Methods and computer software for detecting splice variants

• US 7,962,291 B2
• Filed: 10/02/2006
• Issued: 06/14/2011
• Est. Priority Date: 09/30/2005
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Wang, Hui, Williams, Alan, Clark, Tyson, Cawley, Simon, Blume, John E.
• Primary Examiner(s): Brusca; John S
• Application Number:
  US11/538,054
• Publication Number:
  US 20070148667A1
• Time in Patent Office: 1,716 Days
• Field of Search: None
• US Class Current: 702/20
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

Methods and software products for analysis of alternative splicing are disclosed. In general the methods involve normalizing probe set or exon intensity to an expression level measurement of the gene. The methods may be used to identify tissue-specific alternative splicing events.

Claims

1. A computer-implemented method for identifying exons that are differentially spliced between at least a first and second sample comprising:

(a) obtaining an exon intensity measurement for a first exon in a first gene in said first sample and in said second sample;

(b) calculating a gene level measurement for said first gene in said first sample and in said second sample, wherein the gene level measurement for said first gene is a median normalized intensity value from all exons in said first gene in said first sample and the gene level measurement for said second sample is a median normalized intensity value from all exons in said first gene in said second sample;

(c) calculating a first normalized intensity measurement for said first exon in said first sample and a second normalized intensity measurement for said first exon in said second sample by dividing the intensity measurements obtained in (a) by the gene level measurements obtained in step (b) wherein the exon intensity in said first sample is divided by the gene level measurement in said first sample and the exon intensity in said second sample is divided by the gene level measurement in said second sample;

(d) calculating a splicing index measurement for said first exon, wherein the splicing index is equal to the log₂ of the normalized intensity for said first exon in said first sample divided by the normalized intensity for said first exon in said second sample, and wherein the calculating in steps (b), (c) and (d) is performed by a computer comprising a computer software product; and

(e) identifying said first exon as being differentially spliced if the absolute value of the splicing index obtained in (d) is greater than a threshold value,

wherein exon array background correction is performed by use of a background probe collection.

2. The method of claim 1 where the threshold value is 1-2.

3. The method of claim 1 wherein the threshold value is 2.

4. The method of claim 1 wherein the normalized intensity measurements of step (c) are obtained using the following equation:

5. The method of claim 4 wherein a first value of n_i,j,k is calculated by the computer for a first sample and a second value of n_i,j,k is calculated by the computer for a second sample and the splicing index is calculated by the computer by dividing the value of the n_i,j,k for said first sample by the n_i,j,k for said second sample to obtain a third value and calculating the log₂ of said third value.

6. The method of claim 1 wherein the exon is identified as being overrepresented in said first sample relative to said second sample if the splicing index is greater than zero.

7. The method of claim 1 wherein the exon is identified as being overrepresented in said first sample relative to said second sample if the splicing index is greater than 2.

8. The method of claim 1 wherein the exon is identified as being underrepresented in said first sample relative to said second sample if the splicing index is less than 0.

9. The method of claim 1 wherein the exon is identified as being underrepresented in said first sample relative to said second sample if the splicing index is less than −

2.

10. A computer-implemented method for identifying exons that are differentially spliced between at least a first and second sample comprising:

(a) obtaining an exon intensity measurement for a first exon in a first gene in said first sample and in said second sample;

(b) calculating a gene level measurement for said first gene in said first sample and in said second sample, wherein the gene level measurement of the first sample is a median normalized intensity value from all exons in said first gene in said first sample and the gene level measurement of the second sample is a median normalized intensity value from all exons in the first gene in the second sample;

(c) calculating a first normalized intensity measurement for said first exon in said first sample and a second normalized intensity measurement for said first exon in said second sample using the following equation;

11. The method of claim 10 wherein the threshold value is between about zero and about 2.

12. The method of claim 10 wherein the exon intensity measurements are obtained by hybridizing nucleic acid derived from the sample to an array of probes wherein there is a probeset comprising a plurality of probes for each exon to be interrogated and there are more than 10,000 different probesets.