Detection of microsatellite instability
Methods and systems for detecting MSI are provided. Also provided are methods for enriching human genomic DNA for microsatellite loci. Additionally, an oligonucleotide array for detecting MSI is provided. In some embodiments, the method for detecting MSI comprises detecting sequencing reads from a sample for a plurality of microsatellite loci; determining a metric for repeat length distribution (RLD) for each microsatellite locus; comparing the metric to a threshold value for the microsatellite locus; quantifying the number of microsatellite loci having an RLD metric exceeding the threshold value; and comparing the number of microsatellite loci having an RLD metric exceeding the threshold value to a microsatellite instability (MSI) proportion threshold.
1 . A method for treatment, comprising:
(i) detecting sequencing reads for a plurality of microsatellite loci from a genomic DNA sample derived from a human diagnosed as having colorectal cancer, wherein the genomic DNA sample is cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA), and wherein the sequencing reads are detected using a sequencing device configured to assay for the plurality of the microsatellite loci;
(ii) determining a metric for repeat length distribution (RLD) for each microsatellite locus;
(iii) for each microsatellite locus, comparing the RLD metric to a threshold value for the microsatellite locus, wherein each microsatellite has an independent threshold value, wherein the threshold value has been determined by (i) the repeat length distribution of the microsatellite locus in a MSS sample; (ii) the repeat length distribution of the microsatellite locus in a matched normal sample; (iii) the repeat length distribution of the microsatellite locus in a pre-computed normal background; (iv) the repeat length distribution of the microsatellite locus obtained from a MSS cell line; or (v) a RLD metric at which the microsatellite locus is identified as MSI; and
administering a therapeutically effective amount of at least one antibody and/or at least one small molecule therapeutic agent to an identified human diagnosed as having colorectal cancer and as having microsatellite instability, wherein the identifying of the human diagnosed as having colorectal cancer and as having microsatellite instability comprises: (i) quantifying a number of microsatellite loci having the RLD metric exceeding the threshold value; (ii) comparing the quantified number to a microsatellite instability proportion threshold; and (iii) determining if the quantified number exceeds the microsatellite instability proportion threshold, wherein if the quantified number exceeds the microsatellite instability proportion threshold, the human diagnosed as having colorectal cancer has microsatellite instability;
(iv) wherein the at least one antibody and/or the at least one small molecule is selected from the group consisting of Avastin (Bevacizumab); Bevacizumab; Camptosar (Irinotecan Hydrochloride); Capecitabine; Cetuximab; Cyramza (Ramucirumab); Eloxatin (Oxaliplatin); Erbitux (Cetuximab); 5-FU (Fluorouracil Injection); Irinotecan Hydrochloride; Leucovorin Calcium; Lonsurf (Trifluridine and Tipiracil Hydrochloride); Nivolumab; Opdivo (Nivolumab); Oxaliplatin; Panitumumab; Ramucirumab; Regorafenib; Stivarga (Regorafenib); Trifluridine and Tipiracil Hydrochloride; Vectibix (Panitumumab); Wellcovorin (Leucovorin Calcium); Xeloda (Capecitabine); Zaltrap (Ziv-Aflibercept); and Ziv-Aflibercept; and
wherein the plurality of microsatellite loci are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 microsatellite loci selected from the group consisting of microsatellite loci 1-170:
microsatellite
chromosome
start
end
locus
location
coordinate
coordinate
1
chr1
32936733
32936750
2
chr1
46859627
46859640
3
chr1
149929093
149929109
4
chr1
235344151
235344170
5
chr2
5699709
5699727
6
chr2
5700420
5700434
7
chr2
14638213
14638234
8
chr2
39309548
39309575
9
chr2
47414420
47414447
10
chr2
51061373
51061415
11
chr2
66435743
66435767
12
chr2
95183613
95183636
13
chr2
98998132
98998145
14
chr2
102762122
102762138
15
chr2
129982139
129982152
16
chr2
173266783
173266800
17
chr3
70959190
70959203
18
chr3
123614028
123614044
19
chr4
39500102
39500120
20
chr4
54732045
54732070
21
chr4
151662755
151662771
22
chr5
112877981
112878021
23
chr5
140115261
140115276
24
chr5
181260427
181260439
25
chr6
11714406
11714420
26
chr7
1747883
1747900
27
chr7
143306249
143306274
28
chr8
33498673
33498689
29
chr8
40154248
40154264
30
chr8
58148166
58148186
31
chr8
93925321
93925334
32
chr9
84003046
84003063
33
chr9
126837838
126837854
34
chr10
21518540
21518553
35
chr10
119137173
119137195
36
chr11
5666090
5666107
37
chr11
115176312
115176326
38
chr11
125620870
125620891
39
chr11
125893715
125893728
40
chr12
84892141
84892158
41
chr12
130399009
130399022
42
chr13
50013518
50013540
43
chr14
23183137
23183158
44
chr14
64523982
64523995
45
chr14
73492999
73493015
46
chr16
19118464
19118481
47
chr16
29669203
29669217
48
chr16
58543411
58543424
49
chr17
38995870
38995910
50
chr18
649879
649894
51
chr19
11808381
11808396
52
chr19
21378606
21378625
53
chr19
57257698
57257723
54
chr19
57258437
57258450
55
chr19
57262350
57262363
56
chr20
59922426
59922441
57
chr21
34103315
34103331
58
chrX
2904903
2904920
59
chrX
38805564
38805577
60
chrX
101413944
101413957
61
chrX
102154282
102154298
62
chrX
102658669
102658683
63
chrX
102751949
102751969
64
chrX
145825169
145825191
65
chr1
103663067
103663077
66
chr1
103757205
103757215
67
chr1
116112562
116112575
68
chr1
13782253
13782262
69
chr1
150714123
150714133
70
chr1
151224226
151224235
71
chr1
159062696
159062706
72
chr1
200624913
200624921
73
chr1
221702319
221702330
74
chr1
236897645
236897660
75
chr1
28459218
28459228
76
chr1
52784862
52784872
77
chr1
88983825
88983836
78
chr10
72893710
72893720
79
chr11
105007313
105007323
80
chr11
105008959
105008969
81
chr11
120479927
120479935
82
chr11
63382198
63382209
83
chr11
65501008
65501020
84
chr12
118150956
118150974
85
chr12
119728455
119728465
86
chr12
13211492
13211502
87
chr12
31282674
31282685
88
chr12
54011395
54011406
89
chr12
96286155
96286166
90
chr13
46771334
46771348
91
chr13
49350729
49350739
92
chr13
57725300
57725311
93
chr14
19668967
19668978
94
chr14
53046721
53046733
95
chr15
30113885
30113899
96
chr15
37099551
37099563
97
chr15
41852769
41852780
98
chr15
72572067
72572077
99
chr16
14889234
14889243
100
chr17
1423460
1423474
101
chr17
4539344
4539362
102
chr17
45539269
45539279
103
chr17
46306192
46306202
104
chr17
46523767
46523777
105
chr17
56938993
56939007
106
chr17
58357799
58357806
107
chr18
23529756
23529766
108
chr18
319944
319955
109
chr18
33333178
33333188
110
chr18
9954236
9954249
111
chr19
12463926
12463947
112
chr19
13993876
13993890
113
chr19
44158121
44158134
114
chr19
9251064
9251075
115
chr2
106426090
106426102
116
chr2
108729222
108729232
117
chr2
118096518
118096531
118
chr2
147926116
147926124
119
chr2
151379531
151379545
120
chr2
182942165
182942175
121
chr2
196666794
196666805
122
chr2
202300378
202300388
123
chr2
210315041
210315052
124
chr2
55234233
55234243
125
chr2
63842143
63842158
126
chr2
87757451
87757462
127
chr2
8858645
8858657
128
chr20
53875234
53875245
129
chr20
60012728
60012738
130
chr21
28966883
28966894
131
chr21
32601784
32601798
132
chr22
38683915
38683930
133
chr3
101459057
101459067
134
chr3
113658634
113658645
135
chr3
131014202
131014213
136
chr3
165187860
165187871
137
chr3
170997894
170997904
138
chr3
25719646
25719656
139
chr3
30650379
30650389
140
chr3
51380172
51380179
141
chr5
159099526
159099541
142
chr5
162067984
162067996
143
chr5
177302735
177302747
144
chr5
58974630
58974642
145
chr5
68288684
68288696
146
chr5
78450039
78450050
147
chr5
80675095
80675103
148
chr5
83641432
83641442
149
chr5
88722571
88722584
150
chr6
167040031
167040043
151
chr6
43054238
43054250
152
chr6
49492281
49492291
153
chr6
55874913
55874927
154
chr6
70861991
70862003
155
chr6
88143811
88143822
156
chr7
135619945
135619955
157
chr7
30633896
30633911
158
chr7
54752300
54752311
159
chr7
75193019
75193032
160
chr7
95359943
95359960
161
chr8
23854553
23854565
162
chr8
33499307
33499320
163
chr8
39103749
39103760
164
chr8
7489344
7489353
165
chr8
7822205
7822214
166
chr8
78717503
78717517
167
chr9
107331689
107331701
168
chr9
83969307
83969318
169
chrX
101883466
101883477
170
chrX
18164977
18164992
2 . The method of claim 1 , wherein the plurality of microsatellite loci are at least 58 microsatellite loci selected from the group consisting of microsatellite loci: 1-64.
3 . The method of claim 1 , wherein the plurality of microsatellite loci corresponds to the microsatellite loci:
Chr.
Coordinate
Coordinate
chr1
149929093
149929109
chr1
235344151
235344170
chr1
32936733
32936750
chr1
46859627
46859640
chr10
119137173
119137195
chr10
21518540
21518553
chr11
115176312
115176326
chr11
125620870
125620891
chr11
125893715
125893728
chr11
5666090
5666107
chr12
130399009
130399022
chr12
84892141
84892158
chr13
50013518
50013540
chr14
23183137
23183158
chr14
64523982
64523995
chr14
73492999
73493015
chr16
19118464
19118481
chr16
29669203
29669217
chr16
58543411
58543424
chr18
649879
649894
chr19
11808381
11808396
chr19
21378606
21378625
chr19
57257698
57257723
chr19
57258437
57258450
chr19
57262350
57262363
chr2
102762122
102762138
chr2
129982139
129982152
chr2
14638213
14638234
chr2
173266783
173266800
chr2
39309548
39309575
chr2
47414420
47414447
chr2
5700420
5700434
chr2
66435743
66435767
chr2
95183613
95183636
chr2
98998132
98998145
chr20
59922426
59922441
chr21
34103315
34103331
chr3
123614028
123614044
chr3
70959190
70959203
chr4
151662755
151662771
chr4
39500102
39500120
chr4
54732045
54732070
chr5
140115261
140115276
chr6
11714406
11714420
chr7
143306249
143306274
chr7
1747883
1747900
chr8
33498673
33498689
chr8
40154248
40154264
chr8
93925321
93925334
chr9
126837838
126837854
chr9
84003046
84003063
chrX
101413944
101413957
chrX
102154282
102154298
chrX
102658669
102658683
chrX
102751949
102751969
chrX
145825169
145825191
chrX
2904903
2904920
chrX
38805564
38805577.
4 . The method of claim 1 , wherein the metric for RLD is determined in the absence of a matched normal sample.
5 . The method of claim 1 , wherein the metric of a microsatellite locus is a t-statistic determined using one or more statistical values of the RLD of the human for the microsatellite locus and one or more statistical values of a reference RLD.
6 . The method of claim 5 , wherein the t-statistic is determined based on mean read length and variance in the RLD.
7 . The method of claim 1 , wherein the detecting comprises generating nucleotide sequencing reads from the genomic DNA sample and identifying sequencing reads comprising a microsatellite locus by alignment of the sequencing reads to a set of microsatellite loci DNA sequences; and counting the number of repeat lengths for each sequencing read determined to comprise a microsatellite locus, thereby generating an RLD for each microsatellite locus.
8 . The method of claim 7 , wherein prior to generating nucleotide sequencing reads, the human genomic DNA is enriched for microsatellite loci by hybridizing the human genomic DNA to oligonucleotide probes that hybridize to microsatellite locus-containing DNA.
9 . The method of claim 1 , further comprising:
(i) providing a sample comprising the human genomic DNA fragments; contacting a pool of oligonucleotide probes to the sample;
(ii) capturing DNA fragments comprising microsatellite loci in hybridization complexes with the pool oligonucleotide probes, thereby generating captured DNA;
(iii) separating the hybridization complexes from unbound nucleic acids; eluting the captured DNA from the hybridization complexes;
(iv) sequencing the captured DNA that was eluted from the hybridization complexes;
(v) identifying sequencing reads comprising a microsatellite locus by alignment of the sequencing reads to a set of microsatellite loci DNA sequences; and
(vi) counting the number of repeat lengths for each sequencing read determined to comprise a microsatellite locus, thereby generating an RID for each microsatellite locus.
10 . The method of claim 1 , further comprising:
(i) providing a sample comprising the human genomic DNA fragments;
(ii) amplifying DNA fragments comprising microsatellite loci with oligonucleotide primers specific for a microsatellite locus to produce amplicons;
(iii) sequencing the amplicons;
(iv) identifying sequencing reads comprising a microsatellite locus by alignment of the sequencing reads to a set of microsatellite loci DNA sequences; and
(v) counting the number of repeat lengths for each sequencing read determined to comprise a microsatellite locus, thereby generating an RLD for each microsatellite locus.
11 . The method of claim 1 , wherein the human is treated with at least two antibodies and/or at least two small molecule therapeutic agents.
12 . A method of treatment, comprising:
(i) generating sequencing reads for a plurality of microsatellite loci from a genomic DNA sample derived from a human diagnosed as having colorectal cancer, wherein the genomic DNA sample is cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA), and wherein the sequencing reads are generated using a sequencing device configured to assay for the plurality of the microsatellite loci;
(ii) determining a repeat length distribution (RLD) for each microsatellite locus;
(iii) generating a metric for the RLD for each microsatellite locus;
(iv) for each microsatellite locus, comparing the RLD metric for each microsatellite locus to a threshold value for the microsatellite locus, wherein each microsatellite has an independent threshold value, wherein the threshold value has been determined by (i) the repeat length distribution of the microsatellite locus in a MSS sample; or (ii) the repeat length distribution of the microsatellite locus in a matched normal sample; or (iii) the repeat length distribution of the microsatellite locus in a pre-computed normal background; or (iv) the repeat length distribution of the microsatellite locus obtained from a MSS cell line; or (v) a RLD metric at which the microsatellite locus is identified as MSI; and
(v) administering a therapeutically effective amount of at least one antibody and/or at least one small molecule therapeutic agent to an identified human diagnosed as having colorectal cancer and as having microsatellite instability, wherein the identifying of the human diagnosed as having colorectal cancer and as having microsatellite instability comprises: (i) quantifying a number of detected loci having an RLD metric exceeding the threshold value, (ii) by comparing the quantified number to a locus set proportion threshold; and (iii) determining if the quantified number exceeds the locus set proportion threshold, wherein if the quantified number exceeds the locus set proportion threshold, the human diagnosed as having colorectal cancer has microsatellite instability;
wherein the at least one antibody and/or the at least one small molecule is selected from the group consisting of Avastin (Bevacizumab); Bevacizumab; Camptosar (Irinotecan Hydrochloride); Capecitabine; Cetuximab; Cyramza (Ramucirumab); Eloxatin (Oxaliplatin); Erbitux (Cetuximab); 5-FU (Fluorouracil Injection); Irinotecan Hydrochloride; Leucovorin Calcium; Lonsurf (Trifluridine and Tipiracil Hydrochloride); Nivolumab; Opdivo (Nivolumab); Oxaliplatin; Panitumumab; Ramucirumab; Regorafenib; Stivarga (Regorafenib); Trifluridine and Tipiracil Hydrochloride; Vectibix (Panitumumab); Wellcovorin (Leucovorin Calcium); Xeloda (Capecitabine); Zaltrap (Ziv-Aflibercept); and Ziv-Aflibercept; and
wherein the plurality of microsatellite loci are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 microsatellite loci selected from the group consisting of microsatellite loci 1-170:
microsatellite
chromosome
start
end
locus
location
coordinate
coordinate
1
chr1
32936733
32936750
2
chr1
46859627
46859640
3
chr1
149929093
149929109
4
chr1
235344151
235344170
5
chr2
5699709
5699727
6
chr2
5700420
5700434
7
chr2
14638213
14638234
8
chr2
39309548
39309575
9
chr2
47414420
47414447
10
chr2
51061373
51061415
11
chr2
66435743
66435767
12
chr2
95183613
95183636
13
chr2
98998132
98998145
14
chr2
102762122
102762138
15
chr2
129982139
129982152
16
chr2
173266783
173266800
17
chr3
70959190
70959203
18
chr3
123614028
123614044
19
chr4
39500102
39500120
20
chr4
54732045
54732070
21
chr4
151662755
151662771
22
chr5
112877981
112878021
23
chr5
140115261
140115276
24
chr5
181260427
181260439
25
chr6
11714406
11714420
26
chr7
1747883
1747900
27
chr7
143306249
143306274
28
chr8
33498673
33498689
29
chr8
40154248
40154264
30
chr8
58148166
58148186
31
chr8
93925321
93925334
32
chr9
84003046
84003063
33
chr9
126837838
126837854
34
chr10
21518540
21518553
35
chr10
119137173
119137195
36
chr11
5666090
5666107
37
chr11
115176312
115176326
38
chr11
125620870
125620891
39
chr11
125893715
125893728
40
chr12
84892141
84892158
41
chr12
130399009
130399022
42
chr13
50013518
50013540
43
chr14
23183137
23183158
44
chr14
64523982
64523995
45
chr14
73492999
73493015
46
chr16
19118464
19118481
47
chr16
29669203
29669217
48
chr16
58543411
58543424
49
chr17
38995870
38995910
50
chr18
649879
649894
51
chr19
11808381
11808396
52
chr19
21378606
21378625
53
chr19
57257698
57257723
54
chr19
57258437
57258450
55
chr19
57262350
57262363
56
chr20
59922426
59922441
57
chr21
34103315
34103331
58
chrX
2904903
2904920
59
chrX
38805564
38805577
60
chrX
101413944
101413957
61
chrX
102154282
102154298
62
chrX
102658669
102658683
63
chrX
102751949
102751969
64
chrX
145825169
145825191
65
chr1
103663067
103663077
66
chr1
103757205
103757215
67
chr1
116112562
116112575
68
chr1
13782253
13782262
69
chr1
150714123
150714133
70
chr1
151224226
151224235
71
chr1
159062696
159062706
72
chr1
200624913
200624921
73
chr1
221702319
221702330
74
chr1
236897645
236897660
75
chr1
28459218
28459228
76
chr1
52784862
52784872
77
chr1
88983825
88983836
78
chr10
72893710
72893720
79
chr11
105007313
105007323
80
chr11
105008959
105008969
81
chr11
120479927
120479935
82
chr11
63382198
63382209
83
chr11
65501008
65501020
84
chr12
118150956
118150974
85
chr12
119728455
119728465
86
chr12
13211492
13211502
87
chr12
31282674
31282685
88
chr12
54011395
54011406
89
chr12
96286155
96286166
90
chr13
46771334
46771348
91
chr13
49350729
49350739
92
chr13
57725300
57725311
93
chr14
19668967
19668978
94
chr14
53046721
53046733
95
chr15
30113885
30113899
96
chr15
37099551
37099563
97
chr15
41852769
41852780
98
chr15
72572067
72572077
99
chr16
14889234
14889243
100
chr17
1423460
1423474
101
chr17
4539344
4539362
102
chr17
45539269
45539279
103
chr17
46306192
46306202
104
chr17
46523767
46523777
105
chr17
56938993
56939007
106
chr17
58357799
58357806
107
chr18
23529756
23529766
108
chr18
319944
319955
109
chr18
33333178
33333188
110
chr18
9954236
9954249
111
chr19
12463926
12463947
112
chr19
13993876
13993890
113
chr19
44158121
44158134
114
chr19
9251064
9251075
115
chr2
106426090
106426102
116
chr2
108729222
108729232
117
chr2
118096518
118096531
118
chr2
147926116
147926124
119
chr2
151379531
151379545
120
chr2
182942165
182942175
121
chr2
196666794
196666805
122
chr2
202300378
202300388
123
chr2
210315041
210315052
124
chr2
55234233
55234243
125
chr2
63842143
63842158
126
chr2
87757451
87757462
127
chr2
8858645
8858657
128
chr20
53875234
53875245
129
chr20
60012728
60012738
130
chr21
28966883
28966894
131
chr21
32601784
32601798
132
chr22
38683915
38683930
133
chr3
101459057
101459067
134
chr3
113658634
113658645
135
chr3
131014202
131014213
136
chr3
165187860
165187871
137
chr3
170997894
170997904
138
chr3
25719646
25719656
139
chr3
30650379
30650389
140
chr3
51380172
51380179
141
chr5
159099526
159099541
142
chr5
162067984
162067996
143
chr5
177302735
177302747
144
chr5
58974630
58974642
145
chr5
68288684
68288696
146
chr5
78450039
78450050
147
chr5
80675095
80675103
148
chr5
83641432
83641442
149
chr5
88722571
88722584
150
chr6
167040031
167040043
151
chr6
43054238
43054250
152
chr6
49492281
49492291
153
chr6
55874913
55874927
154
chr6
70861991
70862003
155
chr6
88143811
88143822
156
chr7
135619945
135619955
157
chr7
30633896
30633911
158
chr7
54752300
54752311
159
chr7
75193019
75193032
160
chr7
95359943
95359960
161
chr8
23854553
23854565
162
chr8
33499307
33499320
163
chr8
39103749
39103760
164
chr8
7489344
7489353
165
chr8
7822205
7822214
166
chr8
78717503
78717517
167
chr9
107331689
107331701
168
chr9
83969307
83969318
169
chrX
101883466
101883477
170
chrX
18164977
18164992
13 . The method of claim 1 , wherein the sequencing device is a nanopore sequencing device.
14 . The method of claim 12 , wherein the plurality of microsatellite loci are at least 58 microsatellite loci selected from the group consisting of microsatellite loci: 1-64.
15 . The method of claim 12 , wherein the plurality of microsatellite loci corresponds to the microsatellite loci:
Chr.
Coordinate
Coordinate
chr1
149929093
149929109
chr1
235344151
235344170
chr1
32936733
32936750
chr1
46859627
46859640
chr10
119137173
119137195
chr10
21518540
21518553
chr11
115176312
115176326
chr11
125620870
125620891
chr11
125893715
125893728
chr11
5666090
5666107
chr12
130399009
130399022
chr12
84892141
84892158
chr13
50013518
50013540
chr14
23183137
23183158
chr14
64523982
64523995
chr14
73492999
73493015
chr16
19118464
19118481
chr16
29669203
29669217
chr16
58543411
58543424
chr18
649879
649894
chr19
11808381
11808396
chr19
21378606
21378625
chr19
57257698
57257723
chr19
57258437
57258450
chr19
57262350
57262363
chr2
102762122
102762138
chr2
129982139
129982152
chr2
14638213
14638234
chr2
173266783
173266800
chr2
39309548
39309575
chr2
47414420
47414447
chr2
5700420
5700434
chr2
66435743
66435767
chr2
95183613
95183636
chr2
98998132
98998145
chr20
59922426
59922441
chr21
34103315
34103331
chr3
123614028
123614044
chr3
70959190
70959203
chr4
151662755
151662771
chr4
39500102
39500120
chr4
54732045
54732070
chr5
140115261
140115276
chr6
11714406
11714420
chr7
143306249
143306274
chr7
1747883
1747900
chr8
33498673
33498689
chr8
40154248
40154264
chr8
93925321
93925334
chr9
126837838
126837854
chr9
84003046
84003063
chrX
101413944
101413957
chrX
102154282
102154298
chrX
102658669
102658683
chrX
102751949
102751969
chrX
145825169
145825191
chrX
2904903
2904920
chrX
38805564
38805577.
16 . A method of treatment, comprising:
(i) preparing a nucleic acid library for sequencing, wherein the nucleic acid library for sequencing is enriched for a plurality of microsatellite loci, wherein the nucleic acid library enriched for the plurality of microsatellite loci is prepared by capturing DNA fragments from a sample derived from a human diagnosed as having colorectal cancer with a plurality of oligonucleotide probes that specifically hybridize to microsatellite loci, wherein the genomic DNA sample is cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA);
(ii) sequencing the prepared nucleic acid library with a sequencing device to provide a sequence data set;
(iii) detecting sequencing reads within the sequence data set for the plurality of microsatellite loci;
(iv) determining a metric for repeat length distribution (RLD) for each microsatellite locus of the plurality of microsatellite loci;
(v) for each microsatellite locus, comparing the RLD metric to a threshold value for the microsatellite locus, wherein each microsatellite has an independent threshold value, wherein the threshold value has been determined by (i) the repeat length distribution of the microsatellite locus in a MSS sample; or (ii) the repeat length distribution of the microsatellite locus in a matched normal sample; or (iii) the repeat length distribution of the microsatellite locus in a pre-computed normal background; or (iv) the repeat length distribution of the microsatellite locus obtained from a MSS cell line; or (v) a RLD metric at which the microsatellite locus is identified as MSI; and
(vi) administering a therapeutically effective amount of at least one antibody and/or at least one small molecule therapeutic agent to an identified human diagnosed as having colorectal cancer and as having microsatellite instability, wherein the identifying of the human diagnosed as having colorectal cancer and as having microsatellite instability comprises: (i) quantifying a number of microsatellite loci having an RLD metric exceeding the threshold value; (ii) comparing the quantified number to a microsatellite instability proportion threshold; and (iii) determining if the quantified number exceeds the microsatellite instability proportion threshold, wherein if the quantified number exceeds the microsatellite instability proportion threshold, the human diagnosed as having colorectal cancer has microsatellite instability;
wherein the at least one antibody and/or the at least one small molecule is selected from the group consisting of Avastin (Bevacizumab); Bevacizumab; Camptosar (Irinotecan Hydrochloride); Capecitabine; Cetuximab; Cyramza (Ramucirumab); Eloxatin (Oxaliplatin); Erbitux (Cetuximab); 5-FU (Fluorouracil Injection); Irinotecan Hydrochloride; Leucovorin Calcium; Lonsurf (Trifluridine and Tipiracil Hydrochloride); Nivolumab; Opdivo (Nivolumab); Oxaliplatin; Panitumumab; Ramucirumab; Regorafenib; Stivarga (Regorafenib); Trifluridine and Tipiracil Hydrochloride; Vectibix (Panitumumab); Wellcovorin (Leucovorin Calcium); Xeloda (Capecitabine); Zaltrap (Ziv-Aflibercept); and Ziv-Aflibercept; and
wherein the plurality of microsatellite loci are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 microsatellite loci selected from the group consisting of microsatellite loci 1-170:
microsatellite
chromosome
start
end
locus
location
coordinate
coordinate
1
chr1
32936733
32936750
2
chr1
46859627
46859640
3
chr1
149929093
149929109
4
chr1
235344151
235344170
5
chr2
5699709
5699727
6
chr2
5700420
5700434
7
chr2
14638213
14638234
8
chr2
39309548
39309575
9
chr2
47414420
47414447
10
chr2
51061373
51061415
11
chr2
66435743
66435767
12
chr2
95183613
95183636
13
chr2
98998132
98998145
14
chr2
102762122
102762138
15
chr2
129982139
129982152
16
chr2
173266783
173266800
17
chr3
70959190
70959203
18
chr3
123614028
123614044
19
chr4
39500102
39500120
20
chr4
54732045
54732070
21
chr4
151662755
151662771
22
chr5
112877981
112878021
23
chr5
140115261
140115276
24
chr5
181260427
181260439
25
chr6
11714406
11714420
26
chr7
1747883
1747900
27
chr7
143306249
143306274
28
chr8
33498673
33498689
29
chr8
40154248
40154264
30
chr8
58148166
58148186
31
chr8
93925321
93925334
32
chr9
84003046
84003063
33
chr9
126837838
126837854
34
chr10
21518540
21518553
35
chr10
119137173
119137195
36
chr11
5666090
5666107
37
chr11
115176312
115176326
38
chr11
125620870
125620891
39
chr11
125893715
125893728
40
chr12
84892141
84892158
41
chr12
130399009
130399022
42
chr13
50013518
50013540
43
chr14
23183137
23183158
44
chr14
64523982
64523995
45
chr14
73492999
73493015
46
chr16
19118464
19118481
47
chr16
29669203
29669217
48
chr16
58543411
58543424
49
chr17
38995870
38995910
50
chr18
649879
649894
51
chr19
11808381
11808396
52
chr19
21378606
21378625
53
chr19
57257698
57257723
54
chr19
57258437
57258450
55
chr19
57262350
57262363
56
chr20
59922426
59922441
57
chr21
34103315
34103331
58
chrX
2904903
2904920
59
chrX
38805564
38805577
60
chrX
101413944
101413957
61
chrX
102154282
102154298
62
chrX
102658669
102658683
63
chrX
102751949
102751969
64
chrX
145825169
145825191
65
chr1
103663067
103663077
66
chr1
103757205
103757215
67
chr1
116112562
116112575
68
chr1
13782253
13782262
69
chr1
150714123
150714133
70
chr1
151224226
151224235
71
chr1
159062696
159062706
72
chr1
200624913
200624921
73
chr1
221702319
221702330
74
chr1
236897645
236897660
75
chr1
28459218
28459228
76
chr1
52784862
52784872
77
chr1
88983825
88983836
78
chr10
72893710
72893720
79
chr11
105007313
105007323
80
chr11
105008959
105008969
81
chr11
120479927
120479935
82
chr11
63382198
63382209
83
chr11
65501008
65501020
84
chr12
118150956
118150974
85
chr12
119728455
119728465
86
chr12
13211492
13211502
87
chr12
31282674
31282685
88
chr12
54011395
54011406
89
chr12
96286155
96286166
90
chr13
46771334
46771348
91
chr13
49350729
49350739
92
chr13
57725300
57725311
93
chr14
19668967
19668978
94
chr14
53046721
53046733
95
chr15
30113885
30113899
96
chr15
37099551
37099563
97
chr15
41852769
41852780
98
chr15
72572067
72572077
99
chr16
14889234
14889243
100
chr17
1423460
1423474
101
chr17
4539344
4539362
102
chr17
45539269
45539279
103
chr17
46306192
46306202
104
chr17
46523767
46523777
105
chr17
56938993
56939007
106
chr17
58357799
58357806
107
chr18
23529756
23529766
108
chr18
319944
319955
109
chr18
33333178
33333188
110
chr18
9954236
9954249
111
chr19
12463926
12463947
112
chr19
13993876
13993890
113
chr19
44158121
44158134
114
chr19
9251064
9251075
115
chr2
106426090
106426102
116
chr2
108729222
108729232
117
chr2
118096518
118096531
118
chr2
147926116
147926124
119
chr2
151379531
151379545
120
chr2
182942165
182942175
121
chr2
196666794
196666805
122
chr2
202300378
202300388
123
chr2
210315041
210315052
124
chr2
55234233
55234243
125
chr2
63842143
63842158
126
chr2
87757451
87757462
127
chr2
8858645
8858657
128
chr20
53875234
53875245
129
chr20
60012728
60012738
130
chr21
28966883
28966894
131
chr21
32601784
32601798
132
chr22
38683915
38683930
133
chr3
101459057
101459067
134
chr3
113658634
113658645
135
chr3
131014202
131014213
136
chr3
165187860
165187871
137
chr3
170997894
170997904
138
chr3
25719646
25719656
139
chr3
30650379
30650389
140
chr3
51380172
51380179
141
chr5
159099526
159099541
142
chr5
162067984
162067996
143
chr5
177302735
177302747
144
chr5
58974630
58974642
145
chr5
68288684
68288696
146
chr5
78450039
78450050
147
chr5
80675095
80675103
148
chr5
83641432
83641442
149
chr5
88722571
88722584
150
chr6
167040031
167040043
151
chr6
43054238
43054250
152
chr6
49492281
49492291
153
chr6
55874913
55874927
154
chr6
70861991
70862003
155
chr6
88143811
88143822
156
chr7
135619945
135619955
157
chr7
30633896
30633911
158
chr7
54752300
54752311
159
chr7
75193019
75193032
160
chr7
95359943
95359960
161
chr8
23854553
23854565
162
chr8
33499307
33499320
163
chr8
39103749
39103760
164
chr8
7489344
7489353
165
chr8
7822205
7822214
166
chr8
78717503
78717517
167
chr9
107331689
107331701
168
chr9
83969307
83969318
169
chrX
101883466
101883477
170
chrX
18164977
18164992
17 . The method of claim 16 , wherein the plurality of microsatellite loci are at least 58 microsatellite loci selected from the group consisting of microsatellite loci: 1-64.
18 . The method of claim 16 , wherein the plurality of microsatellite loci corresponds to the microsatellite loci:
Chr.
Coordinate
Coordinate
chr1
149929093
149929109
chr1
235344151
235344170
chr1
32936733
32936750
chr1
46859627
46859640
chr10
119137173
119137195
chr10
21518540
21518553
chr11
115176312
115176326
chr11
125620870
125620891
chr11
125893715
125893728
chr11
5666090
5666107
chr12
130399009
130399022
chr12
84892141
84892158
chr13
50013518
50013540
chr14
23183137
23183158
chr14
64523982
64523995
chr14
73492999
73493015
chr16
19118464
19118481
chr16
29669203
29669217
chr16
58543411
58543424
chr18
649879
649894
chr19
11808381
11808396
chr19
21378606
21378625
chr19
57257698
57257723
chr19
57258437
57258450
chr19
57262350
57262363
chr2
102762122
102762138
chr2
129982139
129982152
chr2
14638213
14638234
chr2
173266783
173266800
chr2
39309548
39309575
chr2
47414420
47414447
chr2
5700420
5700434
chr2
66435743
66435767
chr2
95183613
95183636
chr2
98998132
98998145
chr20
59922426
59922441
chr21
34103315
34103331
chr3
123614028
123614044
chr3
70959190
70959203
chr4
151662755
151662771
chr4
39500102
39500120
chr4
54732045
54732070
chr5
140115261
140115276
chr6
11714406
11714420
chr7
143306249
143306274
chr7
1747883
1747900
chr8
33498673
33498689
chr8
40154248
40154264
chr8
93925321
93925334
chr9
126837838
126837854
chr9
84003046
84003063
chrX
101413944
101413957
chrX
102154282
102154298
chrX
102658669
102658683
chrX
102751949
102751969
chrX
145825169
145825191
chrX
2904903
2904920
chrX
38805564
38805577.