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1.  Universal NGS Library Preparation on the Apollo 324 TM System: Automated Ion Torrent Personal Genome Machine Library Preparation 
The Ion Torrent™ Personal Genome Machine™ (PGM™) is revolutionizing the sequencing world with its “Ion Semiconductor Sequencing”. However, its manual library preparation workflow presents a bottleneck. The Apollo 324™ is a benchtop instrument that fully automates the key steps of the Ion Fragment Library Preparation processing using the PrepXTM PGM Library preparation kit. After a 15-minute setup of reagents on the instrument, the end repair, adapter ligation and size selection are all performed with walk-away operation in just under 3.5 hours. The basic workflow is as follows:
Sample Preparation: Raw DNA samples are sheared using the recommended Covaris® settings to provide a 1 μg (15 mL) sheared DNA sample with a broad size distribution centered at around 200 bp.
Library Preparation: The sheared DNA, reagents, disposables, and AMPure® XP beads are loaded onto the Apollo 324 instrument work surface. The end repair, adapter ligation and size selection are all performed in less than 3.5 hours using AMPure XP beads for both clean-up and size selection. As many as eight library samples can be prepared per run, and two runs can be performed per day.
Library Fragment Quality Assessment: Proper size selection is confirmed by running an Agilent® High Sensitivity Chip. The initial version of the protocol prepares a library fragment with 80% of the total sample between 150-250 bp and an average size of 195 bp, eliminating the need for time-consuming gel size selection. Nick translation and enrichment PCR are typically performed. The resulting DNA library has comparable sequencing results to libraries prepared using the Caliper LabChip® XT system.
PMCID: PMC3630537
2.  Complete Genome Sequence of the Plant Pathogen Erwinia amylovora Strain ATCC 49946▿  
Journal of Bacteriology  2010;192(7):2020-2021.
Erwinia amylovora causes the economically important disease fire blight that affects rosaceous plants, especially pear and apple. Here we report the complete genome sequence and annotation of strain ATCC 49946. The analysis of the sequence and its comparison with sequenced genomes of closely related enterobacteria revealed signs of pathoadaptation to rosaceous hosts.
PMCID: PMC2838050  PMID: 20118253
3.  The genome of the simian and human malaria parasite Plasmodium knowlesi 
Nature  2008;455(7214):799-803.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax​4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
PMCID: PMC2656934  PMID: 18843368
4.  The genome of the social amoeba Dictyostelium discoideum 
Eichinger, L. | Pachebat, J.A. | Glöckner, G. | Rajandream, M.-A. | Sucgang, R. | Berriman, M. | Song, J. | Olsen, R. | Szafranski, K. | Xu, Q. | Tunggal, B. | Kummerfeld, S. | Madera, M. | Konfortov, B. A. | Rivero, F. | Bankier, A. T. | Lehmann, R. | Hamlin, N. | Davies, R. | Gaudet, P. | Fey, P. | Pilcher, K. | Chen, G. | Saunders, D. | Sodergren, E. | Davis, P. | Kerhornou, A. | Nie, X. | Hall, N. | Anjard, C. | Hemphill, L. | Bason, N. | Farbrother, P. | Desany, B. | Just, E. | Morio, T. | Rost, R. | Churcher, C. | Cooper, J. | Haydock, S. | van Driessche, N. | Cronin, A. | Goodhead, I. | Muzny, D. | Mourier, T. | Pain, A. | Lu, M. | Harper, D. | Lindsay, R. | Hauser, H. | James, K. | Quiles, M. | Babu, M. Madan | Saito, T. | Buchrieser, C. | Wardroper, A. | Felder, M. | Thangavelu, M. | Johnson, D. | Knights, A. | Loulseged, H. | Mungall, K. | Oliver, K. | Price, C. | Quail, M.A. | Urushihara, H. | Hernandez, J. | Rabbinowitsch, E. | Steffen, D. | Sanders, M. | Ma, J. | Kohara, Y. | Sharp, S. | Simmonds, M. | Spiegler, S. | Tivey, A. | Sugano, S. | White, B. | Walker, D. | Woodward, J. | Winckler, T. | Tanaka, Y. | Shaulsky, G. | Schleicher, M. | Weinstock, G. | Rosenthal, A. | Cox, E.C. | Chisholm, R. L. | Gibbs, R. | Loomis, W. F. | Platzer, M. | Kay, R. R. | Williams, J. | Dear, P. H. | Noegel, A. A. | Barrell, B. | Kuspa, A.
Nature  2005;435(7038):43-57.
The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes encode ~12,500 predicted proteins, a high proportion of which have long repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal rDNA element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal/fungal lineage after the plant/animal split, but Dictyostelium appears to have retained more of the diversity of the ancestral genome than either of these two groups.
PMCID: PMC1352341  PMID: 15875012
5.  The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129 
Nucleic Acids Research  2003;31(22):6516-6523.
Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium’s nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.
PMCID: PMC275568  PMID: 14602910

Results 1-5 (5)