Guide Home

Phage isolation
and purification

Clone library
construction


Sequencing

Assembly

Post-assembly
analyses

The Guide to Phage Genomics: Isolation, Purification, Cloning, Sequencing, Assembling, Annotating and Analyzing Phage Genomes


PHAGE ISOLATION and PURIFICATION

Isolate and plaque purify phage
Grow up to get ~ 10^12 phage and harvest the lysate

PEG precipitate to concentrate the phage
Isolate the phage particles using CsCl (equilibrium or step gradient)
Pull the phage band and dialyze against a buffer
Extract the phage DNA

CONSTRUCT A CLONE LIBRARY

Do you have <100 ng DNA or potentially problematic regions of DNA (lethal genes or modified bases)?
If yes, then proceed with LASL / RASLs library construction
If no (unlikely with phage genomes), then proceed with conventional library construction

SEQUENCING

Sequence random clones to get 4-10 fold coverage

ASSEMBLY

Software for sequence evaluation and assembly
Raw sequence data to a first assembly
Closing gaps between your assembled contigs

Finding the ends

POST-ASSEMBLY ANALYSES

ORF finding
BLASTing the ORFs
Identifying ORFs by synteny
tRNA
Repeats

Codon Bias
Dinucleotide analysis
Comparative genomics analyses
Placement on the Phage Proteomic Tree


Page created: February 20, 2003
Last modified: February 24, 2003
For questions or comments, e-mail Forest Rohwer forest@sunstroke.sdsu.edu or Matt Sullivan mbsulli@mit.edu