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Alternative splicing results in a single gene coding for multiple protein isoforms, so this task can only be invoked from transcript level data. The algorithm is is based on ANOVA to detect genes with multiple transcripts showing expression changes differently in different biology groups, e.g. a gene has two transcripts: A and B,  transcript A is showing up-regulation in the treated group comparing to the control group, and while B is showing down regulation in treated group.

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The alt-splicing dialog is very similar to ANOVA dialog, since the analysis is based on the ANOVA model specified. To setup an ANOVA model, select first chose factors from the available sample attributes. The factors can be categorical or numeric attribute(s). Click on a check button box to select and click Add factors button to add it a factor to the model (Figure 1).

 

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SubtitleText Alt-splicing dialog, select : selecting factors and alt-splicing factors AnchorName Alt-splicing

Only and only one alt-splicing factor needs to be selected from the ANOVA factors. The ANOVA model performed is based on the factors specified in the dialog,  the  while the transcript ID and transcript ID interaction with alt-splicing factor effects are added automatically into the model :automatically. 

• Transcript ID effect: not all transcripts

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• of a gene

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• are expressed at the same level, so transcript ID is added to the model to account for transcript-to.transcript differences.
• Interaction of transcript ID with alt-splicing factor:

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• that effect is used to estimate whether different transcripts have different expression among the levels of the same factor.

Suppose there is an experiment designed to detect transcripts showing differential expression in two tissue groups: liver vs muscle. The alt-splicing ANOVA dialog allows you to specify the ANOVA model that in this analysis is tissue, the Tissue. The alt-splicing factor is chosen from the ANOVA factor(s), so the alt-splicing factor is also tissue Tissue (Figure 1). Click Next to setup the comparisons (Figure 2) 

 

 

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SubtitleText Alt-splicing ANOVA comparisons setup dialog AnchorName Alt-splicing-contrast

Click Next to setup the comparisons (Figure 2). The levels (i.e. subgroups) of the alt-splicing factor will be displayed in the left panel; click to select a subgroup level name and move it to one of the panels on the right. The fold change calculation on the comparison will use the group in the top panel as the numerator, and the group in the bottom panel as the denominator. Click on Add comparison button to add on a comparison to the comparisons table.

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Click on the Configure to customize Advanced options (Figure 3).

 

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SubtitleText Configuring advanced options when running alt-splicing ANOVA AnchorName Alt-splicing-adv

Low-expression feature and Multiple test correction sections correction sections are the same as the matching GSA advanced option, so see above  GSA advanced options discussion.

Report option

• • User only reliable estimation results: There are situations when a model estimation procedure does not fail outright, but still encounters some difficulties. In this case,  it can even generate p-value and fold change on the comparisons, but they are not reliable, i.e. they can be misleading. Therefore, the default of Use only reliable estimation results is set is set to Yes.

  • Data has been log transformed with base: showing the current scale of the input data on this task.

Alt-splicing ANOVA report

This For this analysis, only genes with more than one transcripts transcript will be included in the calculation. The report format is the same as ANOVA report, each row represent a transcript, and besides statistics information on the specified comparisons, there is also Altalt-splicing information at the right end of the table, this . That information is represented by the p-value of interaction of transcript ID with alt-splicing factor, . Note that the transcripts of the same gene should have the same p-value. Small p-value indicate significance of the gene undergo indicates significant alt-splicing event, by default hence the table is sorted based on this that p-value by default (Figure 4).

 

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SubtitleText Alt-splicing report. Clicking on the column header sorts the table. Panel on the left filters the table AnchorName Alt-splicing report

In the above screenshot exampleexample above (Figure 4), the alt-splicing p-value of gene SLC25A3 is very small which indicating this gene showing alt-splicing based on the tissueindicates that this gene shows preferential transcript expression across tissues. There are 3 splicing variants in this of the gene: NM_213611, NM_005888 and NM_002635.   Fold change clarifies that NM_005888 has much higher expression in the muscle comparing relative to the liver , but (negative fold change, liver as the reference category), while NM_002635 has much lower higher expression in muscle comparing to liver, the fold changes are opposite. When you the liver.

To visualize the difference, click on the Browse to location view icon () to visualize the difference  (Figure 5). The 3rd exon is differentially expressed between NM_005888 and NM_002635. Muscle primarily expresses NM_005888 while liver primarily uses NM_002635.

 

 

 

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SubtitleText Use isoform Isoform proportion track in chromosome view to visualize the alt-splicing genevisualising alternative splicing. Differential usage of exon 3 is highlighted AnchorName Alt-splicing-chrview

 

The 3rd exon is different between NM_005888 and NM_002635. Muscle primarily expressed in NM_005888 while liver primarily expressed in NM_002635. 

 

 

Additional assistance
https://documentation.partek.com/

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