Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Harvard Medical School
3
Genome Sequencing from Single Cells with Ploning
Kun Zhang1* (kzhang@genetics.med.harvard.edu), Adam C. Martiny2, Nikkos B. Reppas1, Sallie W. Chisholm2, and George M. Church1
1Harvard Medical School, Boston, MA and 2Massachusetts Institute of Technology, Cambridge, MA
Currently genome sequencing is performed on cell populations because of the difficulty in preparing sequencing template from single cells. This makes the genome sequences of many difficult-to-culture organisms inaccessible or poorly assembled. We have developed a method that enables genome sequencing from a single cell by performing polymerase cloning (ploning). In this method, we prepare sequencing templates from single cells with real-time multiple displacement amplification (rtMDA), which allows us to tackle the big technical challenge in single-cell whole genome analysis: to detect and suppress spurious amplification while targeting a single molecule of a microbial chromosome.
Experiments on Escherichia coli show that, (1) an amplification magnitude of 109 was achieved by rtMDA, (2) strain-specific genetic signatures were preserved, (3) neither spurious amplification product nor chimeric sequence was detected, (4) an estimated 97% of the target genome could be recovered from a polymerase clone (plone) at the 10X sequencing depth. The remaining regions are not missing, but present at lower copy numbers, and easily recovered by PCR. Since the low-coverage regions seem random, genome coverage can be improved by pooling the sequencing reads from two or more plones of the same type of cells during the assembly stage. Furthermore, we successfully performed ploning on both fresh and frozen Prochlorococcus cells, and obtained nearly complete coverage on both strains (MED4 and MIT9312) we tested. Plones of single cells from an ocean sample (from the Hawaii Ocean Time-series) are being screened for Prochlorococcus cells for genome sequencing. Initial results indicate successful amplification of single Prochlorococcus cells from this sample. After further screening of genome coverage, whole genome shot-gun sequencing will be performed on a few selected plones.
* Presenting author
