Genome analysis of the novel cluster A2 phage Serenity, and an introduction to Single Molecule Real Time (SMRT) Sequencing

Presented by Mitchell GoWashington State University Pullman, WA

In Situ: Fall Semester 2012

In Situ

Isolated 20 novel phages

Serenity

• Found by Isabel Jones in Pullman, WA

• Plaque Morphology:• 1-3 mm in diameter• Turbid and circular

• Siphoviridae

In Silico: Spring 2013Nick Sisneros

Serenity genome• Sequenced at Virginia Commonwealth University

• 454 Pyrosequencing

• 52088 bp long• 62.6% GC content

• Cluster A2 mycobacteriophage

Comparison to other A2 phages

• Jsquared was discovered in Corpus Christi, TX

• Trixie was discovered in Chester, VA

Comparison to other A2 phages

Serenity

Jsquared

Trixie

• Typically see more diversity in second half of the genome

• Serenity and Jsquared are very different from most A2 phages

Additional project• Sequenced Sillygoose, Baxter, Russell, Bear06 and AtticusBane • Using Single Molecule Real Time (SMRT) Sequencing

Introduction to SMRT sequencing

• SMRT sequencing allows for observing DNA polymerase reactions in real time• Records the nucleotides used in DNA replication

• Advantages over 454 Sequencing:• Longer readlengths• Cost less• Phospholinked labels• Zero-mode waveguides (ZMWs)• DNA methylation detection

SMRT vs. 454SMRT 454

Readlengths 3,000 bps 500 bps

Cost $100 $500-$1,000

Fluorescent tags Phospholinked Baselinked

Reaction chamber SMRT Cell/ ZMW DNA Library beads/ PicoTiterPlate device

BP modification detection

5-methylcytosine, N6-methyladnenine, N4-methylcytosine, DNA oxidative damage, etc.

5-methylcytosine

Phospholinked labels • Fluorophores are

phospholinked rather than baselinked

• Each nucleotide has is own color

• Fluorescent tag cleaved by polymerase• Less background light

Baselinked

Phospholinked

454

SMRT

Zero-mode waveguide (ZMW)

Zero-mode waveguide (ZMW)

• Diameter of hole smaller than wavelength of light

• Allows for small illumination volume

• Only illuminates nucleotides that diffuse to bottom of ZMW

• Fluorescent tags cannot emit vertically

SMRT sequencing

• Less background light means clearer results

Real-time detection

•A light pulse is produced at the bottom of each ZMW

•The attached flurophore is released and a colored light is emitted

•Video cameras record light pulses and deliver them to be analyzed

Methylation•Able to detect methylation by the kinetics of DNA replication

•The type of modification can be determined by certain sequences

SMRT sequencingresults for Bear06

•Average read length = 3000 bp

•Filtered reads to enhance assembly

•Coverage after filtering = 540x

DNA Master with uncorrected file

DNA Master with corrected file

Likely start site Tape measure gene

Tape measure gene

Bear06

Checking the genome

Verifying genome start sites

• Start sites are highly conserved in cluster A3 phages

Bear06 SMRT results• Cluster A3 mycobacteriophage

52,412 bp, 64% GC content

• Annotation 89 putative genes, 3 tRNA genes Function predicted for 24/89 genes

•Possible guanine methylation At GGCNA

Conclusions• Serenity’s genome is 52088 base pairs

long and has a GC content of 62.6%

• There are 95 genes in which only approximately 21% had a potential function.

Conclusions/ future plans• SMRT Sequencing was successfully

used to sequence mycobacteriophages

• When compared to 454, SMRT delivers:• Longer readlengths, less cost, wider

range detection of nucleotide modifications

• Look into possible guanine methylation in Bear06, other mycobacteriophages and Mycobacterium smegmatis

Acknowledgements• Howard Hughes Medical Institute SEA-PHAGES

• School of Biological Sciences and School of Molecular Biosciences at Washington State University

• Dr. Patrick Carter, Dr. William Davis, Dr. McKenna Kyriss, Stacy Hathcox, Steven Micheletti, and Dr. Julie Stanton

• Nick Sisneros & Pacific BioSciences®

Thank you

Questions?

In Situ: TEM results

Migo94 Sillygoose

Bear06

AtticusBane

Baxter Russell

Serenity genome• Sequenced at Virginia Commonwealth University

• 454 Pyrosequencing

• 52088 bp long• 62.6% GC content

• Cluster A2 mycobacteriophage

How SMRT sequencing works

Light Source

Dichroic

Obj

ectiv

e Le

ns

SMRT Cell(Multiplexed ZMWs)

Color Separation

Primary AnalysisBase Calling

Start site

Phage Atticus Bane

Verifying genome start site