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Based on the comparison of different sequencing strategies in six small marine microbial genome, the paper evaluated the utility and cost-effectiveness of a hybrid sequencing approach using 3730xl Sanger sequecing and 454 run to generate higher-quality lower-quality lower-cost assemblies compared to current Sanger sequencing strategies alone. For the genome more than 3Mb with many sequencing gaps and hard stops, the sequence strategy of 5.3X Sanger sequencing plus two 454 runs is the best choice.

Proc Natl Acad Sci U S A. 2006 Jul 13; [Epub ahead of print] Books, LinkOut

A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes.

Goldberg SM, Johnson J, Busam D, Feldblyum T, Ferriera S, Friedman R, Halpern A, Khouri H, Kravitz SA, Lauro FM, Li K, Rogers YH, Strausberg R, Sutton G, Tallon L, Thomas T, Venter E, Frazier M, Venter JC.
J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850;
Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850;
Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093-0202.

Since its introduction a decade ago, whole-genome shotgun sequencing (WGS) has been the main approach for producing cost-effective and high-quality genome sequence data. Until now, the Sanger sequencing technology that has served as a platform for WGS has not been truly challenged by emerging technologies. The recent introduction of the pyrosequencing-based 454 sequencing platform (454 Life Sciences, Branford, CT) offers a very promising sequencing technology alternative for incorporation in WGS. In this study, we evaluated the utility and cost-effectiveness of a hybrid sequencing approach using 3730xl Sanger data and 454 data to generate higher-quality lower-cost assemblies of microbial genomes compared to current Sanger sequencing strategies alone.

1. Sanger Sequencing for Genome:
Introduced Time: > 10 yrs
Cost: $8000-$10000 / M
for small marine microbes, 5.3X Sanger Sequencing Data is OK.

2. About 454 GS20 Sequencing Platform:
from 454 Life Science
100 times faster
length: ~100 bp

Six Marine Microbial Genomes:
1. Janibacter sp. HTCC2649,
2. Erythrobacter litoralis HTCC2594,
3. Prochlorococcus marinus str. MIT9211,
4. Pseudoalteromonas tunicata D2,
5. Vibrio angustum S14,
6. Psychromonas sp. CNPT3

Results:
1. 5.3X Sanger sequencing data + one/two 454 sequencing run, Gaps closed, N50 Contigs sequence increase.
2. for additional data of 454, the number of gaps decreased, however, the total amount of missing sequence increased in many cases.
3. the addition of 454 data did not increase the quality of assembly in repetitive areas of any of the six genomes.
4. Sequencing Strategy:
5X Sanger WGS + 3X addtional Sanger WGS:
a. small genome size (<3Mb)
b. a small number of gaps
c. high levels of repetitive structure including physical ends
5X Sanger WGS + Two 454 Runs:
a. large genome size
b. many sequencing gaps
c. many hard stops

Materials and Methods:
1. Laboratory:
454 processing:
a. the methods and protocals by Rothberg.
b. 5 ug of microbial DNA for the library preparation:
i) double-stranded
ii) nonamplified
iii) nondegraded
iv) DNA fragments of > 1.5Kb
v) had an OD 260/280 reading of >= 1.8
vi) concentration of >= 50ng/ul
vii) suspended in TE buffer (10mM Tris/1mM EDTA, pH 8.0)
High-throughput AB 3730xl Sanger sequencing processing:
a. Library construction : BstXI
b. Clone picking and inoculaiton : Q-bot or Q-Pix colony-picking robots
c. DNA template preparation : Thermo CRS
d. Sequencing reactions : Biomek FX (average read length >800bp)
2. Informatics:
Pseudoread Creation:
a. by 454's Newbler assember (www.454.com/enabling-technology/the-software.asp)
b. contigs of < 2 reads deep were break apart. (see fig.4),
average depth = 21.7,
the distance between consecutive pseudoreads = 9.35 (890-600)/(32-1)
Assembly :
Assembly Analysis :

? hard stop : high GC Content (> 60%) and Strong Secondary Structure.

Note:
de novo: In general usage, de novo is a Latin expression meaning 'afresh', 'anew', 'beginning again'.

In Banking, a de novo bank is defined as a state member bank that has been in operation for five years or less. Commercial banks that have been in existence for five years or less and convert to Fed membership are also subject to the de novo bank application and supervision standards. -DeNovobanks.com

In Bioinformatics, de novo is a form of sequencing, as in "peptide de novo sequencing".

In Biology, de novo synthesis refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to their being recycled after partial degradation. For example, de novo synthesis of nucleotides, as opposed to the salvage pathway.

In Financial terminology, numbers reported by newly founded companies (especially the financial services industry) are qualified as "de novo," to distinguish them from older companies. For example, "growth de novo" means growth of newly started companies.

In Law, the expression trial de novo literally means "new trial". It is most often used in certain legal systems that provide for one form of trial, then another if a party remains unsatisfied with the decision.

In Medicine and genetics a de novo mutation is one which neither parent possessed or transmitted. It first appeared in the DNA of the affected individual.

from Wikipedia