universtity of auckland - bioinformatics institute base ... · universtity of auckland -...
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![Page 1: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/1.jpg)
IntroductionPhred
Base calling projectProblems and outlook
Universtity of Auckland - Bioinformatics InstituteBase calling project
Gabriele Härtinger
Supervisors: David Bryant, Jari Kaipio
10/12/09
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 2: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/2.jpg)
IntroductionPhred
Base calling projectProblems and outlook
Guide
IntroductionPCRSanger method
PhredOverviewAlgorithms
Base calling projectNon-negative least-squaresMatlab
Problems and outlook
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 3: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/3.jpg)
IntroductionPhred
Base calling projectProblems and outlook
PCRSanger method
PCR
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 4: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/4.jpg)
IntroductionPhred
Base calling projectProblems and outlook
PCRSanger method
PCR
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 5: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/5.jpg)
IntroductionPhred
Base calling projectProblems and outlook
PCRSanger method
Sanger method
source:http://departments.oxy.edu/biology/Stillman/bi221/092200/lecture_notes.htm
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 6: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/6.jpg)
IntroductionPhred
Base calling projectProblems and outlook
PCRSanger method
Sanger method
source: http://dnasequencing.wordpress.com/
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 7: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/7.jpg)
IntroductionPhred
Base calling projectProblems and outlook
PCRSanger method
Chromatogram
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 8: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/8.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred
I standard program for base calling
I reads DNA sequencer trace data
I calls bases
I assigns quality values to the bases
I writes the base calls and quality values to output files
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 9: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/9.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred
I standard program for base calling
I reads DNA sequencer trace data
I calls bases
I assigns quality values to the bases
I writes the base calls and quality values to output files
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 10: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/10.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred
I standard program for base calling
I reads DNA sequencer trace data
I calls bases
I assigns quality values to the bases
I writes the base calls and quality values to output files
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 11: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/11.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred
I standard program for base calling
I reads DNA sequencer trace data
I calls bases
I assigns quality values to the bases
I writes the base calls and quality values to output files
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 12: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/12.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred
I standard program for base calling
I reads DNA sequencer trace data
I calls bases
I assigns quality values to the bases
I writes the base calls and quality values to output files
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 13: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/13.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred - Algorithms
I With simple Fourier methods Phred examine the four basetraces.
I Check the region surrounding each point in the data set inorder to predict a series of evenly spaced predicted locations.
I Examine each trace to find the centers of the actual, orobserved, peaks.
I Control every of the four traces separate, many peaks overlap.I Dynamic programming algorithm match the predicted peaks
locations with the observed peaks.Some peaks will be exclude and some will splitted.
I Unmatched observed peaks will compared with the predictedpeak and if found inserted.
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 14: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/14.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred - Algorithms
I With simple Fourier methods Phred examine the four basetraces.
I Check the region surrounding each point in the data set inorder to predict a series of evenly spaced predicted locations.
I Examine each trace to find the centers of the actual, orobserved, peaks.
I Control every of the four traces separate, many peaks overlap.I Dynamic programming algorithm match the predicted peaks
locations with the observed peaks.Some peaks will be exclude and some will splitted.
I Unmatched observed peaks will compared with the predictedpeak and if found inserted.
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 15: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/15.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred - Algorithms
I With simple Fourier methods Phred examine the four basetraces.
I Check the region surrounding each point in the data set inorder to predict a series of evenly spaced predicted locations.
I Examine each trace to find the centers of the actual, orobserved, peaks.
I Control every of the four traces separate, many peaks overlap.I Dynamic programming algorithm match the predicted peaks
locations with the observed peaks.Some peaks will be exclude and some will splitted.
I Unmatched observed peaks will compared with the predictedpeak and if found inserted.
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 16: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/16.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred - Algorithms
I With simple Fourier methods Phred examine the four basetraces.
I Check the region surrounding each point in the data set inorder to predict a series of evenly spaced predicted locations.
I Examine each trace to find the centers of the actual, orobserved, peaks.
I Control every of the four traces separate, many peaks overlap.
I Dynamic programming algorithm match the predicted peakslocations with the observed peaks.Some peaks will be exclude and some will splitted.
I Unmatched observed peaks will compared with the predictedpeak and if found inserted.
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 17: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/17.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred - Algorithms
I With simple Fourier methods Phred examine the four basetraces.
I Check the region surrounding each point in the data set inorder to predict a series of evenly spaced predicted locations.
I Examine each trace to find the centers of the actual, orobserved, peaks.
I Control every of the four traces separate, many peaks overlap.I Dynamic programming algorithm match the predicted peaks
locations with the observed peaks.Some peaks will be exclude and some will splitted.
I Unmatched observed peaks will compared with the predictedpeak and if found inserted.
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 18: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/18.jpg)
IntroductionPhred
Base calling projectProblems and outlook
OverviewAlgorithms
Phred - Algorithms
I With simple Fourier methods Phred examine the four basetraces.
I Check the region surrounding each point in the data set inorder to predict a series of evenly spaced predicted locations.
I Examine each trace to find the centers of the actual, orobserved, peaks.
I Control every of the four traces separate, many peaks overlap.I Dynamic programming algorithm match the predicted peaks
locations with the observed peaks.Some peaks will be exclude and some will splitted.
I Unmatched observed peaks will compared with the predictedpeak and if found inserted.
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 19: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/19.jpg)
IntroductionPhred
Base calling projectProblems and outlook
Non-negative least-squaresMatlab
Base calling
project:base calling in another way
Deconvolve the peaks with thenon-negative least-squares method.
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 20: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/20.jpg)
IntroductionPhred
Base calling projectProblems and outlook
Non-negative least-squaresMatlab
Base calling
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 21: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/21.jpg)
IntroductionPhred
Base calling projectProblems and outlook
Non-negative least-squaresMatlab
Non-negative least-squares
I least-squares
y = A x + e ⇒ e = y − A x
min ||e||2 = min ||y − A x ||2 =1N
N∑k=1
e2k ← minimize
I non-negative least-squares method
minx≥0||e||2 = min
x≥0||y − A x ||2 =
1N
N∑k=1
e2k ← minimize
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 22: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/22.jpg)
IntroductionPhred
Base calling projectProblems and outlook
Non-negative least-squaresMatlab
Non-negative least-squares
I least-squares
y = A x + e ⇒ e = y − A x
min ||e||2 = min ||y − A x ||2 =1N
N∑k=1
e2k ← minimize
I non-negative least-squares method
minx≥0||e||2 = min
x≥0||y − A x ||2 =
1N
N∑k=1
e2k ← minimize
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
![Page 23: Universtity of Auckland - Bioinformatics Institute Base ... · Universtity of Auckland - Bioinformatics Institute Base calling project GabrieleHärtinger Supervisors: David Bryant,](https://reader030.vdocuments.site/reader030/viewer/2022040712/5e16a5955c6af901ae15616c/html5/thumbnails/23.jpg)
IntroductionPhred
Base calling projectProblems and outlook
Non-negative least-squaresMatlab
Matlab - LS
0 20 40 60 80 100 120−0.2
0
0.2
0.4
0.6
0.8
1
1.2
Blue: true xGreen: ChromatogramRed: regularized LS
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
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IntroductionPhred
Base calling projectProblems and outlook
Non-negative least-squaresMatlab
Matlab - NNLS
0 20 40 60 80 100 120−0.2
0
0.2
0.4
0.6
0.8
1
1.2
Blue: NNLSGreen: ChromatogramRed: regularized LS
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
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IntroductionPhred
Base calling projectProblems and outlook
Problem
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Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
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IntroductionPhred
Base calling projectProblems and outlook
Outlook
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Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project
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IntroductionPhred
Base calling projectProblems and outlook
Acknowledgement
A big THANKS to Allen, David, Jari, Peter
and to all of the Bioinformatics Institute.
Gabriele Härtinger Universtity of Auckland - Bioinformatics Institute Base calling project