Identifying and correcting an allelic ladder signal for DNA analysis

• US 9,920,362 B2
• Filed: 12/30/2010
• Issued: 03/20/2018
• Est. Priority Date: 12/30/2010
• Status: Active Grant

• Current Assignee: Analogic Corporation
• Original Assignee: Analogic Corporation
• Inventors: Lai, Ching Ming
• Primary Examiner(s): Vanni, G. Steven
• Application Number:
  US13/977,316
• Publication Number:
  US 20130273529A1
• Time in Patent Office: 2,637 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/10 Signal processing, e.g. from mass spectrometry [MS] or from PCR
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for determining an allelic ladder signal for DNA analysis includes obtaining a measured allelic ladder signal for an allelic ladder substance, which includes a plurality of fragments, obtaining a reference set of expected fragment sizes of fragments of the ladder substance, and generating a signal identifying whether a peak for a fragment size of the measured ladder signal is a true peak of the ladder substance based on the reference set of expected fragment sizes, wherein the allelic ladder signal for DNA analysis includes the true peaks identified in the signal.

Claims

1. A method for determining an allelic ladder signal for DNA analysis, comprising:

receiving a measured allelic ladder signal for an allelic ladder substance, which includes a plurality of fragments, wherein the measured allelic ladder signal is an electrical signal;

obtaining, from a memory device, a reference set of expected fragment sizes of fragments of the ladder substance;

comparing, with a processor, each fragment size of a peak of the measured allelic ladder signal with each of the expected fragment sizes to generate an electrical signal indicating of an absolute value of a difference between each of the fragment sizes of each of the peaks of the measured allelic ladder signal and each of the expected fragment sizes;

determining, with the processor and for each of the fragment sizes of the peaks of the allelic ladder signal, whether a fragment size of a peak of the peaks of the measured allelic ladder signal is within a predetermined fragment size range of each of the expected fragment sizes from the electrical signal indicating the absolute value;

identifying, with the processor and for each peak with a fragment size within the predetermined fragment size range of an expected fragment size, an expected fragment size closest to the fragment size of the peak from the electrical signal indicating the absolute value;

determining, with the processor, peaks with identified expected fragment sizes closest to the fragment sizes of the peaks as true peaks and all other peaks as false peaks; and

generating, with the processor, the allelic ladder signal for DNA analysis with the true peaks and not the false peaks, wherein the allelic ladder signal for DNA analysis is an electrical signal.

2. The method of claim 1, wherein the false peaks are discarded from the measured allelic ladder signal.

3. The method of claim 1, further comprising:

determining the measured ladder signal is missing a peak.

4. The method of claim 3, further comprising:

generating a peak for the missing peak; and

including the generated peak in the allelic ladder signal for DNA analysis.

5. The method of claim 4, wherein generating the peak includes interpolating fragment sizes of neighboring peaks to generate the missing peak.

6. The method of claim 5, further comprising:

determining an interpolation coefficient used to interpolate the missing peak based on a corresponding fragment size in the reference set.

7. The method of claim 4, further comprising:

determining an allele number for a sample DNA fragment size based on the allelic ladder signal for DNA analysis.

8. The method of claim 7, wherein the sample DNA and the allelic ladder substance are concurrently processed in different lanes of a sample carrier.

9. The method of claim 1, wherein the reference set of expected fragment sizes of the fragments in the ladder includes an average of the fragment sizes from a plurality of ladder signals previously successfully verified.

10. The method of claim 9, further comprising:

updating the average based on a newly acquired and successfully verified ladder signal.

11. A method, comprising:

receiving, from an optical channel of an optical detector of an optical reader, a measured allelic ladder signal for an allelic ladder substance, which includes a plurality of fragments;

retrieving from a memory device a reference set of expected fragment sizes of fragments of the allelic ladder substance;

comparing, with a processor, each of the expected fragment sizes with each fragment sizes of each peak of the measured allelic ladder signal;

determining, with the processor, for a peak of the measured allelic ladder signal absolute values between a fragment size of each peak and each of the expected fragment sizes;

identifying, with the processor, and for the fragment, a first expected fragment size corresponding to a smallest deviation of the deviations for a peak;

comparing, with the processor, the smallest absolute value for each peak with a first predetermined threshold corresponding to the first expected fragment size;

identifying, with the processor, a peak as a true peak in response to the smallest absolute value for a peak satisfying the first predetermined threshold corresponding to the first expected fragment size;

identifying, with the processor, for each peak, a second expected fragment size corresponding to a second absolute value for each peak;

comparing, with the processor, the second absolute value for the each peak with a second predetermined threshold corresponding to the second expected fragment size;

identifying, with the processor and for a peak as a false peak in response to the second absolute value for a peak failing to satisfy the second predetermined threshold corresponding the second expected fragment size; and

correcting the measured allelic ladder signal for DNA analysis by removing the false peak from the measured allelic ladder signal, which results in a corrected allelic ladder signal that includes only true peaks.

12. The method of claim 11, further comprising:

determining an allele number for a sample DNA fragment size with the corrected allelic ladder signal; and

sequencing the sample DNA with allele numbers determined for a plurality of DNA fragment sizes in the DNA sample.

13. The method of claim 11, further comprising:

identifying, with the processor, an expected fragment size with no identified true peak; and

creating, with the processor, a peak for the expected fragment size with no identified true peak by interpolating neighboring peaks using the expected fragment size as an interpolation coefficient,

wherein the corrected allelic ladder signal includes both true peaks and the interpolated peaks.

14. The method of claim 13, further comprising:

determining a new reference set of expected fragment sizes of fragments of the allelic ladder substance from the measured and corrected signal.

15. The method of claim 14, wherein the new reference set of expected fragment sizes of fragments of the allelic ladder substance includes an average fragment size for every true peak in the ladder signal.

16. The method of claim 15, further comprising:

updating the reference set and its average after each performance of the method over a plurality of performances.

17. The method of claim 15, wherein the average is a weighted average, with a first weight for a current run and a second weight for a previous run, and the first weight is greater than the second weight.