Download Filtering Restriction Fragment Size on Affymetrix SNP Arrays

Filtering Restriction Fragment Size on Affymetrix SNP
Arrays for Copy Number or LOH Analysis using Partek
Genomics Suite for use with FFPE Samples
Overview
Many samples available to medical researchers are Formalin-Fixed, ParaffinEmbedded (FFPE). However, this storage method can increase the rate of breaks
in nucleic acids. The high density of Affymetrix arrays allows larger restriction
fragments, which are more likely to contain breaks to be preferentially removed
from the analysis. This removal will increase the resulting signal to noise ratio
within the sample and can result in cleaner data. For a more complete description
of various sample preparation techniques, please consult the technical note from
Affymetrix, entitled “Copy Number and Genotype Analysis of FFPE-extracted
DNA”.
Note – The workflows described below are enabled in Partek version 6.4 or later. Please contact [email protected] to
upgrade to the most current version of Partek. Partek screenshots shown below may vary from that which is displayed in the
most current version of Partek.
Filtering Instructions
Step 1 - Importing Probe Intensities from CEL Files
•
Open the Copy Number Workflow within Partek® Genomics Suite™
(Partek GS) by selecting it from the Workflow drop down in the upper
right corner of the software
•
Under Import Study Samples, select Load Allele Intensities (Figure 1)
Filtering Restriction Fragment Size on Affymetrix SNP Arrays for Copy Number or LOH Analysis
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Figure 1: Using the Copy Number Workflow
Figure 1: Using the Copy Number Workflow in Partek GS
•
Select the drop down option to Import from Affymetrix CEL files
(Figure 2)
Figure 2: Configuring the Open Allele Intensity dialog
•
Browse to the folder containing the CEL files and move all CEL files to
import into the right hand pane. You may include more CEL files from a
different folder by simply browsing to a second folder, once you’ve added
files from the first folder (Figure 3)
Filtering Restriction Fragment Size on Affymetrix SNP Arrays for Copy Number or LOH Analysis
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Figure 3: Selecting CEL files to import
•
Once all files are contained within the right hand pane, select Next to
continue the workflow (Figure 3)
•
Browse to the appropriate sample information file (Figure 4); there are
several ways to create this file, “Creating a Sample Information File”
user’s guide can be found on the tutorial page, contact Partek support for
more information
Figure 4: Selecting the Sample Information file
•
Define the appropriate path and file name for the resulting file that will
contain the combined probe intensities (Figure 4)
•
Select Import to continue (Figure 4)
Step 1 is complete with the creation of the copy number intensity spreadsheet
(Figure 5).
Filtering Restriction Fragment Size on Affymetrix SNP Arrays for Copy Number or LOH Analysis
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Figure 5: Viewing the Copy Number Intensity Spreadsheet
Step 2 - Filtering Larger Restriction Fragments from the Analysis
•
To change the distribution of restriction fragments used in the analysis,
from the Filter menu, select Filter Columns > Filter by Fragment
Length (Figure 6)
Figure 6: Filtering by Fragment Length
•
Partek might prompt you to specify a file containing a list of fragment
sizes (Figure 7). If this file is not found, the software will automatically
Filtering Restriction Fragment Size on Affymetrix SNP Arrays for Copy Number or LOH Analysis
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download it from the Partek servers. Note: the SNP6 file works for both
SNP5 and SNP6
Figure 7: Specifying the File Location
•
Once the file is completely downloaded, then you’ll be prompted to set the
minimum and maximum fragment length in base pairs that you’d like to
use for the analysis. A histogram of the overall distribution is displayed to
aid in your selection. You can set the min and max by either typing in
values or using the slider buttons (Figure 8)
Figure 8: Setting the Min and Max Fragment Length
Question: What should I use for min & max values?
Unfortunately, there are not universal answers that apply to all cases. In general,
you should monitor the distribution of the fragments on agarose gels prior to
hybridization on the array and use the size information from the gels to optimize the
limits used for analysis. If no physical size data is available, removing fragments
above 500 or 700 base pairs is a recommended starting point.
Step 2 is complete when the intensity spreadsheet shows a limited number of
columns. The spreadsheet below shows 380K markers, down from 1.3 M in Figure
5 (these data were generated from SNP5). Also, notice that a horizontal yellow bar
Filtering Restriction Fragment Size on Affymetrix SNP Arrays for Copy Number or LOH Analysis
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has been introduced showing that the spreadsheet has been filtered on columns
(Figure 9).
Figure 9: Viewing the Filtered Spreadsheet
Step 3 - Creating Copy Number Estimates from Intensities
•
From the Copy Number workflow, select Create Copy Number from
Intensities. You’ll be prompted to select either a Paired or Unpaired
analysis (Figure 10). The rest of this tutorial shows the steps associated
with an unpaired analysis
Figure 10: Choosing a Study Type
•
Next, specify the data spreadsheet that contains the recently imported and
filtered allele intensities (Figure 11). Note: spreadsheets in Partek are
numbered, so consult the left hand pane of the main Partek user interface
to see the number association with the intensity spreadsheet
•
Specify the baseline file used for unpaired analysis. You can either use
the pre-made baseline file provided by Partek derived from 270 HapMap
samples or create your own from your samples.
Filtering Restriction Fragment Size on Affymetrix SNP Arrays for Copy Number or LOH Analysis
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•
Specify the name and location for the output file, which will contain the
copy number estimates (Figure 11)
Figure 11: Specifying the Copy Number Baseline Input and Output
Step 3 is complete with the creation of a second spreadsheet containing copy
number estimates (Figure 12).
Figure 12: Viewing the 2nd Spreadsheet of Copy Number Estimates
Filtering Restriction Fragment Size on Affymetrix SNP Arrays for Copy Number or LOH Analysis
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Step 4 -Visualizing Copy Number
•
From the Copy Number workflow, select QA/QC > Chromosome View
(Figure 13)
Figure 13: Viewing the Chromosome View
A two track view is created. The lower track will contain a heat map with one row
for every sample in the spreadsheet. The upper track shows a detailed view of the
selected sample in the lower track. The selected sample can be identified by a bar
box around that sample. You can change the selected sample by clicking on
another sample. Multiple samples can be selected by shift or control clicking
combinations. The detailed view on the upper track has raw intensities displayed in
the background in grey and summarized copy number estimates displayed in
colored dots in the foreground.
Step 5 - Create Table of Segments
•
From the table of copy number intensities, run the Segmentation
algorithm from the Copy Number Analysis Section of the Copy Number
workflow (Figure 14)
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Figure 14: Selecting the Segmentation algorithm
•
Use the defaults in the segmentation dialog. Details on each parameter are
available in the question mark icons. (Figure 15)
Figure 15: Configuring the Segmentation dialog
A table of segments will be created. This table has one break per row per sample.
Each segment in each sample is displayed in its own row with detailed information
about that segment displayed (Figure 16).
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Figure 16: Viewing the Segmentation Spreadsheet
Step 6 – Finding & Visualizing Shared Segments
•
With a table of individual segments created, find common segments by
selecting Find Regions in Multiple Samples in the Copy Number
Analysis section of the Copy Number workflow (Figure 17)
Figure 17: Selecting Finding Regions from the Workflow
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In the Find Regions in Multiple Samples dialog, you can define the minimum
number of samples for a segment to be displayed. Check the Merge adjacent
regions that pass the above criteria box and click OK, then a second table of shared
regions per sample will be created and a new result file will be written (Figure 18).
Figure 18: Configuring the Find Regions dialog
•
To visualize common segments across multiple samples, select Plot
Detected Regions from the Copy Number Analysis section of the Copy
Number workflow (Figure 19)
Figure 19: Selecting the Plot Detected Regions option
A karyoview will be presented with common segments visualized (Figure 20).
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Figure 20: Viewing the Karyoview
Step 7 - Adding Gene Annotations
Gene annotation information can be added to a spreadsheet by selecting Find
Overlapping Genes in the Copy Number Analysis section of the Copy Number
workflow (Figure 21)
Figure 21: Selecting Find Overlapping Genes from the Workflow
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•
Select the source of gene information in the Report overlap with
database drop down menu (Figure 22). Common segments can be
extended by a user-definable number of basepairs to include nearby genes.
Figure 22: Configuring the Output Overlapping Features dialog
The resulting annotation information can be included either in the currently selected
table but in a new column, or in an entirely new spreadsheet with each overlapping
gene on its own row (Figure 23).
Figure 23: Viewing the Annotation Information in a new spreadsheet
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