5400 Fragment Analyzer

The 5400 Fragment Analyzer redefines the bounds of automated QC analysis of nucleic acids. Capable of interfacing with world leading robotic systems, analysis of DNA and RNA samples can now be performed around-the-clock.

AATI Scientist looking at data

What is ProSize Data Analysis Software?

ProSize Data Analysis Software is a powerful software program used with the Fragment Analyzer, ZAG DNA Analyzer System, 5400 Fragment Analyzer, and Femto PulseSystem for the analysis of raw nucleic acid data. Developed to analyze a wide range of nucleic acids including: genomic DNA, NGS libraries, plasmids, microsatellites, PCR amplicons, restriction enzyme digests, mRNA, small RNA, and total RNA.

Designed with the end user in mind, the software provides quick and easy visual analysis of DNA and RNA quality automatically reporting the size and concentration of DNA and RNA fragments upon opening of the raw data files. Results are displayed as both electropherogram traces and as digital gel images along with a Peak Table displaying size, concentration, percent concentration, and molarity all on the same screen. Quality metrics such as GQN, DQN, RQN, and DV200 are also reported.

Data analysis is aided by the ability to export size, concentration, and quality number data in a generic CSV file format for effortless spreadsheet analysis or Adobe PDF sample reports containing user specified information. Users can also export electropherogram traces and digital gel images in JPEG, BMP, or PNG image formats for use in a variety of projects.

In addition, ProSize provides the convenience of 21 CFR 11 data tracking and digital signature generation regulated by the U.S. Food and Drug Administration (FDA). Applicable to all industries under the review of the FDA, 21 CFR delineates the requisite criteria for electronic documents and signatures to be considered equivalent to hard copies.

Analysis with ProSize

ProSize provides easy and flexible data analysis with the option of Advanced Settings for unique circumstances. The mode of analysis is automatically selected based on the reagent kit utilized, but the Advanced Settings tab allows for selection between twelve different nucleic acid analysis options. Selecting the specific type of nucleic acids allows for unique software parameters to be applied to each separation for data processing. Based on the data analysis mode chosen, quantification analysis is determined and additional fields may be displayed.

  • DNA - For anlyzing double stranded DNA (dsDNA) fragment samples with discrete Gaussian peaks.
  • gDNA - For analyzing dsDNA samples containing peaks, smears, non-Gaussian peaks and/or uneven baselines. This mode is commonly used for genomic DNA (gDNA).
  • RNA (Eukaryotic) - Calculates the ratio of 18S and 28S ribosomal RNA peaks.
  • RNA (Prokaryotic) - Calculates the ratio of the 16S and 23S ribosomal RNA peaks.
  • RNA (Plant) - Calculates the ratio of the 18S and 25S ribosomal RNA peaks.
  • mRNA - For analyzing messenger RNA (mRNA) samples.
  • Small RNA - For analyzing micro RNA (miRNA) samples. Calculates %miRNA, small RNA (pg/µL) and size in nucleotide (nt).
  • NGS - For analyzing dsDNA samples containing peaks, smears, non-Gaussian peaks and/or uneven baselines. This mode is commonly used for Next Generation Sequencing (NGS) fragment library analysis.
  • Mutation Analysis - This mode is used in combination with the Mutation Discovery Kit.
  • Restriction Digest - For analyzing standard dsDNA fragment samples where discrete, Gaussian peaks are present. This mode can be used in analyzing samples cleaved with restriction enzymes.
  • High Sensitivity - Changes the display from ng/µL to pg/µL for NGS analysis only.
  • Linear Interpolation - This mode uses two markers-one above and one below a target fragment. The size is based on linear interpolation between the upper and lower marker.

Benefits of ProSize

User Friendly Software
Intuitive user interface designed to help researchers analyze samples allowing them to quickly move to downstream applications.
Enhance Electropherograms
Flexibility for representing and illustrating the electropherogram appearance allows the user to strengthen areas of interest.
Export Data
Data and images can be easily exported as a CSV file or a project report for further data analysis. The user has the option of including all the data or selecting only the information of interest.
Compare Samples
Compare samples from the same run or different files is easily achieved in ProSize. Overlays allow for direct comparison of sizing, peak height, and separation profile.
Objective QC
Quality analysis of a sample is easily achieved with a numerical quality number. The DQN, GQN, RQN, and DV200 are reported. The threshold for the DQN and GQN can be set by the user allowing greater flexibility with degraded samples.
Eliminate Bottlenecks
Batch process 100's of samples simultaneously to eliminate laboratory bottlenecks.

Data Displayed by ProSize

ProSize automatically presents nucleic acid analysis data in three ways: electropherogram, digital gel image, and a peak table. The digital gel image and electropherogram provide clear visualizations of size and concentration data, with exact values reported in the peak table. Electropherograms and digital gel images can have several basic adjustments made, including:

  • Zoom & Auto fit
  • Peak Label (Peak ID, Migration Time, Peak Height, Corrected Peak Area, Size, Average Size, or Concentration)
  • Show/Hide peak start and end point lines
  • Add Annotations
  • Change color and adjust intensity of digital gel image

ProSize Features

ProSize includes many additional features that allow the user to individualize their data analysis and electropherograms to fit their particular needs. The ability to adjust settings and compare runs in one software program saves time and enhances data analysis.

Overlay Samples

Comparison of samples within the same run or between different runs can easily be achieved with ProSize. A "Quick Overlay" of samples from the same run only requires a simple click on the gel image and the overlays appear in the electropherogram area. Comparisons between different run files are carried out in the Overlay Screen. Up to 384 samples from the same kit can be compared in both the digital gel and electropherogram view. The comparison can then be saved as a Project and exported in a data report or as a PDF.

Smear Analysis

Selection of a particular size range to get an average size of the smear and total concentration can be accomplished with the Smear Analysis tab. This is particularly useful when the concentration of a specific size range within a smear is more important than the entire smear or the sample has a non-Gaussian shape. Eight different smear ranges can be selected and applied to individual samples or the entire run. The results for each range are displayed in the smear table that can be exported in a CSV format or printed as PDF report.

Flag Analysis

Flag analysis allows the user to select specific criteria to be met within the data and generates a binary output where 0 = false and 1 = true. This is useful for applications like genotyping where the presence or absence of specific fragments needs to be scored. Flag criteria can be based on the following:

  • Boolean expression
  • Size (bp or nt)
  • Concentration (ng/µL or nmol/µL)
  • Peak Height (RFU)

Results are listed in the Flag Analysis Table and can be exported.

Advanced Flag Analysis

The Advanced Flag Analysis tab contains settings to enable the user to select even more specific criteria to be met within the data and generate a table with the results. The Advanced Flag criteria can be based on size, concentration, peak height, RFU, and/or corrected peak area and can be applied to all or to individual wells. A plate of individual flag conditions can be defined, saved and loaded for future plates.

DNA Quality Number (DQN) & Genomic DNA Quality Number (GQN)

ProSize uses two dynamic quality metrics for DNA quality. The DQN and GQN provide researchers with objective quality metrics adaptable to shifting QC demands. The DQN was developed for use with large DNA fragments, such as those encountered during PacBio library preparation, where the average library size ranges from 20 Kb to 40 Kb. Specific to genomic DNA assessment, the GQN provides reliable analysis of gDNA quality for numerous downstream applications, notably NGS.

The quality of isolated FFPE DNA samples can vary greatly among different samples and species. Illumina developed workflow guidelines based on the GQN value to determine the minimal sample input required for successful library preparation.

RNA Quality Number (RQN)

ProSize uses an RNA Quality Number (RQN) as a quality metric indicator. The RQN is based on a scale from 1 to 10 where 1 represents completely degraded total RNA and 10 represents fully intact total RNA. A large RQN value indicates a higher quality of RNA in the sample. ProSize considers the entire electropherogram including the 5S and Fast Region where the small RNA separates, as well as the sharpness of the 28S and 18S ribosomal peaks, the low baseline between the peaks, the ratio of the 28S/18S ribosomal peaks, and the degradation in front of the 18S peak when calculating the RQN. The RQN and 28S/18S ribosomal ratio are automatically reported, allowing for easy evaluation of total RNA quality.

ProSize has a dedicated plant mode that can be selected under the Advanced Settings Tab for evaluating plant RNA. Plant RNA is more complex because of the chloroplastic RNA. To address the complexity of plant RNA, the plant mode in ProSize also considers the 5S and Fast Region where the 5S and 8S chloroplastic peaks lie, as well as the sharpness of the 25S and 18S ribosomal peaks, the low baseline between the peaks, the ratio of the 25S/18S ribosomal peaks, and the degradation in front of the 18S peak when calculating the plant RQN.

All of the RNA Kits have a Total RNA Exclusion Region Tab in ProSize. This tab allows the user to choose a range to exclude from the RQN calculation. This is particularly useful for RNA samples tand Fast region or genomic DNA contamination. ProSize allows users to remove contamination from RQN calculations for a more accurate analysis.


The quality of isolated FFPE RNA samples can vary greatly among different samples and species. Agilent - formerly AATI collaborated with Illumina to develop the DV200 metric enabling accurate assessment of FFPE RNA quality. The DV200 metric measures the percentage of RNA fragments > 200 nucleotides to determine the minimal RNA input required for successful library preparation.

CRISPR Plugins

There are three CRISPR Plugins for ProSize.

  1. CRISPR Main Plugin enables a user to enter the expected PCR and cleavage fragment sizes. The expected cleaved fragments are automatically highlighted, determined molarity (nmole/L), sized, and the % Cleaved is reported and can be exported as a PDF report.
  2. CRISPR Pooled Cell Line (PS) Plugin for ProSize enables users to enter expected PCR and cleavage fragment sizes, and automatically score, predict zygosity, and highlight detected cleaved fragments and calculate % Mutated of the sample. The data can be exported as a PDF report of CSV file for further analysis.

Batch Processing

High throughput labs will benefit from the ability to Batch Process an entire day's analysis in one easy procedure. A list of runs within a specified folder, with user defined parameters are exported to an output folder in varying forms such as PDF. The user has the option of selecting various data including: peak table, flag analysis, smear analysis, electropherogram, and gel images. This feature allows high throughput users to streamline their workflow.


The Quantification Tab assigns how the sample is quantified by ProSize (Upper Marker, Lower Marker, or Ladder), defines the working concentration of the marker or ladder reference, and the applied dilution factor for calculating the final sample concentration. The dilution factor can be changed if more or less sample or ladder is added than recommended in the Kit manuals. For users who quantify the ladder prior to use for exact concentration results, the final concentration can be changed.

Exportable Data

The immense amount of data provided by ProSize is efficiently handled with the option of creating project reports, exporting data for spreadsheet analysis, or batch data processing. This offers the user the option of including all the data or selecting only the information of interest to a file path of choice.

  • Peak Table
  • Smear Analysis Table
  • Quality Table
  • Flag Criteria Analysis Table
  • Electropherogram

Molarity Calculation

Determination of nucleic acid fragment or smear molarity is crucial to numerous applications. ProSize Data Analysis Software automatically calculates molarity from either Peak Maxima or Average Size and reported in either pmole/L or nmole/L, depending upon researcher preferences. Users who prefer a visual representation of molarity have the option of changing the Y-axis units from RFU to the molarity units previously specified, pmole/L or nmole/L. Accordingly, % Concentration can be determined as a function of molarity or concentration.

Additional Features

  • Larger electropherogram image
  • Manual adjustment of electropherogram/digital gel image during analysis
  • Analyze select regions of the electropherogram with the Inclusion region function
  • Adjust resolution of exported electropherograms for publication quality images
  • Quickly select a start time to display on the X-axis
  • Efficiently annotate the digital gel image and/or the electropherogram
  • Add your company logo to exported reports
  • Quickly open the most recent run files

ProSize Specifications

Windows 7 or Windows 10 Operating System
32- bit or 64-bit processor
Intel Core 2 or faster processor
4 GB of RAM
500 GB available hard-disk space
1280 x 1024 or 1280 x 800 screen resolution
PDF format Adobe
CSV Microsoft Excel, OpenOffice
JPEG, BMP, PNG Any suitable program