SoftGenetics - Software PowerTools for Genetic Analysis

 

 

 

 

 

NGS replacement for MLPA® and Similar Analysis Techniques
with the NextGENe® software’s Batch CNV Tool.

NextGENe’s Batch CNV Tool provides capability for CNV detection by comparing a batch of samples to one another all at once, without requiring controls samples. The Batch CNV Tool was developed in collaboration with Dr. Bekim Sadikovic and colleagues, Western University, Pathology and Laboratory Medicine London Health Sciences Centre, and St. Joseph's Health Care London, Ontario, Canada users.

 

The use of NGS and NextGENe software's Batch CNV Tool for screening dramatically increases laboratory throughput while significantly reducing the cost of MLPA Analysis. This robust tool can be used to replace Multiplex Ligation-dependent Probe Amplification and similar techniques, typically performed by capillary electrophoresis (CE) fragment analysis, with High-Throughput or Next Generation Sequencing. The HTS approach to CNV does not use the problematic steps of traditional MLPA reactions, which are challenging to optimize. Steps in the MLPA protocol include: DNA quantification, denaturation, and ligation, which must be optimized to produce reliable results. In addition, the MLPA laboratory procedure is labor intensive.

 

The Batch CNV Tool allows users to take advantage of NGS attributes of:

  • Higher throughout
  • Lower costs
  • Simplicity

“Detection of copy number variants is often performed using multiplex ligation-dependent probe amplification (MLPA). While it is sensitive, this method can be slow and labor intensive. High Throughput Sequencing (HTS) using targeted panels offers a different approach that is simpler, cheaper, and faster. Using a simple coverage normalization method with compatible data, it is possible to generate CNV calls with HTS and outperform the use of MLPA and Sanger sequencing” [1].


The choice of the library preparation method is very important for this analysis. Any method that produces inconsistent coverage from sample to sample will result in poor sensitivity and specificity. Samples prepared using SeqCap EZ Choice (Roche NimbleGen, Inc.) have been shown to work well with the tool.


The tool calculates the pileup coverage at each position in the targeted panel, normalizes it by a constant value in each sample, and reports the normalized values.

The normalization procedure is:

  1. Get the total coverage in each sample (adding the coverage at each position in the ROI bed file).
  2. Divide these values by the average across all samples to get the normalization factor for each sample.
  3. Divide the coverage at each position by the normalization factor for that sample.
  4. When reporting a value for a given region, reports the average of all bases within that region.

Figure 1: Table of Results

Any regions with an average normalized value outside the set thresholds (0.8 and 1.2 by default) will be highlighted as a CNV (Figures 2 and 3). The region names are taken from the 4th column of the BED file used to define the regions. Sub-region results (for example, within an exon) can be visualized (figure 4) in order to see potential sub-exon copy number variations.

 

 

Figure 2: Global plot with samples in rows and regions in columns.
Red squares indicate a deletion call while green squares indicate a duplication call

 

Figure 3: Results across several regions for a given sample.
A duplication of the GARS gene is shown.

 

 

Figure 4: Results within an exon (that was called as a duplication) in a single sample.
The normalized coverage (blue) is consistently above the cutoff for a duplication call at 1.20.

 

For further information please review or download the following Application Note:
Using NextGENe® Batch CNV Tool as an NGS replacement for MLPA® and Similar Analysis Techniques

 

Pricing & Trial Version:
Request 30-day trial/Price Quote

 

Reference Material:
Download/View NextGENe® Brochure
NextGENe User Manual
Annotated Reference Genomes

NextGENe Batch CNV tool (View) (view on )

ASHG 2016 Poster: CLINICAL NGS PIPELINE OUTPERFORMS A COMBINED APPROACH USING SANGER SEQUENCING AND MLPA IN TARGETED GENE PANEL ANALYSIS

 

Reference: [1] Schenkel, Laila C., et al. "Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis.” The Journal of Molecular Diagnostics (2016).

 

Trademarks property of their respective owners

SoftGenetics - Software PowerTools for Genetic Analysis

 

SoftGenetics - Software PowerTools for Genetic Analysis

 

 

SOFTWARE


Review all of our software products

 

 

 

 

 

 

 

 

 

SoftGenetics - Software PowerTools for Genetic Analysis

 

 

 

SoftGenetics Software PowerTools for Genetic Analysis provides current up-to-date information and pricing on all products.
All requests are secure.

 

SoftGenetics - Software PowerTools for Genetic Analysis

 

 

EXPERTISE


Request trial versions of our software and tap into our knowledge base

 

 

Trial Software

All of our software products are available for review and trial periods. Please use link to request a trial version.

 

Request Trial >>

 

Online Training

Our software professionals are available to you for online training of any of our products. Please contact an expert now.

 

Request Training >>

 

Webinars

SoftGenetics provides online webinars to assist you in the growing knowledge of our products. Visit our Analysis Corner.

 

View Webinars >>

 

SoftGenetics - Software PowerTools for Genetic Analysis

 

 

SEND US A MESSAGE


Representatives are always ready to assist you


Name *
Email *
Phone

###
-
###
-
####
Message *

 

SoftGenetics - Software PowerTools for Genetic Analysis

100 Oakwood Avenue | Suite 350 | State College, PA 16803 | USA
Phone: 814-237-9340 or 888-791-1270
Email: info@softgenetics.com

 

© 2020 SoftGenetics, LLC. All rights reserved

Trademarks are property of their respective owners

Research use only (RUO)

 

Web design by Lovett Creations