De Novo Microbial Sequencing

Rapid de novo sequencing of microbes

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Infectious disease research app note

Application Notes & Publications

Read application notes and peer-reviewed publications to learn how Ion Torrent™ sequencing has empowered advances in de novo sequencing.

Data Set

Download a data set generated on the Ion S5™ System and see the results for yourself.

Ion S5 System

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 sequencing offers important new opportunities for the discovery and characterization of viral, bacterial, and fungal organisms. For researchers characterizing the genomic structures of microbes, de novo sequencing and assembly of 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 a fast, simple, affordable 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 S5 System, and the Ion PGM™ System with Ion Hi-Q™ chemistry, sequencing assembly metrics are better than ever, with up to a 90% decrease in indel error rates for microbial sequencing (Figure 1), giving you a fast path to whole-genome sequencing.

Figure 1. Decrease in indel false positives for microbial de novo assembly, using the Ion PGM™ Hi-Q™ Sequencing Kit instead of the Ion PGM™ Sequencing 200 Kit v2. Template preparation for 200-base read fragment libraries was performed using the Ion PGM™ Template OT2 200 Kit. Indel error rate per 100 kb analysis performed with SPAdes 3.1 delta-score filter.

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Construct de novo fragment libraries

Ion Xpress™ Plus Fragment Library Kit

The Ion Xpress Plus Fragment Library Kit provides rapid and flexible enzyme-based library construction in as little as 2 hours for gDNA and amplicon libraries, with significantly higher yields and lower bias than other library construction techniques.

Thermo Scientific MuSeek Library Preparation Kit

The MuSeek™ Library Preparation Kit provides a fast and simple transposon-based method for preparing high-quality genomic DNA libraries for Ion Torrent sequencing. The kit utilizes MuA transposase for fragmentation and simultaneous tagging of the target DNA, enabling library construction in as little as 80 minutes.

Thermo Scientific ClaSeek Library Preparation Kit

The ClaSeek™ Library Preparation Kit is designed for fast and convenient construction of an amplification-free NGS fragment library from DNA sample input as low as 5 ng.

Mate-pair sequencing

For de novo genome assembly, a mate-pair approach enables varying sizes of large insert 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% of sequence reads are wasted due to the insert being a concatemer instead of a single fragment.

The NxSeq Mate Pair Kit for Ion Torrent, available as a custom kit from Lucigen, Inc., generates mate-pair libraries with an insert size of 2–8 kb and uses Chimera Code™ and Junction Code™ technology* to help ensure that the correct ends of the insert are sequenced (Table 1).

  NxSeq Mate Pair Kit for Ion Torrent Competitor Kit A Competitor Kit B
Library size supported User-defined, 2-8 kb 2-12 kb range, 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 NxSeq Mate Pair Kit for Ion Torrent 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.

How the NxSeq Mate Pair Kit for Ion Torrent works
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De novo microbial sequencing workflow for the Ion S5 System


1 Construct
Library

Your choice of fragment library preparation kits, with the addition of a mate-pair library, enables low-cost sample preparation for de novo sequencing of microbial genomes.
Read more

2 Prepare
Template

The Ion Chef™ System provides simple, high-throughput template preparation with only minutes of hands-on time.
Read more

3 Run
Sequence
S5

The Ion S5 System enables high quality, rapid de novo sequencing with 400 or 200 bp sequencing.

4 Analyze
Data

Primary data analysis is performed using Torrent Suite™ Software. DNASTAR™ Lasergene™ Genomics Suite provides an easy-to-use interface for genome assembly.

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.

By expanding the functionality of the current on-board software, plugins offer a powerful means to manage a full range of additional applications and analyses. Torrent Suite Software allows you to customize your analysis at the end of each run through the use of different plugins—de novo assembly, resequencing, and metagenomics applications are included in the software—which are available for download in the Torrent Browser Plugin Store.

DNASTAR - Ion Torrent: De Novo Bacterial Genome

DNASTAR - SeqMan NGen software enables you to assemble reads from any major next-gen sequencing platform, including Ion Torrent platforms, with just a few simple steps.

This video demonstrates a de novo bacterial genome assembly of Ion Torrent data and post-assembly analysis, including annotating the consensus via a BLAST search and reviewing the depth of coverage.

 

Application notes and literature

Ion S5 System infectious disease application note
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
Ion Hi-Q™ chemistry for the Ion PGM™ System

Publications

The Ion PGM System is cited in more than 500 peer-reviewed publications about small-genome sequencing, making it the leading system for de novo assembly of small genomes.

Veras AAO, Sá PHCG, Pinheiro KC, Graças DA, Baraúna RA, et al. (2014). Efficiency of Corynebacterium pseudotuberculosis 31 Genome Assembly with the Hi-Q Enzyme on an Ion Torrent PGM Sequencing Platform. J Proteomices Bioinform 7: 374-378. DOI: 10.4172/jpb.1000342

Soni I, Chakrapani H, Chopra S. (2015). Draft Genome Sequence of Methicillin-SensitiveStaphylococcus aureus ATCC 29213 Genome Announc 3(5): e01095-15. DOI: 10.1128/genomeA.01095-15

Holmes A, Allison L, Ward M, Dallman TJ, Clark R, Fawkes A, Murphy L, Hanson M (2015). Utility of Whole-Genome Sequencing of Escherichia coli O157 for Outbreak Detection and Epidemiological Surveillance J Clin Microbiology 53: 3565-3573. DOI: 10.1128/JCM.01066-15

Orsini M, Mangone I, DiPasquale A, Perticara S, Sacchini L, Cito F, Iannetti S, Marcacci M, Ancora M, Calistri P, Di Giannatale E, Cammà C (2015). Draft Genome Sequences of 19 Salmonella enterica Serovar Typhimurium [4,5:i:] Strains Resistant to Nalidixic Acid from a Long-Term Outbreak in Italy Genome Announc 3(4):e00911-15. DOI: 10.1128/ genomeA.00911-15

Da Silva Sanotos AC, Rodrigues J. (2015). Draft Genome Sequence of Shiga Toxin-ProducingEscherichia coli Strain D92/09 Genome Announc 3(4): e00805-15. doi:10.1128/genomeA.00805-15

Mattos-Guaraldi AL, Guimarães LC, Santos CS, Veras AAO, Carneiro AR, Soares SC, Ramos JN, Souza C, Vieira VV, Hirata R, Jr, Azevedo V, Pacheco LGC, Silva A, Ramos RTJ (2015). Draft Genome Sequence of Corynebacterium striatum1961 BR-RJ/09, a Multidrug-Susceptible Strain Isolated from the Urine of a Hospitalized 37-Year-Old Female Patient Genome Announc 3(4):e00869-15. DOI: 10.1128/genomeA.00869-15

Small-genome sequencing

Learn about how Ion Torrent sequencing has enabled customers in their microbiology research.

WB31127
Ion 16S™ Metagenomics Solution

*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 ]