fivefold coverage (approximately9500 clones sequenced from both insertends and an average sequence read lengthof 460 bp) yields P, = e-5 = 0.0067, or 0.67percent unsequenced. If L is genome lengthand n is the number of random sequencesegments done, the total gap length is Le-",and the average gap size is L/n. Fivefoldcoverage would leave about 128 gaps aver-aging about 100 bp in size.

To approximate the random model dur-ing actual sequencing, procedures for libraryconstruction (15) and cloning (16) weredeveloped. Genomic DNA from H. influen-zae Rd strain KW20 (17) was mechanicallysheared, digested with BAL 31 nuclease toproduce blunt ends, and size-fractionated byagarose gel electrophoresis. Mechanicalshearing maximizes the randomness of theDNA fragments. Fragments between 1.6and 2.0 kb in size were excised and recov-ered. This narrow range was chosen to min-imize variation in growth of clones. In ad-dition, we chose this maximum size to min-imize the number of complete genes thatmight be present in a single fragment, andthus might be lost as a result of expressionof deleterious gene products. These frag-ments were ligated to Sma I-cut, phos-phatase-treated pUC18 vector, and the li-gated products were fractionated on an aga-rose gel. The linear vector plus insert bandwas excised and recovered. The ends of thelinear recombinant molecules were repairedwith T4 polymerase, and the moleculeswere then ligated into circles. This two-

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What Genomes Done Before?

• What Genomes Had Been Sequenced?

How Were Genomes Being Sequenced?

• How Were Genomes Being Sequenced?

What is new here?

• What did they do that was new in terms of sequencing?

Organisms Chosen

• What is H. Influenzae?

• Why did they choose it?

Sequencing Details?

• Outline of sequencing approach?

Shotgun Sequencing

• What is shotgun sequencing?

• How does it work?

Shotgun Sequencing

• What is shotgun sequencing?

• How does it work?

• How can you complete a genome this way?

Lander Waterman

P0=e-m

P0 = probability a base is

not sequenced

m = coverage

?

P0=e-m

P0 = probability a base is

not sequenced

WHY DOES RANDOMNESS

MATTER?

Show Curve

00.10.20.30.40.50.60.70.80.9

1

0 2000 4000 6000 8000 10000 12000Perc

ent U

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ed

Number of Reads

Lander Waterman Curve

100 Bp 500 bp 1000 bp

Other issues

• Why do they talk about ESTS?

• Why do they need a database?

Assembly

Assembly

Assembly Method

• TIGR Assembler

• Smith Waterman

• Paired Ends

Assembly Results

• Contigs

• Sequencing Gaps

• Physical Gaps

Gap Filling

Gap Filling

• DNA Hybridization

• Peptide Links

• Phage lambda libraries

• PCR

Accuracy Checks?

Accuracy Checks?

• Frameshift in protein sequences vs. Homologs

• Coverage > 1x

• Few ambiguities

• Comparison to H. Influenza sequence in DBs

Cost

Cost

• 0.48$ / finished bp

Annotation

• Structural

• Functional

Annotation

• Structural

• Functional

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on February 8, 2018

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