Dna Fragment Analysis Software Mac

Getting Started (Download tutorial, Contact us for demo account!)

Background

Capillary gel electrophoresis (CE) is a proven sensitive high-throughput, high-resolution system for nucleic acid analysis. Other than Sanger sequencing, it was traditionally applied as “fragment analysis” for AFLP, MLPA, and SNP detection, etc.

  1. Network licenses for the most common sequence analysis software like Mac Vector, Sequencher, Vector NTI are available from the Penn Molecular Profiling Group. Facility staff will consult with users whenever there are problems, and will make suggestions to improve sequencing results for.
  2. Peak Scanner, is a free program developed by ABI to aid in fragment analysis. It only runs on Windows. STRand, is a free program developed to aid in fragment analysis. Genographer  To open the electropherogram file, you must acquire trace viewing software. One option is to use an ABI program called EditView (available from our main page).

Software to align DNA, RNA, protein, or DNA + protein sequences via pairwise and multiple sequence alignment algorithms including MUSCLE, Mauve, MAFFT, Clustal Omega, Jotun Hein, Wilbur-Lipman, Martinez Needleman-Wunsch, Lipman-Pearson and Dotplot analysis.

In most recent CRISPR/Cas9 genome editing studies, multiple CE-based assays have been developed and reported as a fast, sensitive, precise and cost effective approach for mutation detection, such as IDAA (Indel Detection by Amplicon Analysis), Fluorescent PCR, CRISPR-STAT, etc. The CE instruments with Sanger sequencing capability (e.g. Thermo Fisher 3130 and 3730 series) are different from the ones without the capability (e.g. AATI Fragment Analyzer). The former instruments can reach single base resolution. The mutation screening assays (IDAA, Fluorescent PCR, CRISPR-STAT), developed on these types of instruments, are with simple protocol (two-step: PCR-CE) independent on enzyme cleavage . It reported that the sensitivity and resolution is comparable to NGS with an indel detection sensitivity ≈0.1% (Lonowski et al., 2017) , which can be applied in in basic research and more challenging genome editing applications such as therapeutic indel profiling.

Moreover, it has been reported as “using CE as an analytical tool fundamentally changes the scale and complexity of experimental design” (Gardner et al., 2016 ). Based on this publication, high-throughput CE assays based on fluorescent labeled oligonucleotides can be designed to in-depth characterize/discovery/engineer/screen nucleic acid metabolic enzymes (ligases, polymerases, exonuclease, RNase H2, etc.), including study on DNA repair, recombination, restriction and modification and RNA metabolism. It also claimed “Restriction endonuclease site specificities and reaction parameters can be determined using a panel of fluorescently labeled substrates containing variable recognition sequences.” Furthermore, it has been applied as reagent quality control assays.

GeneMapper is the supplier (Thermo Fisher) software to run the instrument and process the data. Peak Scanner is a free alternative software from the same supplier for data processing. There are also several similar softwares on the market. From 2017, free cloud version of Peak Scanner is available from Thermo Fisher. There is no limiting factor for primary CE data process anymore (baselining, peak detection and sizing). However, none of them can fully satisfy the data analysis (fragment labeling and quantification, especially targeting efficiency calculation for genome editing study) requirements for above new applications. The bottleneck is quantitative post-processing data from instruments instead of experiments.

The solution provided here is not a me-too solution for GeneMapper. Instead, it is an add-on (or secondary) data processing to adapt for broad analytical applications . Users can keep using their predefined peak detection settings (algorithms and parameters) to ensure the data processing integrity. The software fills the gap between the instrument output raw data to the final scientific insights. It can significantly speed up data analysis and reduce turnaround time.

The videos in Video Gallery are good resources for you to understand the software and data analysis.

The algorithms embedded in the SaaS solution are universal. Contact us if you need customized solution from other brands’ CE instruments.

Dna Fragment Analysis Software Mac 2017

Requirements

CSV output files from GeneMapper or Peak Scanner or GeneMarker software are required for fragment analysis.

Two analysis algorithms

Two analysis algorithms are available for data analysis, Quantitative fragment (peak) percentage is calculated based on peak area.

  • Single basepair resolution: For analysis with single basepair difference, for example, genome eiditing with 1bp insertion or deletion, dA tailing efficiency evaluation.
  • Standard algorithm: For all the other cases, including the case with continous fragments grouping for anaysis.

Sample naming rule

There are no specfic requirements for sample names if you do not use the statistical analysis feature.

Sample naming rule for automatic statistical analysis and plotting is following:

  • Use ”-” as a separator for each sample name.
  • Prefix is highly recommended to be 2-digit or 3-digit index, e.g. from 01, 02 to 96. This is convenient for sample display in a certain order.
  • Sample replicates are grouped by three ways: prefix only, suffix only, prefix and suffix.
  • Examples of sample name with grouped by prefix only (recommended): 01-sample1-20C, 02-sample1-20C, 03-sample1-20C.
  • Examples of sample name with grouped by suffix only: sample1-20C-1, sample1-20C-2, sample1-20C-3.
  • Examples of sample name with grouped by both prefix and suffix (recommended): 04-sample1-20C-1, 05-sample1-20C-2, 06-sample1-20C-3.

Standard Algorithm

Analysis Settings

  • Based on the data review, design fragment analysis set. Click ”New Analysis Set” to input design information. For the new users, please refer to FAQ for more details of analysis set design. Contact us if you need help for design.
  • Click “New Fragment Analysis”.
  • Fill in the required information as following:
    • Fragment dye selection: The dye you used for your oligos. For example, FAM is a blue dye (choose B here).
    • Fragment height threshold: Any fragments with peak height lower than the threshold (exclusive) are considered as instrument noises instead of real signal. The noises fragments are filtered out of the further analysis. It is recommended to set the threshold in the range of 100 to 500 for dye B, depending on the experimental data. Refer to the following site for threshold setting up.
    • Fragment analysis set: Choose your predefined analysis set.
  • Click “Create Fragment Analysis” to get the results.

Results review

Pcr Fragment Analysis

Results review

  • If you see some waring message like the following, you need to investigate your analysis set design:
    Warning: 75 % of tested samples ( 9/ 12 ) with fragments above threshold (100 ) but not selected by analysis set.
  • You can always re-analyze the fragments with current settings by click “Reparse Fragments” button. When you update the analysis set contents (not only the name), click this button to re-analyze the data.
  • Important: Click the “Results Table” to view the results by sample.The results and figures (including statistical analysis) will be displayed.

Single Basepair Analysis

Fragment

Analysis settings

  • Adjust the analysis settings at GeneMapper or Peak Scanner to have correct fragment/peak calling for the single basepair analysis. Contact us if you need help for settings.
  • Click “Single Basepair Analysis”.
  • Fill in the required information as following:
    • Fragment dye selection: Same description as “standard algorithm”.
    • Fragment height threshold: Same description as “standard algorithm”.
    • Reference/control sample name: If you have replicated run controls, choose only one to input here.
    • Reference/control sample position: Choose ”Low” if your reference sample has smaller size of basepairs.
  • Click “Create Fragment Analysis” to get the results.

Results review

  • The selected fragments are labeled as ”Low” and “High”, all the other fragments are labeled as “Invalid”.
  • You can search the table by “Invalid” to get all non-selected fragments to ensure that there are no critical fragments with wrong classification.
  • You can always re-analyze the fragments with current settings by click “Reparse Fragments” button.
  • Important: Click the “Results Table” to view the results by sample.The results and figures (including statistical analysis) will be displayed.

Analysis Set Design

Definition

  • Each analysis set includes one or multiple predefined regions for data analysis.
  • Each region is defined to capture/label one or multiple fragments/peaks.
  • Region center and radius are defined in terms of peak size (in bp).
  • For each region, the analysis range is from 'center' - 'radius' to 'center' + 'radius'.
  • Ensure the selected fragment fall inside the range of designed region.
  • The region range cannot be overlapped between adjacent regions.
  • Only one kind of dye (B, Y, O, G) can be input for each analysis set. Contact us if you need multiplex analysis.
  • Figure A: Example of analysis design, each region is designed to select top 1 (in terms of peak height) fragment in the designed range. Input as fragment number = 1.
  • Figure B: Example of analysis design, each region is designed to select top several (in terms of peak height) fragments in the designed range. For this case, if the fragment number is 4 or more, all fragments are selected. If the fragment number is 3, only the top 3 fragments are selected.
  • An analysis set can includes several regions like the cases shown in figure A and B.

Design

Restriction Fragment Analysis

  • Click “New Analysis Set”.
  • Example of analysis set with single fragment selection for each region.

Dna Fragment Analysis Software Mac Pro

  • Example of analysis set with different number of fragments selection for each region.
  • Click “Save” to create an analysis set.

References

Instructions about instruments:

Instructions about instrument software:

  • Peak Scanner™ Software 2.0 User Guide

Reports about applications:

Molecular diagnostics CRISPR–Cas9 genome editing
    Indel Detection by Amplicon Analysis (IDAA) Fluorescent PCR CRISPR-STAT
Nucleic acid enzyme characterization

GeneMapper Software for fragment analysis. This replaces GeneScan/Genotyper. We provide two stand alone Dell PC workstations both equipped with the latest version of Genemapper, free for our customers located on site at 21 Sachem Street, Office Room 148, New Haven, Ct.

GeneMarker This software has been designed and created in order to provide genetic researchers with a biologist friendly genotyping analysis tool, it is much less expensive than GeneMapper.

STRand It is very convenient and easy to use. The set-up is not intuitive, but their support staff is very helpful.

FREE-Peak Scanner A new easy to use app tool from Thermo-Fisher/Applied Biosystems available through Cloud Connect services. This software allows users to view, edit, analyze, print and export fragment analysis data from files generated by Applied Biosystems instruments.

FREE-Microstaillite Analysis an app tool from Thermo-Fisher/Applied Biosystems available through Cloud Connect services.

Formatting and File conversion Software

Gm Convert For everybody who uses GeneMapper, this piece of software should make life easier (as far as data formatting is concerned anyway). This is an executable (os x, windows) and Python command line (all platforms) program to re-array data output by GeneMapper (AppliedBiosystems).

Formatomatic Population Genetic File Creator and Converter, this program adds quite a few formatting options to other available toolkits.

3730 Conversion Utility For modifying 3730 files to work with Genescan, click the link to download the program.

Repeat Search, Primer Design and PCR utilities

MsatCommander msatcommander is a python program written to locate microsatellite (SSR, VNTR, &c) repeats within fasta-formatted sequence or consensus files. msatfinder will search for all di-, tri-, tetra-, penta-, and hexa-nucleotide repeats (with options to search for fewer repeat types and combinations of repeat types).

BioPHP microsatellite repeat finder

Perfect Microsatellite Repeat Finder- open source application will help locate microsatellite repeats.
Primer3Plus pick primers from a DNA sequence
PerlPrimer open-source PCR primer design

Data Transformation and Analysis Software

Data Transformation and Preliminary Analysis:
Msat
MsatToolkit
Genalex

Multi-Purpose:
Arlequin
Genepop (web version) or Genepop

Specialized:
Assignment and Hybridization
Structure
NewHybrids

Landscape Genetics
R: http://lib.stat.cmu.edu/R/CRAN/
R Geneland package (installation within R) instructions

Relatedness
Cervus
Kingroup

Multi-Purpose ‘Borrowed’ Software:

PAST is a free, easy-to-use data analysis package originally aimed at paleontology but now also popular in ecology and other fields. Inspired by PALSTAT, it includes common statistical, plotting and modeling functions