Systems and methods for probe design to detect the presence of simple and complex indels

• US 10,535,420 B2
• Filed: 03/15/2013
• Issued: 01/14/2020
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Schmidt, Jeanette Pruzan, Davies, Christopher, Wong, Brant
• Primary Examiner(s): Woitach, Joseph
• Application Number:
  US13/844,567
• Publication Number:
  US 20140274728A1
• Time in Patent Office: 2,496 Days
• Field of Search: None
• 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 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Abstract

Methods and systems for the determination of a collection of relevant single nucleotide polymorphisms (SNP) probe compatible insertion/deletion probes across a genome to determine probes that can detect a variety of insertions and deletions.

Claims

1. A method of providing a genotyping array for genotyping a plurality of complex or multi-base indels within one or more sample genomes, the method comprising:

accessing a data set of one or more genome databases, said data set comprising information regarding genomic locations of the plurality of complex or multi-base indels (insertion/deletion) and sequences flanking at least one direction of the indels;

designing a plurality of indel probes to generate a plurality of designed indel probes for inclusion on a genotyping array by using one or more computer processors coupled to one or more memories configured to provide the one or more computer processors with instructions to, for each complex or multi-base indel of the plurality of complex or multi-base indels;

(a) access or generate a probe sequence that is complementary to a target sequence flanking the complex or multi-base indel;

(b) compare a deletion allele interrogation base to an insertion allele interrogation base in an indel identification sequence, the deletion allele interrogation base and the insertion allele interrogation base being referred to collectively as the interrogation bases, and,

(c) in response to the interrogation bases being different and being in different detection channels, create and output a single indel probe adapted for using the interrogation bases to differentiate the deletion allele and the insertion allele; and

(d) in response to the compared interrogation bases being a same interrogation base, incorporate the same interrogation base into the probe sequence and set new interrogation bases to be compared based on a next downstream base in a deletion allele sequence and a next downstream base in an insertion allele sequence and repeat (b)-(c) until either an interrogation distance threshold would be exceeded or the interrogation bases are different and are in different detection channels, and, when the interrogation bases are different and are in different detection channels, create and output a single indel probe adapted for using the interrogation bases to differentiate the deletion allele and the insertion allele;

synthesizing a plurality of indel probes of the genotyping array according to the plurality of designed indel probes; and

providing, with the genotyping array, labelled nucleotide bases corresponding to the interrogation bases for adding to a corresponding probe of the plurality of synthesized probes after the corresponding probe is hybridized with a sample during a genotyping assay, interrogation bases corresponding to some of the plurality of synthesized indel probes having different interrogation distances than do interrogation bases corresponding to others of the plurality of synthesized indel probes, a complex or multi-base indel of the plurality of complex or multi-base indels being genotyped by detecting labels corresponding to the labelled nucleotide bases.

2. The method of claim 1 wherein the instructions comprise instructions to:

(e) in response to the compared interrogation bases being different and being in the same detection channel, determine allele-specific oligonucleotides (ASO) for creating-two different indel probes, one for the insertion allele and one for the deletion allele by moving one base downstream in an insertion allele sequence and one base downstream in a deletion allele sequence to incorporate the compared interrogation bases, respectively, into each indel probe;

(f) identify a new interrogation base for a first probe that measures a first of the alleles;

(g) move downstream one base in a sequence of a second of the alleles to incorporate a base one downstream of the compared interrogation bases into a second probe that measures a second of the alleles;

(h) identify a new interrogation base for the second probe and compare to the new interrogation base for the first probe;

(i) repeat (g) and (h) until the new interrogation bases are the same or an interrogation distance threshold is exceeded;

(j) when an interrogation distance threshold is first exceeded, switch the second probe to the first probe and repeat (f), (g), and (h) until an identical interrogation base is found or an interrogation distance threshold is exceeded; and

(k) output the first and second probes as allele-specific oligonucleotides indel probes.

3. The method of claim 1 wherein the instructions comprise instructions to:

in response to the compared bases being different and being in the same detection channel, determine allele-specific oligonucleotides (ASO), creating two ASO probes comprising an insertion probe and a deletion probe for two different alleles by;

sliding one base downstream and incorporate the previous compared base into each ASO probe; and

for both the insertion and deletion probes, using a next base as a new interrogation base;

wherein determining final allele-specific probes requires the addition of only a single additional base into the deletion probe and the insertion probe.

4. A method for genotyping a plurality of complex or multi-base indels within one or more sample genomes comprising:

for each synthesized indel probe of a plurality of synthesized indel probes, adding one or more nucleotides or oligonucleotides including a label to the synthesized indel probe after hybridization with a sample, the one or more nucleotides or oligonucleotides being added corresponding to an interrogation base; and

genotyping a complex or multi-base indel of the plurality of complex or multi-base indels by detecting a respective label corresponding to a respective one or more nucleotides or oligonucleotides added to a respective synthesized indel probe of the plurality of synthesized indel probes;

wherein the plurality of synthesized probes have been designed by steps comprising;

accessing a data set of one or more genome databases, said data set comprising information regarding genomic locations of the one or more complex or multi-base indels (insertion/deletion) and sequences flanking at least one direction of the indels;

for each of the plurality of indel probes to be designed, designing the indel probe by;

(a) generating a probe sequence that is complementary to a target sequence flanking the complex or multi-base indel, and comparing interrogation bases in an indel identification sequence for a deletion allele probe and for an insertion allele probe; and

(b) if the compared interrogation bases are different and are in different detection channels, creating and outputting a single indel probe using the interrogation bases to differentiate the indel alleles and END;

(c) if the compared interrogation bases are different and in the same detection channel, EXIT (a)-(c);

(d) if the compared interrogation bases are the same, repeating beginning at step (a), but setting the interrogation base one base downstream of the previously compared interrogation base as a current compared interrogation base and increasing an interrogation distance counter by one until a base after the indel identification sequence differs from a prior base of the indel and when the interrogation bases, after the downstream setting, are different and are in different detection channels, creating and outputting a single indel probe using the interrogation bases to differentiate the deletion allele and the insertion allele; and

(e) repeating steps a-c until (b OR c) is TRUE or until an interrogation distance threshold is exceeded;

wherein designing results in a plurality of designed indel probes such that interrogation bases corresponding to some of the plurality of designed indel probes have different interrogation distances than do interrogation bases corresponding to others of the plurality of designed indel probes.

5. The method of claim 4 wherein indel probes are compatible with a system and analysis software using SNP probes to identify single nucleotide polymorphisms (SNPs) by detecting the identity of an interrogation base added to the SNP probes.

6. The method of claim 4 wherein creating and outputting a single SNP probe further comprises:

storing data representing the single SNP probe on a tangible media either immediately or after design of a plurality of SNP probes.

7. The method of claim 4 wherein creating and outputting a single SNP probe further comprises:

creating an oligonucleotide probe and configuring the oligonucleotide probe with other probes for use in an oligonucleotide array.

8. The method of claim 4 further comprising:

if the compared bases are different and in the same detection channel, EXIT (a)-(c) and determine allele-specific oligonucleotides (ASO), creating two different indel probes for the insertion allele and the deletion allele by;

sliding one base downstream and incorporating the previous interrogation base into each ASO probe; and

identifying a new interrogation base for the probe that measures the deletion;

sliding downstream one base the probe that measures the insertion until an identical interrogation base is found;

determining final allele-specific indel probes.

9. The method of claim 8 further comprising:

if no nearby identical interrogation base can be found, sliding the deletion or insertion probe one base downstream and retrying a number of times until an interrogation distance threshold is exceeded.

10. The method of claim 4 further comprising:

if the compared bases are different and in the same detection channel, EXIT (a)-(c) and determine allele-specific oligonucleotides (ASO), creating two different indel probes for the insertion allele and the deletion allele by;

sliding one base downstream and incorporating the previous interrogation base into each ASO probe; and

identifying a new interrogation base for the probe that measures the insertion;

sliding downstream one base the probe that measures the deletion until an interrogation base in the same detection channel is found;

determining final allele-specific indel probes.

11. The method of claim 10 further comprising:

if no nearby interrogation base in the same channel can be found, sliding deletion (or insertion) probe one downstream and retrying a number of times until an interrogation distance threshold is exceeded.

12. The method of claim 11 further comprising:

if the interrogation distance threshold is exceeded or if either the insertion or deletion probe has an interrogation distance greater than a threshold, repeating using the other probe and comparing resulting ASO indel probes.

13. The method of claim 4 further comprising:

if the compared bases are different and in the same detection channel, EXIT (a)-(c) and determine allele-specific oligonucleotides (ASO), creating two probes for two different alleles by;

sliding one base downstream and incorporating the previous compared base into each ASO probe; and

for both the insertion and deletion probes, using the next base as the new interrogation base;

determining final allele-specific probes wherein effectively requiring only the addition of a single additional base into the ASO probe for both the deletion and the insertion probe.

14. The method of claim 4 further comprising:

creating indel probes from both left and right sides.

15. The method of claim 4 wherein indel probes are designed so that each indel is represented by a collection of indel probes.