Molecular Typing

iSEQ Biochemistry Overview

The iSEQ application uses reference sequences as a comparative measure to identify microbes, viruses, or other haploid organisms with the discriminatory power down to a single nucleotide. An optimized protocol has been developed based on the MassCLEAVE™ chemistry to enable accurate identification.

The iSEQ assay is PCR based and allows for the amplification of a target region of interest. Following SAP treatment, the samples are subject to in vitro transcription and base-specific enzymatic cleavage of 500 – 800 bp PCR products of the target region of interest. The process is homogeneous and does not require purification of the PCR product or subsequent analytes. The workflow is automatable and thus amenable to high throughput. After sample conditioning, the cleavage mixture is resolved and data is acquired by the MassARRAY MALDI-TOF Mass Spectrometry. A protocol overview is provided in the figure below.

Sensitive

• Enables two levels of amplification, PCR and in vitro transcription

Cost-effective

• Miniaturization in 384 well format
• Requires low amount of starting material (2 - 5 ng DNA)

Accurate

• Offers 4 data points per amplicon

Key benefits of iSEQ process:

iSEQ Software Overview

The iSEQ software suite enables rapid, efficient plate design, automatic data analysis, and sample identification. The Plate Editor enables you to custom design and store your microplate formats with your samples, reference sequences, and reactions. All plate formats are automatically stored in the database for later retrieval. The Plate Editor software interface is depicted in Figure 2.

Figure 2. Plate Editor software interface. Click to Enlarge

During analysis, the experimental mass pattern of the sample is automatically compared to an in silico reference mass signal pattern derived from an individual or a set of reference sequences. Differences between the expected and the observed mass signal pattern are interpreted and enable identification of the organism and detection of any sequence variation.

Identification results are automatically retrieved in less than an hour. The iSEQ software generates a report containing ID results listing the best matching reference sequences for each target region of interest, as well as, sequence variations and new sequences. A graphical software interface of the ID results and mass spectra is provided in Figure 3.

Figure 3. Data Analysis software interface

» Click to Enlarge	» Click to Enlarge

In addition to the iSEQ software, we have developed a Simulation Software to enable the user to simulate peak patterns for reference sequences and to determine the optimal number of reactions to run. To download the latest version of the simulation tool, click the CONTACT tab at the top to register.