Transcript
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
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