De Novo Microbial Sequencing
Close the gap in de novo sequencing
with Ion TrueMate Library Kits
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Adopting next-generation sequencing (NGS) in your lab is now simpler than ever before with industry-leading speed and affordability, with the flexibility to do multiple sequencing applications on a single system.
Whole-genome shotgun sequencing offers important new opportunities for the discovery and characterization of microbial organisms. For researchers characterizing the genomic structures of microbes, de novo sequencing and assembly of complete genomes is an important step. These basic research projects require deep coverage across the genome and high quality data. Ion Torrent™ semiconductor sequencing has revolutionized de novo sequencing for microbial research. By democratizing sequencing through simple, low-cost system that is designed to deliver accurate results in less than a day, more and more sequencing projects that were previously unattainable due to budget or time constraints are now feasible. With 400-base pair sequencing on the Ion PGM System, sequencing assembly metrics are better than ever, giving you a fast path to whole-genome sequencing.
For de novo genome assembly, a mate-pair approach enables large inserts from genomic libraries to be accommodated. Mate-pair sequencing approaches work by using a pair of tags with a given insert space that accommodates long inserts of several kilobases in length. One of the challenges with traditional mate-pair sequencing is that a relatively high number of chimeric molecules can form during the circularization/ligation steps, resulting in pairs of reads from different regions of the genome, creating what is called a “false mate”. These “false mates” mean that as many as 50% (or more) of sequence reads are wasted due to the insert being a concatemer instead of a single fragment.
The Ion TrueMate Library Kit generates mate-pair libraries with an insert size of 2–8 kb and uses “ChimeraCode™” and “Junction Code™” technology* to help ensure that the correct ends of the insert are sequenced (Table 1).
|Ion TrueMate Library Kit||Competitor Kit A||Competitor Kit B|
|Library size supported||User-defined, 2-8 kb||Ranges from 2-12 kb, with 2-5 kb more frequently observed||2-15 kb, difficult to control size|
|Chimeras||Largely prevented, most chimeras detected||Numerous chimeras, no chimera detection||Numerous chimeras, no chimera detection|
|Mate pair efficiency||>90%||~5%||~5%|
How the Ion TrueMate Library Kit works
A mate-pair library is a specific type of DNA fragment library derived from fragments separated by several kilobases of intervening sequence. The resulting single end sequencing read consists of two juxtaposed sequence tags (a pair), each from opposite ends of the same long user-defined DNA fragment. The distance between reads on the original fragment is much longer than standard paired-end sequencing protocols, which makes it ideal for applications such as de novo genome sequencing, gap closure and genome finishing, repetitive genomes, and detection of structural variation.
De Novo Microbial Sequencing Workflow for the Ion S5 System
De Novo Microbial Sequencing Application Notes, Literature & Publications
Application Notes and Literature
Ion S5 System infectious disease application note
Significantly improved de novo assembly of small genomes using the Ion TrueMate™ Plus Library Kit on the Ion PGM™ System
Microbial Solutions Brochure
Discover how German scientists leveraged the Ion PGM Sequencer to get answers when faced with a serious public health outbreak (shiga toxin-producing E. coli outbreak in northern Germany)
The Ion PGM™ System, with 400-base read length chemistry, enables routine high-quality de novo assembly of small genomes
The Ion PGM System is cited in more than 450 peer-reviewed publications about small genome sequencing, making it the leading system for de novo assembly of small genomes.
Petrof, E., et al. (2013). Stool substitute transplant therapy for the eradication of Clostridium difficile infection: 'RePOOPulating' the gut Microbiome 2013, 1:3. DOI: 10.1186/2049-2618-1-3
Hassan, S. S., et al. (2012). Complete genome sequence of Corynebacterium pseudotuberculosis biovar ovis strain P54B96 isolated from antelope in South Africa obtained by rapid next generation sequencing technology Stand Genomic Sci 7(2): 189-199. DOI: 10.4056/sigs.3066455
Yergeau, E., et al. (2012). Next-generation sequencing of microbial communities in the Athabasca River and its tributaries in relation to oil sands mining activities Appl Environ Microbiol 78(21): 7626-7637. DOI: 10.1128/AEM.02036-12
On Top of Outbreaks
Hear Dag Harmsen, M.D. from the University of Münster discuss how the Ion PGM™ System provides fast and accurate whole-genome “shotgun” sequencing of microbes for disease research on retrospective samples from outbreaks.
De Novo Microbial Sequencing Informatics Solutions
Torrent Suite™ Software provides the tools that take you from raw sequence data to informative results, including optimized signal processing, base calling, sequence alignment, and variant analysis. Post run, sequencing data are available for download with a simple right-click. Reports are also easily browsed, with expandable analysis plots and straightforward tables that summarize key results to help ensure that sequencing runs are of high quality.
De Novo sequencing assembly specify workflows in third party software packages such as the DNAStar® - SeqMan NGen® software package automate genome assembly & closure. The DNAStar® - SeqMan NGen® software package offers both rapid reference-guided and de novo genome assembly while minimizing data analysis time (<2 hours with 10.5 GM of RAM).
Learn more about how this software can simplify your data analysis.
For Research Use Only. Not for use in diagnostic procedures.
*Lucigen is the registered trademark owner of ChimeraCode and JunctionCode; no ownership or sponsorship is implied herein.
†Mellmann A, Harmsen D, Cummings CA et al. (2011) Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS One 6, e22751; Rohde H, Qin J, Cui Y et al. (2011) Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N Engl J Med 365, 718-724; Sherry NL, Porter JL, Seemann T et al. (2013) Outbreak investigation using high-throughput genome sequencing within a diagnostic microbiology laboratory. J Clin Microbiol. 2013 Feb 13. [Epub ahead of print ]
The Ion TrueMate™ Plus Library Kit generates user-defined mate-pair insert libraries and the Ion Xpress™ Plus Fragment Library Kit enables low-cost fragment sample preparation for de novo assembly of small genomes.
The Ion Chef™ System® provides simple, high-throughput template preparation with only minutes of hands-on time. The Ion OneTouch™ 2 System provides simple, 15-minute template preparation for 400 & 200 bp sequencing runs.
The Ion PGM™ System with the Ion PGM™ Hi-Q™ Sequencing Kit enables high quality, rapid de novo sequencing with 400 or 200 bp sequencing.
Primary data analysis is performed using Torrent Suite™ Software. DNASTAR™ Lasergene™ Genomics Suite provides an easy-to-use interface for genome assembly.
Dunlap, C., et al. (2013). "Genomic analysis and secondary metabolite production in Bacillus amyloliquefaciens." Biological Control, 64(2), February 2013. DOI: 10.1016/j.biocontrol.2012.11.002
Petrof, E., et.al. (2013). “Stool substitute transplant therapy for the eradication of Clostridium difficile infection: ‘RePOOPulating’ the gut”. Microbiome 2013, 1:3 doi:10.1186/2049-2618-1-3
Hassan, S., et.al. (2012). “Complete genome sequence of Corynebacterium pseudotuberculosis biovar ovis strain P54B96 isolated from antelope in South Africa obtained by rapid next generation sequencing technology”. Stand. Genomic Sci. 2012 7:2. doi:10.4056/sigs.3066455.
Antwerpen, M., et. al. (2012). “Draft Genome Sequence of Bacillus anthracis BF-1, Isolated from Bavarian Cattle”. J. Bacteriol. 2012, 194(22):6360. DOI: 10.1128/JB.01676-12.
Yergeau, E., et. al. (2012). “Next-Generation Sequencing of Microbial Communities in the Athabasca River and Its Tributaries in Relation to Oil Sands Mining Activities” Appl. Environ. Microbiol. November 2012 vol. 78 no. 21 7626-7637. DOI: 10.1128/AEM.02036-12
Adlakha, N., et al. (2013). "Draft Genome Sequence of the Paenibacillus sp. Strain ICGEB2008 (MTCC 5639) Isolated from the Gut of Helicoverpa armigera." Genome Announc 1(1). DOI: 10.1128/genomeA.00026-12
Agarwal, L. and H. J. Purohit (2013). "Genome Sequence of Rhizobium lupini HPC(L) Isolated from Saline Desert Soil, Kutch (Gujarat)." Genome Announc 1(1). DOI: 10.1128/ genomeA.00071-12
Planet, P., et al. (2013), "Bordetella holmesii: initial genomic analysis of an emerging opportunist." Pathogens and Disease. DOI: 10.1111/2049-632X.12028
Roy, A. S., et al. (2013). "Draft Genome Sequence of Pseudomonas aeruginosa Strain N002, Isolated from Crude Oil-Contaminated Soil from Geleky, Assam, India." Genome Announc 1(1). DOI: 10.1128/ genomeA.00104-12
Debroy, S., et al. (2013). "Draft Genome Sequence of the Nitrate- and Phosphate-Accumulating Bacillus sp. Strain MCC0008." Genome Announc 1(1). DOI: 10.1128/ genomeA.00189-12
Silva, A., et al. (2012). "Complete genome sequence of Corynebacterium pseudotuberculosis Cp31, isolated from an Egyptian buffalo." J Bacteriol 194(23): 6663-6664. DOI: 10.1128/ JB.01782-12
Manzoor, S., et al. (2013). "First Genome Sequence of a Syntrophic Acetate-Oxidizing Bacterium, Tepidanaerobacter acetatoxydans Strain Re1." Genome Announc 1(1). DOI: 10.1128/ genomeA.00213-12
Joshi, M. N., et al. (2013). "Draft Genome Sequence of the Halophilic Bacterium Halobacillus sp. Strain BAB-2008." Genome Announc 1(1). DOI: 10.1128/ genomeA.00222-12
Debroy, S., et al. (2013). "Draft Genome Sequence of a Phosphate-Accumulating Bacillus sp., WBUNB004." Genome Announc 1(1). DOI: 10.1128/ genomeA.00251-12