pathway ranking tool
DESCRIPTION
Pathway Ranking Tool. Dimitri Kosturos Linda Tsai SoCalBSI, 8/21/2003. Project Overview. Project Overview. BioDiscovery, Inc. at Marina del Rey Analyzing microarray data on pathway level instead of individual gene level Methods: -Enrichment Analysis -Permutational Statistics - PowerPoint PPT PresentationTRANSCRIPT
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Pathway Ranking ToolDimitri Kosturos Linda TsaiSoCalBSI, 8/21/2003
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Project OverviewBioDiscovery, Inc. at Marina del ReyAnalyzing microarray data on pathway level instead of individual gene levelMethods:-Enrichment Analysis-Permutational Statistics-S. Metric-Multivariate test
Project Overview
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Validation of statistical methods2 data sets: Brain Tumor, Interferon-gamma.Sources of annotation: BioCarta, Kegg, Gene Ontology.Project Overview, cont.
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phenotypemicroarrayalgorithmpathwayProject Flowchart
Dimitri,(Computer Scientist)
Linda(biologist)
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GeneSight is a data analysis softwareFeature:-Statistical significance testing-Multiple Data Visualizations -Automated gene annotation-Complete result reports-Pathway analysis (?)Research and Development in GeneSight
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Glioblastoma multiforme(GBM) is the most malignant of the glial tumors, classified as grade IV.
Many brain tumors are currently incurable.Average survival time: 1 yearBiology of Brain Tumor
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Oncogenes: promote normal cell growth Tumor suppressor genes: retard cell growth http://www.med.harvard.edu/publications/On_The_Brain/Volume4/Number2/SP95Awry.htmlBad Genes Foment Trouble
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Interferon is a class of cytokines that mediate antiviral, antiproliferative, antitumor activites, etc.IFN gamma is produced by T lymphocytes in response to mitogens or to antigens. IFNs bind to their receptors and initiate JAK-STAT signaling cascade.Biology of Interferon
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http://www.grt.kyushu-u.ac.jp/eny-doc/pathway/ifn_gamma.htmlBiology of Interferon, cont.
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Grouping related genes together into pathways(A)BioCartaEx: p53 Signaling Pathway(B)KEGGEx:Citrate cycle (TCA cycle)Grouping genes into structured, controlled vocabularies (ontologies) Gene Ontology-Biological Process. Ex: angiogenesis, apoptosis-Molecular Function. Ex: DNA binding activity-Cellular Component.Ex: nucleus, mitochondria
Gene Annotations
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Traditional method of ranking gene pathwaysSteps:1. Mann-Whitney Test: obtain list of probe sets that satisfy a certain p-value.2. Cluster analysis: see how many of listed probe occur in a cluster (pathway).
Example:Original data: 12,625 genes. Select genes p-value narrow to 927 genes.2.Cluster those 927 genes into clusters.
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Mann-Whitney Test, Denovo Glioblastoma p
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How Affy. Microarray Chips Workhttp://www.ucl.ac.uk/oncology/MicroCore/HTML_resource/Norm_Affy1.htmBest results: Genes hybridize perfectly with Perfect Match, and not at all with Mismatch. PM: Perfect Match MM: Mismatch
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Example of GeneSight PlotDataTheoretical Tumor Expression Levels (Log Transformed)Notice column replicates, Probe Set replicates.
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Given Data SetsGiven two data sets: Brain Tumor, IFN-Brain Tumor Data Set has 5+ tumor types,however, only 2 Tumor types were used (Denovo Glioblastoma, Progressive Glioblastoma)IFN- Data Set: the entire data set was used.
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What and why?Goal: write a prototype extension to GeneSight that uses permutational statistics to develop a custom distribution for a given Microarray data set.Overall significance: the software provides a list of (potentially) significant pathways that enables researchers to focus their work.
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What is permutational statistics?Choose different Control and Experiment groupings (permute).
By iterating through an adequate number of permutations, we can determine if a pathway is likely to be significant (p-value).
(In this context.)
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Permutational Stats.There are two versions of the S. Metric currently implemented.S. Metric I =S. Metric II =M = Number of Genes flagged as significantTotal = Total number of Genes in the Pathway
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(Layman's) How Statistics WorksDataStatisticP-ValuePermute HereS. Metric I, IIAfter all permutations are done, calculate the p-Value
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AlgorithmTake at least 10,000 unique permutations. A unique permutation is determined by a Permute class. For each condition For each permutation For each gene Calc. Mean diff. Calc. T-stat End For For each pathway store the statistic End for End for calcPvalue(stored statistic) End For pValue
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LimitationsComputational Power (Memory, CPU)Required number of replicates (8,8)
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Output of result
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Validation of pathway analysisMethod 1Problem: lack of insignificant pathways????
Sheet1
Computer algorithm classified as significant pathwaysComputer algorithm classified as insignificant pathways
Linda's Selection of significant pathwaysTrue PositiveFalse Negative
Linda's Selection of insignificant pathwaysFalse PositiveTrue Negative
Sheet2
Sheet3
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Validation of pathway analysisMethod 2Comparision of Prediction Methods024681012141616111621263136414651566166717681869196# of Pathways in BioCarta sorted by P-value# of identified significant pathways
Best algorithm
Random
Worst
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ResultBrain Tumor-BioCarta
Chart1
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# of pathways in BioCarta sorted by P-value
# of sig. identified pathways
D. glioblastoma
bioCarta-D. Glioblastoma
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bioCarta-D. Glioblastoma
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insig. SMI
insig. SMII
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Number of Pathways in BioCarta
Number of Identified pathways
D. glioblastoma-BioCarta Insignificant
KEGG-D.Glioblastoma
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# of identified pathways
D. Glioblastoma sort2
CC-D. Glioblastoma
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5959595953535353
6060606053535353
6161616153535353
6262626253535353
6363636353535353
6464646453535353
6565656553535353
6666666653535353
6767676753535353
6868686853535353
6969696953535353
7070707053535353
7171717153535353
7272727253535353
7373737353535353
7474747453535353
7575757553535353
7676767653535353
7777777753535353
7878787853535353
7979797953535353
CC-D. Glioblastoma
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
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112800
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123100
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133100
133100
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143100
143100
153100
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163100
163100
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173100
183100
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183100
183200
183300
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193600
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204000
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304200
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314200
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324200
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334300
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364400
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414600
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444700
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505000
505000
505000
505000
505000
515100
525200
535300
545400
555500
565600
575700
585800
595900
606000
616100
626200
636300
646400
656500
666600
676700
686800
696900
707000
717100
727200
737300
747400
757500
767600
777700
787800
797900
SMI
SMII
DG0.001
DG0.01
#of terms in biological process
# of terms identified
D. Glioblastoma-BP(GO)
MF-D. Glioblastoma
0000
0000
0000
0000
0000
0100
0100
0100
0100
0100
0100
0100
0100
0100
1100
2100
2100
2100
2100
2100
2100
2100
2100
3100
3100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
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4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4200
5300
5300
5300
5300
5300
5300
5300
5300
5300
5400
5400
5400
5400
5400
5400
5500
5500
5500
5500
5500
5500
5500
5500
5500
5600
5700
5700
5700
5700
5700
5800
5800
5800
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5800
5800
5800
5800
5800
5800
5800
5800
5800
5800
5800
5800
5800
5800
5800
5800
5800
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5800
5800
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5800
5800
5800
5800
5800
5800
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
5900
51000
51000
51000
51000
51000
51000
51000
51000
51000
51000
51000
51000
61000
61100
61100
61100
61100
61100
61100
61100
61100
61100
61100
61100
61100
61100
71100
71100
71100
71100
71100
71100
71100
71100
71100
71100
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71100
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71100
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71100
71100
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71100
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71100
71100
71100
71100
71100
71100
71100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
81100
91100
91100
91200
91200
91200
91200
101200
101200
101200
101200
101200
101200
101200
101200
101200
101200
111200
111200
111200
111200
111200
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111200
121200
121200
121200
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121200
121200
121200
131200
131200
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141200
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141200
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141300
141300
141300
141300
141300
141300
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141400
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161700
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181800
181800
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181800
181800
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201800
201800
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201800
201900
202000
202000
202000
202000
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202100
202100
202100
202100
202100
202200
202200
202300
202400
202400
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202500
202600
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202700
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202700
202700
202700
202700
202700
202700
202700
202700
202700
202700
202800
202800
202800
202900
202900
203000
203000
203000
203000
203000
203000
203000
203000
203000
203000
203000
203000
213000
213000
213000
213000
213000
213000
223000
223000
223000
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223000
223000
223000
223000
223000
233000
233000
233100
233200
233200
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253900
263900
273900
283900
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283900
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283900
284000
284100
284100
284100
284100
284100
284100
284100
284100
284100
284100
284100
284100
284100
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284100
284100
284100
284100
284100
284100
284100
284100
284100
284100
284100
294100
304100
304100
304100
304200
304300
304400
304500
304500
304600
304600
304600
304600
304600
304600
314600
314600
314600
324600
324600
324600
324600
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364700
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394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
394900
404900
404900
404900
414900
414900
414900
424900
424900
424900
424900
424900
424900
424900
424900
424900
424900
424900
424900
424900
424900
424900
424900
434900
434900
434900
434900
434900
444900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
454900
464900
474900
484900
484900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
494900
504900
504900
504900
504900
504900
514900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
524900
525000
525100
525100
525100
525100
525100
525100
525100
525100
525100
525100
525100
525200
525200
525200
525200
525300
525300
525300
525300
525300
525300
525300
525300
525300
525300
525300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
535300
insig SMI
insig SMII
insig DG0.001
insig DG0.01
#of terms in biological process
# of terms identified
D. Glioblastoma- insignificant BP(GO)
BioCarta-P. Glioblastoma
SM-ISMIIDG0.001DG0.01
000
000
000
000
000
000
000
001
011
021
031
041
042
043
043
043
053
053
053
053
053
053
053
053
053
053
053
053
063
063
073
074
074
074
074
074
074
074
074
075
075
076
076
076
076
076
176
277
377
477
577
677
777
877
977
1077
1178
1278
1378
1478
1578
1678
1778
1778
1788
1788
1798
1798
1798
1798
1798
1798
1798
1798
1798
1798
17108
17108
17108
17108
17108
17108
17108
17108
17108
17108
17108
17108
17108
17118
17118
17118
17118
17118
17118
17118
17118
17128
17128
17129
17129
17129
17129
17129
17129
17139
17139
17139
17139
17139
17149
17149
17159
17159
17159
17159
17159
171610
171710
171710
171710
171710
171710
171710
171710
171710
171710
171710
171810
171810
181810
181810
181811
181811
181811
181811
181811
181811
181811
181811
181811
181811
181811
181811
181811
181811
181811
181812
181813
181813
181813
181814
181814
181814
181814
181814
181814
181815
181816
181816
181816
181816
181816
181816
181816
181816
181817
181817
181817
181817
181817
181817
181817
181818
181819
181819
181819
181819
181819
181819
181819
181819
181820
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
181821
191921
202021
212121
BioCarta-P. Glioblastoma
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1700
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1800
1900
2000
2100
SMII
DG0.001
DG0.01
# of terms in CC
# of identified terms
D. glioblastoma-CC (GO)
KEGG-P.Glioblastoma
SM-I(sort2)SMIIDG0.001DG0.01
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
000
010
010
010
010
010
010
010
010
010
020
020
020
020
020
020
020
020
020
020
020
020
020
020
020
020
021
021
021
021
021
021
021
021
021
021
021
021
022
022
022
022
022
022
022
022
022
022
022
032
032
032
032
032
032
032
032
033
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
034
035
035
035
035
035
035
035
035
035
035
035
035
035
035
035
035
035
035
035
035
036
036
036
036
036
036
036
036
036
036
036
036
037
038
038
038
038
038
038
038
038
038
038
038
038
038
038
048
048
048
048
058
058
058
058
058
058
058
058
058
058
058
058
158
258
358
458
558
658
758
858
958
1058
1158
1258
1358
1458
1558
1658
1758
1858
1958
2058
2058
2058
2058
2058
2058
2058
2058
2058
2058
2058
2058
2058
2058
2058
2058
2068
2068
2068
2068
2068
2068
2068
2068
2068
2068
2068
2068
2078
2078
2078
2088
2088
2088
2088
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2098
2198
2298
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
2398
23108
23108
23108
23108
23108
23108
23108
23108
23108
23108
23108
23108
23108
23108
23108
24108
24108
24108
24118
24118
24118
24118
24118
24118
24118
24118
24118
24118
24118
24118
24118
25118
25128
25128
25128
25128
25138
25138
25149
251410
251510
261610
261610
261610
261610
261610
261610
261610
261610
261610
261610
261610
261610
261610
261610
261710
261710
261710
261710
261811
261912
261912
261912
261912
261912
261912
261912
261912
261913
261913
261914
261914
261914
261914
261914
261914
261914
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261915
261916
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
261917
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
262017
272017
272017
272017
272017
272017
272017
272017
272017
272017
272117
272117
272117
272117
272117
272117
272117
272117
272217
272317
272317
272317
272317
272317
272417
272417
272518
272519
272520
272520
272620
272620
272620
272720
272720
272820
272820
272820
282820
282920
282920
282920
292920
292920
302920
302920
302920
302920
302920
302920
302920
302920
302920
302920
302920
302920
302920
302920
302920
302920
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302921
302922
302923
302923
302923
302923
302923
302923
302923
302923
302923
302923
302924
302924
302924
302924
302924
302924
302924
302924
302924
302924
302924
302924
302925
302925
302925
302925