hhmi talk final
TRANSCRIPT
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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
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In Situ: Fall Semester 2012
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In Situ
Isolated 20 novel phages
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Serenity
• Found by Isabel Jones in Pullman, WA
• Plaque Morphology:• 1-3 mm in diameter• Turbid and circular
• Siphoviridae
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In Silico: Spring 2013Nick Sisneros
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Serenity genome• Sequenced at Virginia Commonwealth University
• 454 Pyrosequencing
• 52088 bp long• 62.6% GC content
• Cluster A2 mycobacteriophage
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Comparison to other A2 phages
• Jsquared was discovered in Corpus Christi, TX
• Trixie was discovered in Chester, VA
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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
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Additional project• Sequenced Sillygoose, Baxter, Russell, Bear06 and AtticusBane • Using Single Molecule Real Time (SMRT) Sequencing
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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
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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
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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
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Zero-mode waveguide (ZMW)
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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
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SMRT sequencing
• Less background light means clearer results
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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
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Methylation•Able to detect methylation by the kinetics of DNA replication
•The type of modification can be determined by certain sequences
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SMRT sequencingresults for Bear06
•Average read length = 3000 bp
•Filtered reads to enhance assembly
•Coverage after filtering = 540x
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DNA Master with uncorrected file
DNA Master with corrected file
Likely start site Tape measure gene
Tape measure gene
Bear06
Checking the genome
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Verifying genome start sites
• Start sites are highly conserved in cluster A3 phages
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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
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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.
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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
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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®
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Thank you
Questions?
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In Situ: TEM results
Migo94 Sillygoose
Bear06
AtticusBane
Baxter Russell
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Serenity genome• Sequenced at Virginia Commonwealth University
• 454 Pyrosequencing
• 52088 bp long• 62.6% GC content
• Cluster A2 mycobacteriophage
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How SMRT sequencing works
Light Source
Dichroic
Obj
ectiv
e Le
ns
SMRT Cell(Multiplexed ZMWs)
Color Separation
Primary AnalysisBase Calling
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Start site
Phage Atticus Bane
Verifying genome start site