Difference between revisions of "Ksherbina Week 11"

Revision as of 05:55, 11 November 2013

Paper for Journal Club:

Stephens, R.S., Kalman, S., Lammel, C., Fan, J., Marathe, R., Aravind, L., Mitchell, W., Olinger, L., Tatusov, R., Zhao, Q., Koonin, E. V., Davis, R.W. (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282: 754-759. doi: 10.1126/science.282.5389.754

10 Biological Terms from Paper

• Biovar - A strain differentiated from other strains by biochemical or other non-serological means.

Abedon, S. T. (1998) Supplemental Lecture (98/04/14 update). <http://www.mansfield.ohio-state.edu/~sabedon/biol3010.htm>. Accessed 10 November 2013.

• Contig - Copies of pieces of DNA that represent the overlapping regions of a chromosome.

U.S. Department of Energy Human Genome Project (2013) The Human Genome Project Information Archive 1990-2003. <http://web.ornl.gov/sci/techresources/Human_Genome/glossary.shtml>. Accessed 10 November 2013.

• Hydrodynamic shearing - A technique that fragments DNA molecules by forcing them through a small orifice or the bore of a small-diameter tube at high velocity.

Joneja, A. & Huang, X. (2009) Supplementary Material For: A device for automated hydrodynamic shearing of genomic DNA. BioTechniques 46: 553–556.

• Serology - A type of laboratory medicine that analyzes blood serum for signs of infection by looking at antigen-antibody interactions in vivo.

Serology. (n.d.). Medical-Dictionary. <http://medical-dictionary.thefreedictionary.com/serology>. Accessed 10 November 2013.

Outline of the Article

Importance of sequencing the Chlamydia trachomatis genome

• C. trachomatis causes several diseases in human beings, including trachoma, which leads to blindness.
• C. trachomatis infections in humans may increase the risk of HIV infection.
• At the time the article was released, little was known about the two developmental stages of C. trachomatis (i.e. the elementary body and the reticulate body) as well as the bacterial pathogen's physiology and genetics.

Method used to sequence the genome of Chlamydia trachomatis strain D/UW-3/CX

• Chlamydial elementary bodies (EBs) were isolated from infected host cells using sonication on ice and then centrifuged to create pellets that were suspended in 5 mL of Hank's balanced salt solution.
• After two washes through centrifugation, extracellular DNA was removed by incubating the cells in 5µg/mL of DNase and RNase at 37°C for 30 min.
• The cells were purified using 30%, 40%, 45%, and 50% Renografin and then suspended in HBSS or SPG and stored at -70°C.
• After hydrodynamic shearing, the fragmented DNA was cloned into M13 phage.
• 28,458 sequencing reactions were performed using dye-labeled primers with the ABI Catalyst 800 Turbo robot followed by 4,688 dye-terminator reactions.
• 23 contigs from 4 to 164 kbp were observed using the Phrap and Phred software.
• Physical gaps in the genome were closed by either sequencing a PCR product that spans the gap or by using custom oligonucleotide primers.
• The plasmid that was sequenced from strain D/UW-3/CX had two less codons that previously sequenced chlamydial plasmids.
• Two methods were used to validate the assembled sequence: the predicted restriction digest map of the sequence as compared to the physical genome map of NotI and SgrAI and the restriction map and fragment sizes were analyzed after digesting oligonucleotides spaced 15kbp apart with BamHI .
• The results showed that the strain contains a 1,042,519 base pair chromosome and a 7493 bp plasmid.

Methods used to annotate the genome

Analysis of predicted chlamydial proteins and their function

Model Organism Database

http://efaecalis.mlst.net/ http://liferay.csb.univie.ac.at/portal/web/chlamydiaedb http://chlamydiae.com/twiki/bin/view/Main/Links/GeneLinks