AUTOMATED NUCLEIC ACID REPEAT COUNT CALLING METHODS

1 . A computer-implemented method for determining the number of CGG repeats in a DNA comprising a CGG-rich region, the method comprising:

a) receiving, by one or more processors, DNA size and abundance data of DNA amplification products generated from the DNA comprising the CGG-rich region by using a primer set comprising a first primer recognizing the CGG-rich region and a second primer recognizing a region outside of the CGG-rich region;

b) generating, by the one or more processors, a set of sample data by sampling the DNA size and abundance data at a sampling frequency;

c) generating, by the one or more processors, a set of low-pass data by applying a low-pass filter to the set of sample data;

d) generating, by the one or more processors, a set of band-pass data by applying a band-pass filter to the set of sample data;

e) identifying, by the one or more processors, one or more peaks in the low-pass data;

f) identifying, by the one or more processors, one or more peaks in the band-pass data; and

g) identifying, by the one or more processors, a final peak representing a number of CGG repeats in the CGG-rich region based on the one or more peaks in the low-pass data and the one or more peaks in the band-pass data.

2 . The computer-implemented method of claim 1 , further comprising resolving the DNA amplification products to generate the DNA size and abundance data prior to step a).

3 . The computer-implemented method of claim 2 , wherein the resolving is carried out by capillary electrophoresis.

4 . The computer-implemented method of claim 1 , further comprising converting, by the one or more processors, the DNA size and abundance data from a time domain to a base-pair length domain prior to step b).

5 . The computer-implemented method of claim 4 , wherein a DNA ladder is used to convert the DNA size and abundance data from the time domain to the base-pair length domain.

6 . The computer-implemented method of claim 1 , wherein the sampling frequency is equal to four samples per base-pair.

7 . The computer-implemented method of claim 1 , wherein the band-pass filter has a low cutoff frequency of 2/13 multiplied by the sampling frequency and a high cutoff frequency of 2/11 multiplied by the sampling frequency.

8 . The computer-implemented method of claim 1 , wherein the low-pass filter has a cutoff frequency of 1.0*10 −5 multiplied by the sampling frequency.

9 . The computer-implemented method of claim 1 , wherein the low-pass filter and the band-pass filter are zero-phase finite impulse response (FIR) filters implemented using a Hamming window.

10 . The computer-implemented method of claim 1 , wherein generating the set of sample data by sampling the DNA size and abundance data at the sampling frequency comprises:

generating a linear interpolation of the DNA size and abundance data; and

sampling the linear interpolation of the DNA size and abundance data at the sampling frequency.

11 . The computer-implemented method of claim 1 , wherein the set of sample data comprises a signal representing a combination of a CGG series of the CGG-rich region and a full-length amplicon of the DNA comprising the CGG-rich region, the set of band-pass data comprises a signal representing the CGG series of the CGG-rich, and the set of low-pass data comprises a signal representing the full-length amplicon of the DNA comprising the CGG-rich region.

12 . The computer-implemented method of claim 1 , wherein identifying the final peak representing the number of CGG repeats in the DNA comprising the CGG-rich region comprises:

removing peaks from the one or more peaks in the low-pass data having a width less than 4.5 base-pairs and a height less than a threshold value;

removing peaks from the one or more peaks in the band-pass data having a width less than 4.5 base-pairs and a height less than the threshold value;

removing peaks from the one or more peaks in the band-pass data having a height less than a height of an adjacent peak having a larger base-pair length;

in response to a peak of the one or more peaks in the low-pass data having a height less than a height of a peak of the one or more peaks in the band-pass data that is within 3 base-pairs of the peak of the one or more peaks in the low-pass data, setting a center of the peak of the one or more peaks in the low-pass data to a center of the peak of the one or more peaks in the band-pass data, and setting a boundary of the peak of the one or more peaks in the low-pass data to a union of the peak of the one or more peaks in the low-pass data and the peak of the one or more peaks in the band-pass data;

merging peaks of the one or more peaks in the low-pass data and the one or more peaks in the band-pass data that have base-pair lengths greater than 165 base-pairs and that are within 30 base-pairs of each other; and

merging peaks of the one or more peaks in the low-pass data and the one or more peaks in the band-pass data that are within 15 base-pairs and that are more than a factor of 2 different in height, wherein a remaining peak of the one or more peaks in the low-pass data is the final peak.

13 . The computer-implemented method of claim 1 , wherein the DNA comprising a CGG-rich region is the 5′-UTR of the fragile X mental retardation 1 gene (FMR1).

14 . The computer-implemented method of claim 1 , wherein the DNA comprising a CGG-rich region is the 5′-UTR of the fragile X mental retardation 2 gene (FMR2).

15 . The computer-implemented method of claim 1 , wherein the first primer comprises at least four CGG or CCG repeats.

16 . The computer-implemented method of claim 1 , wherein the primer set further comprises a third primer recognizing a region outside of the CGG-rich region that is on the opposite side as the region recognized by the second primer.

17 . A computer-implemented method for determining a genotype associated with Fragile X syndrome in an individual, the method comprising:

a) performing DNA amplification reaction using a primer set comprising a first primer recognizing the CGG-rich region on the 5′ UTR of the FMR1 gene and a second primer recognizing a region outside of the CGG-rich region on the 5′ UTR of the FMR1 gene;

b) resolving the DNA amplification products to obtain DNA size and abundance data;

c) applying a low-pass filter and a band-pass filter to the DNA size and abundance data to identify a peak representing a number of CGG repeats in the CGG-rich region on the 5′ UTR of the FMR1 gene; and

d) determining the genotype of the individual based on the identified peak.

18 . The computer-implemented method of claim 17 , wherein resolving is carried out by capillary electrophoresis.

19 . The computer-implemented method of claim 17 , further comprising converting, by the one or more processors, the DNA size and abundance data from a time domain to a base-pair length domain prior to step c).

20 . The computer-implemented method of claim 19 , wherein a DNA ladder is used to convert the DNA size and abundance data from the time domain to the base-pair length domain.

21 . The computer-implemented method of claim 17 , wherein the method further comprises sampling the DNA size and abundance data at a sampling frequency, and wherein applying the low-pass filter and the band-pass filter to the DNA size and abundance data comprises applying the low-pass filter and the band-pass filter to the sampled DNA size and abundance data.

22 . The computer-implemented method of claim 21 , wherein the sampling frequency is equal to four samples per base-pair.

23 . The computer-implemented method of claim 21 , wherein the band-pass filter has a low cutoff frequency of 2/13 multiplied by the sampling frequency and a high cutoff frequency of 2/11 multiplied by the sampling frequency.

24 . The computer-implemented method of claim 21 , wherein the low-pass filter has a cutoff frequency of 1.0*10 −5 multiplied by the sampling frequency.

25 . The computer-implemented method of claim 21 , wherein sampling the DNA size and abundance data at the sampling frequency comprises:

generating a linear interpolation of the DNA size and abundance data; and

sampling the linear interpolation of the DNA size and abundance data at the sampling frequency.

26 . The computer-implemented method of claim 17 , wherein the low-pass filter and the band-pass filter are zero-phase finite impulse response (FIR) filters implemented using a Hamming window.

27 . The computer-implemented method of claim 17 , wherein the DNA size and abundance data comprises a signal representing a combination of a CGG series of the FMR1 gene and a full-length amplicon of the 5′ UTR of the FMR1 gene, the set of band-pass data comprises a signal representing the CGG series of the FMR1 gene, and the set of low-pass data comprises a signal representing the full-length amplicon of the 5′ UTR of the FMR1 gene.

28 . The computer-implemented method of claim 17 , wherein identifying the peak representing the number of CGG repeats in the CGG-rich region on the 5′ UTR of the FMR1 gene comprises:

removing peaks from the one or more peaks in an output of the low-pass filter having a width less than 4.5 base-pairs and a height less than a threshold value;

removing peaks from the one or more peaks in an output of the band-pass filter data having a width less than 4.5 base-pairs and a height less than the threshold value;

removing peaks from the one or more peaks in the output of the band-pass filter having a height less than a height of an adjacent peak having a larger base-pair length;

in response to a peak of the one or more peaks in the output of the low-pass filter having a height less than a height of a peak of the one or more peaks in the output of the band-pass filter that is within 3 base-pairs of the peak of the one or more peaks in the output of the low-pass filter, setting a center of the peak of the one or more peaks in the output of the low-pass filter to a center of the peak of the one or more peaks in the output of the band-pass filter, and setting a boundary of the peak of the one or more peaks in the output of the low-pass filter to a union of the peak of the one or more peaks in the output of the low-pass filter and the peak of the one or more peaks in the output of the band-pass filter;

merging peaks of the one or more peaks in the output of the low-pass filter and the one or more peaks in the output of the band-pass filter that have base-pair lengths greater than 165 base-pairs and that are within 30 base-pairs of each other; and

merging peaks of the one or more peaks in the output of the low-pass filter and the one or more peaks in the output of the band-pass filter that are within 15 base-pairs and that are more than a factor of 2 different in height, wherein a remaining peak of the one or more peaks in the output of the low-pass filter is the final peak.

29 . The computer-implemented method of claim 17 , further comprising determining whether the individual is a carrier for fragile X syndrome based on the genotype of the individual, wherein a number of CGG repeats in the CGG-rich region on the 5′ UTR of the FMR1 gene between 5-44 repeats is indicative of a normal allele, a number of CGG repeats in the CGG-rich region on the 5′ UTR of the FMR1 gene between 45-54 repeats is indicative of a an intermediate allele, a number of CGG repeats in the CGG-rich region on the 5′ UTR of the FMR1 gene between 55-200 repeats is indicative of a premutation allele, and wherein a number of CGG repeats in the CGG-rich region on the 5′ UTR of the FMR1 gene greater than 200 repeats is indicative of a full mutation allele.

30 . A computer-implemented method for determining the number of nucleic acid repeats in a DNA comprising a nucleic acid repeat region, the method comprising:

a) receiving, by one or more processors, DNA size and abundance data of DNA amplification products generated from the DNA comprising the nucleic acid repeat region by using a primer set comprising a first primer recognizing the nucleic acid repeat region and a second primer recognizing a region outside of the nucleic acid repeat region;

b) generating, by the one or more processors, a set of sample data by sampling the DNA size and abundance data at a sampling frequency;

c) generating, by the one or more processors, a set of low-pass data by applying a low-pass filter to the set of sample data;

d) generating, by the one or more processors, a set of band-pass data by applying a band-pass filter to the set of sample data;

e) identifying, by the one or more processors, one or more peaks in the low-pass data;

f) identifying, by the one or more processors, one or more peaks in the band-pass data; and

g) identifying, by the one or more processors, a final peak representing a number of nucleic acid repeats in the nucleic acid repeat region based on the one or more peaks in the low-pass data and the one or more peaks in the band-pass data.

31 . A non-transitory computer-readable storage medium comprising computer-executable instructions for carrying out any one of the computer-implemented methods of claim 1 .

32 . A system comprising a processor configured to carry out any one of the computer-implemented methods of claim 1 .