Supporting Information
Figure S 1. Amplicon libraries on 1% agarose gel. (A) Final product of ligation method. Both indicated
bands were purified. (B) Duplicate libraries (DA1/DA2) generated using the DA method. (C) DA
titration with 200 and 400 ng. (D) PE products after PCR1 and PCR2. (E) RNA titration using standard
PE3 PCR protocol for high amount RNA samples (100 ng and 50 ng) and an extended protocol for
low amount RNA samples (10–1 ng, see Methods). Bands are shown after PCR2. Bands between
500–600 bp in length (indicated by white arrows) were excised and gel-purified.
B
95
10
10-2
CDR3 frequency [%]
25
Reliably detected CDR3s: 12,069
100
95
75
25
10-3
Frequency range : 1.7 - 3.3 ´ 10-4
Abundance range: 25,536–5
Replicate DA1
Replicate DA2
97%
97.1%
100
75
50
25
0
D
14000
10
8000 10000 12000 14000
12000
10000
8000
10-1
10-2
10
-3
Frequency range : 1.7 - 2.9 ´ 10
DA2
8000
6000
4000
2000
2000 4000 6000 8000
14000
8000 10000 12000 14000
12000
-4
10000
Abundance range: 23,135–4
0
C
0
Replicate DA2
Replicate DA2
50
Replicate DA1
50
0
10-1
Replicate DA1
Unique CDR3s present in all compared datasets [%]
75
0
Reads mapping to reliably detected CDR3s [%]
A
100
8000
DA1
r=0.93
8000
p<0.01
6000
Reliably detected CDR3s: 12,318
4000
100
101
102
103
Number of unique CDR3s
104
100
101
102
103
Number of unique CDR3s
104
2000
2000 4000 6000 8000
0
5000
Figure S 2. DA duplicates (DA1/DA2, technical replicates) yield highly comparable HTS datasets. (A)
Reliably detected CDR3s were determined as described in Fig. 2 using a 95% reliable detection cutoff. On average, ≈12,200 CDR3s were reliably detected. (B) Their corresponding average frequency
and abundance ranges are shown (≈1.7–3.3x10-4% and ≈24,000–5 reads per unique CDR3,
respectively). (C) Reliably detected CDR3s corresponded to ≈97% of total sequencing reads. (D) The
Spearman rank correlation coefficient was high (r=0.93).
B
95
10
10-1
50
0
10-2
CDR3 frequency [%]
25
Reliably detected CDR3s: 11,661
100
95
75
DA
DA
10-3
Frequency range : 1.7 - 3.3 ´ 10-4
Abundance range: 25,536–5
C
DA
PE
96.9%
95.7%
100
75
50
25
0
D
8000 10000 12000
10
0
12000
10000
8000
PE
10-1
6000
4000
50
PE
PE
Unique CDR3s present in all compared datasets [%]
75
0
Reads mapping to reliably detected CDR3s [%]
A
100
10-2
2000
2000
4000
6000
8000 10000 12000
25
10
-3
12000
Frequency range : 1.2 - 4.5 ´ 10-4
10000
Abundance range: 15,380–6
DA
0
Reliably detected CDR3s: 11,297
100
101
102
103
Number of unique CDR3s
r=0.9
8000
p<0.01
6000
4000
104
100
101
102
103
Number of unique CDR3s
104
2000
2000 4000
6000
2000
4000
6000
8000
Figure S 3. Pairwise comparison of HTS datasets from antibody repertoire libraries prepared using
the DA and PE method. (A) Reliably detected CDR3s were determined as described in Fig. 2 using a
95% reliable detection cut-off. On average, ≈11,500 CDR3s were reliably detected. (B) Their
corresponding average frequency and abundance ranges are shown (≈1.5–3.9x10-4 % and ≈20,000–5
reads per unique CDR3, respectively). (C) Reliably detected CDR3s corresponded to ≈96% of total
sequencing reads. (D) The Spearman rank correlation coefficient was high (r=0.90).
B
95
10
10-1
50
10-2
Frequency range : 1.9 - 8.2 ´ 10
0
CDR3 frequency [%]
25
Reliably detected CDR3s: 8,831
100
95
75
Ligation
Ligation
10-3
-4
Abundance range: 9,254–4
C
Ligation
DA
96.5%
95.4%
100
75
50
25
0
D
10000
10
6000
6000
4000
10-2
10
-3
2000
2000
10000
6000
8000
r=0.86
6000
102
103
p<0.01
4000
Reliably detected CDR3s: 8,570
101
6000
10000
Ligation
Number of unique CDR3s
4000
8000
Abundance range: 25,536–12
6000
100
10000
DA
Frequency range : 1.7 - 7.9 ´ 10-4
25
8000
8000
DA
50
0
6000
0
10-1
DA
Unique CDR3s present in all compared datasets [%]
75
0
Reads mapping to reliably detected CDR3s [%]
A
100
104
100
101
102
103
Number of unique CDR3s
104
2000
2000
4000
6000
2000
4000
6000
Figure S 4. Pairwise comparison of HTS datasets from antibody repertoire libraries prepared using
the ligation and DA method. (A) Reliably detected CDR3s were determined as described in Fig. 2
using a 95% reliable detection cut-off. On average, ≈8,700 CDR3s were reliably detected. (B) Their
corresponding average frequency and abundance ranges are shown (≈1.8–8.1x10-4% and ≈17,000–8
reads per unique CDR3, respectively). (C) Reliably detected CDR3s corresponded to ≈96% of total
sequencing reads. (D) The Spearman rank correlation coefficient was high (r=0.86).
B
95
10
10-1
50
10-2
Frequency range : 1.9 - 6.1 ´ 10-4
CDR3 frequency [%]
25
0
Ligation
Ligation
Reliably detected CDR3s: 9,111
100
95
75
10
-3
Abundance range: 9,254–3
C
Ligation
PE
96.7%
93.3%
100
75
50
25
0
D
10000
10
6000
10-2
PE
Frequency range : 1.2 - 1.3 ´ 10
10000
6000
8000
102
103
6000
10000
r=0.79
6000
p<0.01
4000
Reliably detected CDR3s: 7,925
101
4000
8000
Ligation
Number of unique CDR3s
6000
2000
2000
-3
10-3
100
10000
4000
Abundance range: 15,380–17
25
8000
8000
PE
50
0
6000
0
10-1
PE
Unique CDR3s present in all compared datasets [%]
75
0
Reads mapping to reliably detected CDR3s [%]
A
100
104
100
101
102
103
Number of unique CDR3s
104
2000
0
2000
4000
2000
4000
6000
0
Figure S 5. Pairwise comparison of HTS datasets from antibody repertoire libraries using the ligation
and PE method. A) Reliably detected CDR3s were determined as described in Fig. 2 using a 95%
CDR3 reliable detection cut-off. On average, ≈8,700 CDR3s were reliably detected. (B) Their
corresponding average frequency and abundance ranges are shown (≈1.6–3.7x10-4% and ≈12,500–
10 per unique CDR3, respectively). (C) Reliably detected CDR3s corresponded to ≈95% of total
sequencing reads. (D) The Spearman rank correlation coefficient was high (r=0.79).
B
100
10-1
75
10-3
100
95
25
0
10-1
100
75
10-1
Frequency range : 1.1 - 2.6 ´ 10
Abundance range: 24,670–6
50
10
10
10
10
Number of unique CDR3s
96.3%
0
10
4
10
10000
15000
10000
10000
5000
5000
10000
15000
10000
Frequency range : 1.2 - 3 ´ 10
Abundance range: 15,380–4
10
0
10
1
10
2
-4
10
3
15000
PE3
5000
15000
10
Number of unique CDR3s
PE2
10000
15000
r=0.93
10000
p<0.01
5000
10000
3
95.9%
25
10-4
Reliably detected CDR3s: 13,167
2
96.2%
75
-4
-2
10-3
25
1
PE3
PE3
PE3
50
0
PE2
10-4
Reliably detected CDR3s: 13,013
100
95
0
PE1
100
D
10-2
10-3
C
PE2
50
CDR3 frequency [%]
10
75
0
Frequency range : 1.1 - 2.6 ´ 10-4
Abundance range: 20,939–5
10-4
Reliably detected CDR3s: 12,948
PE2
Unique CDR3s present in all compared datasets [%]
10-2
25
0
PE1
PE1
50
Reads mapping to reliably detected CDR3s [%]
A
100
95
10000
PE1
10000
5000
10000
15000
10000
r=0.94
r=0.93
p<0.01
p<0.01
5000
4
5000
10000
5000
10000
5000
10000
Figure S 6. Variations in the PE protocol have minimal effects on HTS datasets. PE1 and PE2 were
prepared with 8 cycles in the last annealing temperature step of PCR1 (16 total cycles) and either
amplified with 12 (PE1) or 8 (PE2) total cycles in PCR2, resulting in an overall number of 28 and 24
cycles, respectively. For PE3, 4 cycles were switched from PCR2 to PCR1, having 12 cycles in the
last annealing temperature step of PCR1 (20 total cycles) and a total of 8 cycles in PCR2, resulting in
an overall cycle number of 28 (equal cycle number to PE1 and standard conditions used for ligation
and DA). (A) Reliably detected CDR3s were determined as described in Fig. 2 using a 95% CDR3
reliable detection cut-off. On average, ≈13,000 CDR3s were reliably detected. (B) Their
corresponding average frequency and abundance ranges are shown (≈1.1–2.7x10-4% and ≈20,000–5
per unique CDR3, respectively). (C) Reliably detected CDR3s corresponded to ≈96% of sequencing
reads. (D) The Spearman rank correlation coefficients were high (r≈0.93).
A
B
100
95
10
10
10
-2
0
-1
100 ng
100 ng
50
10-2
10-3
25
Frequency range : 1.1 - 2.3 ´ 10-2
Abundance range: 13,000–281
Reliably detected CDR3s: 837
100 ng
50 ng
10 ng
82.5%
50
25
46.3%
48.8%
49.1%
44.9%
44.2%
Reliably detected CDR3s: 835
75
10-3
Abundance range: 10,771–213
D
2500
100
1500 2000 2500
2000
1500
10
-1
10-2
10-3
25
Frequency range : 1.2 - 2.3 ´ 10-2
1500
Frequency range : 1.1 - 2.7 ´ 10-2
1500
1000
1500
100
10 ng1500
10
-2
10-3
25
5 ng
5 ng
50
1500
50 ng1500
1000
1500
100
100 ng1500
10-2
10-3
25
1 ng
1 ng
50
Frequency range : 1.2 - 1.2 ´ 10-2
1500
1500
1500
1000
1000
1000
p<0.01
1000
1500
p<0.01
0
500
r=0.77
p<0.01
1000
1500
1000
r=0.41
p<0.01
1000
r=0.84
p<0.01
1000
r=0.92
500 ng1500
1000
1000
r=0.39
1500
p<0.01
0
500
1500
r=0.78
1000
p<0.01
1000
1500
r=0.39
p<0.01
1000
1500
p<0.01
0
500
0
500
1000
2000
2000
1000
r=0.91
p<0.01
1500
Abundance range: 8,197–85
1500
r=0.92
1500
1000
500
2000
2000
10-1
75
1500
r=0.84
1500
Frequency range : 1 - 2.9 ´ 10-2
p<0.01
0
2000
2000
Abundance range: 18,636–535
Reliably detected CDR3s: 629
1000
r=0.76
1500
1000
r=0.38
5 ng 1500
1500
2000
2000
10-1
75
500
2000
2000
Abundance range: 17,175–445
Reliably detected CDR3s: 676
1500
1 ng 1000
500 1000 1500
10 ng
10 ng
50
10-2
CDR3 frequency [%]
25
0
50 ng
50
0
1 ng
75
10-1
75
0
100
95
5 ng
100
50 ng
Unique CDR3s present in all compared datasets [%]
Frequency range : 1.2 - 2.4 ´ 10
Abundance range: 15,380–320
75
0
100
95
-3
Reliably detected CDR3s: 767
Reads mapping to reliably detected CDR3s [%]
10-2
25
0
100
95
500 ng
500 ng
50
0
100
95
500 ng
100
10-1
75
0
100
95
C
100
1000
1500
r=0.85
p<0.01
1000
1500
r=0.78
p<0.01
1000
1500
r=0.41
p<0.01
1000
1500
p<0.01
1000
Reliably detected CDR3s: 2,189
0
10
1
10
2
10
10
3
Number of unique CDR3s
10
4
10
0
10
1
10
2
10
3
10
4
Number of unique CDR3s
Figure S 7. Comparison of HTS datasets from antibody repertoire libraries prepared using the PE
method and lowering the amounts of total RNA input (500–1 ng). Including the 1 ng dataset into the
comparison of 500–5 ng datasets reduced the number of reliably detected CDR3s and CDR3 rank
correlation (Fig. 4, Table S1). (A) Reliably detected CDR3s were determined as described in Fig. 2
using a 95% reliable detection cut-off. On average, ≈1,000 CDR3s were reliably detected. (B) Their
corresponding average frequency and abundance ranges are shown (≈1.1–2.3x10-2% and ≈14,000–
300 per unique CDR3, respectively). (C) Reliably detected CDR3s corresponded to ≈52% of total
sequencing reads. (D) The Spearman rank correlation coefficients ranged between r=0.92 and
r=0.38.
B
95
10-1
10-2
Frequency range : 5.5 ´ 10-1 - 5.7 ´ 10-4
VDJ frequency [%]
25
Reliably detected VDJs: 31,343
0
100
95
50
10
-3
Abundance range: 4,772–5
82.5%
50
25
0
D
30000
10-1
20000
Frequency range : 5.7 ´ 10-1 - 5.3 ´ 10-4
10000
10000
30000
101
102
40000
30000
Abundance range: 4,304–4
3
10
10
4
100
105
101
102
103
104
10
5
100
100
95
75
DA1 trimmed
10-1
DA1 trimmed
50
10-2
Frequency range : 1.5 - 4.7 ´ 10-4
VDJ frequency [%]
25
Reliably detected VDJs: 13,267
100
95
10-3
25
0
10000
20000
DA1 trimmed
DA2 trimmed
87.3%
87.9%
100
75
50
25
0
H
10000
15000
15000
10000
10000
DA2 trimmed
10
-1
DA2 trimmed
DA2 trimmed
50
Abundance range: 15,952–5
20000
G
100
75
10-2
Frequency range : 1.5 - 5.4 ´ 10-4
10-3
Abundance range: 14,380–5
5000
5000
10000
15000
10000
10000
15000
r=0.9
10000
DA1 trimmed
Reliably detected VDJs: 13,726
102
p<0.01
10000
10000
Number of unique VDJs
F
101
r=0.68
DA1 untrimmed
20000
E
100
20000
40000
Reads mapping to reliably detected VDJs [%]
0
40000
DA2 untrimmed
Number of unique VDJs
Unique VDJs present in all compared datasets [%]
76.9%
75
30000
10-3
10
0
DA2 untrimmed
Reliably detected VDJs: 39,416
0
0
DA1 untrimmed
40000
10-2
25
C
100
DA2 untrimmed
75
DA2 untrimmed
Unique VDJs present in all compared datasets [%]
50
DA1 untrimmed
DA1 untrimmed
75
Reads mapping to reliably detected VDJs [%]
A
100
p<0.01
5000
103
Number of unique VDJs
104
105
100
101
102
103
104
10
5
Number of unique VDJs
5000
10000
5000
10000
Figure S 8. VDJ primer trimming reduces VDJ diversity. The effect of primer trimming on VDJ diversity
was assessed using the DA method duplicate (DA1/DA2). Primer trimming of VDJ regions was
performed as detailed in Methods. (A/E) Reliable detected VDJs were determined as described in Fig.
2 using a 95% VDJ reliable detection cut-off. On average, ≈35,400 and ≈13,500 VDJs were reliably
detected for non-trimmed and trimmed HTS datasets, respectively. (B/F) Their corresponding average
frequency and abundance ranges are shown. (C/G) Reliably detected VDJs corresponded to
≈80/≈88% of total sequencing reads. (D/H) The Spearman’s rank correlation coefficients were r=0.68
and r=0.90 for non-trimmed and trimmed HTS datasets, respectively.
Table S 1. RNA titration (500–5 ng) using the PE method. On average, ≈6,200 CDR3s were reliably
detected, which extended over average frequency and abundance ranges of ≈1.1–2.5x10-3% and
≈15,000–34 reads per unique CDR3, respectively. Reliably detected CDR3s corresponded to ≈92% of
total sequencing reads. The corresponding Spearman rank correlation coefficients are shown in Fig.
4.
HTS dataset
(PE)
500 ng
100 ng
50 ng
10 ng
5 ng
Average
Number of reliably
detected CDR3s
Frequency
range [%]
Abundance
range
6,363
6,327
6,333
5,858
6,155
6,207
1.2–2.4x10-3
1.1–2.5x10-3
1.2–2.5x10-3
1.1–2.7x10-3
1.0–2.2x10-3
15,380–32
13,000–30
10,771–23
17,175–44
18,636–40
14,992–34
1.1–2.5x10-3
Reads mapping to
reliably detected
CDR3s [%]
93.4
91.0
92.4
92.1
90.5
91.9
Table S 2. The 30 highest ranked CDR3 amino acid sequences of all RNA titration datasets are shown in a color-coded manner with respective frequencies.
Color code based on CDR3s from the 500 ng sample: Top 1–5: bright fill, white font; Top 6–10: bright fill, black font; Top 11–15: Dark fill, white font; Top 16–
20: grey fill, white font; Top 21–25: pastel fill, black font; Top 26–30: yellow fill, black font. Clones present in 5 out of 6 datasets are marked in red font.
Clones, present in less than five out of the six datasets are not color-indicated. The mean Spearman rank correlation coefficient of datasets 500–1ng is
r=0.81. Freq.: frequency of clones in percent.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
500 ng
Sequence
ARWEYYAMDY
ARLNYYGNLFV
ARHAYYDQTEVSFVY
ARLDLFAY
ARHHRYAYYFDY
ASYRYGWYFDV
ARDVGYGNYFVY
ARSGKVRNAMDY
ARYRLGTYFDY
ASYVYGYYFDY
TREGDSPFAY
ATYDGYYFDY
ARLIYGSSYIVDF
ARGGISVDY
TTGFAY
AAYYYGSSYGFAY
ARLGYYGWFAY
ARSGSSSGYFDY
AREARTTARFAY
ARLGYYGYFAY
ARLGYYGGFAY
ARDKGRVFFDY
ATYDGYCFDY
ARWELGRAWFAY
ARDYGSSHFDH
ARSGGYGNYLAWFAY
ARFPMDY
ARGGHDFSLDY
SRHYYSTPYYAMDY
ARDSFLITRALDY
Freq.
1.156
0.802
0.528
0.449
0.433
0.420
0.388
0.342
0.296
0.288
0.261
0.254
0.251
0.250
0.226
0.225
0.222
0.220
0.215
0.215
0.207
0.203
0.200
0.193
0.186
0.183
0.182
0.182
0.179
0.171
100 ng
Sequence
ARWEYYAMDY
ARLNYYGNLFV
ARHAYYDQTEVSFVY
ARHHRYAYYFDY
ARDVGYGNYFVY
ASYRYGWYFDV
ARLDLFAY
ARSGKVRNAMDY
ATYDGYYFDY
ARYRLGTYFDY
ASYVYGYYFDY
ARLIYGSSYIVDF
ARLGYYGWFAY
ARGGISVDY
ARSGSSSGYFDY
ARLGYYGYFAY
AREARTTARFAY
ARLGYYGGFAY
TREGDSPFAY
ARDKGRVFFDY
ATYDGYCFDY
TTGFAY
AAYYYGSSYGFAY
TRGGNWEDFDY
ARFPMDY
SRHYYSTPYYAMDY
ARDSFLITRALDY
ARGGHDFSLDY
ARWELGRAWFAY
ARSGGYGNYLAWFAY
Freq.
1.070
0.890
0.630
0.474
0.443
0.410
0.403
0.341
0.288
0.286
0.268
0.253
0.252
0.249
0.249
0.249
0.242
0.238
0.231
0.226
0.224
0.217
0.215
0.212
0.194
0.192
0.190
0.189
0.187
0.183
50 ng
Sequence
ARWEYYAMDY
ARLNYYGNLFV
ARHAYYDQTEVSFVY
ARDVGYGNYFVY
ARLDLFAY
ARHHRYAYYFDY
ASYRYGWYFDV
ARSGKVRNAMDY
ATYDGYYFDY
ARYRLGTYFDY
ASYVYGYYFDY
ARGGISVDY
ARLGYYGWFAY
ARLGYYGYFAY
ARSGSSSGYFDY
TTGFAY
ARLGYYGGFAY
ARLIYGSSYIVDF
ATYDGYCFDY
AREARTTARFAY
TREGDSPFAY
ARDKGRVFFDY
ARLDYYGYLNY
AAYYYGSSYGFAY
ARFPMDY
TRGGNWEDFDY
ARWELGRAWFAY
ARLSYYGRFAY
AKNYYYGYVAY
ARQGYYGKGDY
Freq.
1.153
0.927
0.594
0.467
0.463
0.457
0.374
0.348
0.332
0.311
0.289
0.273
0.267
0.262
0.258
0.250
0.246
0.241
0.229
0.228
0.228
0.218
0.211
0.209
0.207
0.198
0.192
0.191
0.185
0.185
10 ng
Sequence
ARWEYYAMDY
ARLNYYGNLFV
ARHAYYDQTEVSFVY
ARHHRYAYYFDY
ARLDLFAY
ARDVGYGNYFVY
ASYRYGWYFDV
ARSGKVRNAMDY
ARLIYGSSYIVDF
ATYDGYYFDY
ASYVYGYYFDY
ARGGISVDY
ARLGYYGWFAY
ARYRLGTYFDY
TTGFAY
AREARTTARFAY
ATYDGYCFDY
ARLGYYGYFAY
ARSGSSSGYFDY
TRGGNWEDFDY
AAYYYGSSYGFAY
ARLGYYGGFAY
ARWELGRAWFAY
TREGDSPFAY
ARDSFLITRALDY
ARDKGRVFFDY
ARLDYYGYLNY
ARFPMDY
AKNYYYGYVAY
SRHYYSTPYYAMDY
Freq.
1.055
0.811
0.576
0.518
0.449
0.426
0.354
0.314
0.276
0.276
0.263
0.263
0.262
0.262
0.253
0.252
0.252
0.244
0.242
0.231
0.213
0.209
0.202
0.202
0.200
0.191
0.191
0.188
0.183
0.182
5 ng
Sequence
ARWEYYAMDY
ARLNYYGNLFV
ARHAYYDQTEVSFVY
ARHHRYAYYFDY
ARDVGYGNYFVY
ARLDLFAY
ASYRYGWYFDV
ARSGKVRNAMDY
ARWELGRAWFAY
ARSGSSSGYFDY
ATYDGYYFDY
ARDINYDIYWYFDV
ARGGISVDY
ASYVYGYYFDY
ARYRLGTYFDY
ARLGYYGGFAY
ARLGYYGYFAY
TRGGNWEDFDY
ARLGYYGWFAY
TREGDSPFAY
ATYDGYCFDY
ARDYGSSHFDH
TTGFAY
ASYRYAWYFDV
ARLIYGSSYIVDF
ARFPMDY
AREARTTARFAY
ARSGGYGNYLAWFAY
ARDKGRVFFDY
ARLDYYGYLNY
Freq.
1.021
0.916
0.635
0.497
0.415
0.414
0.391
0.319
0.277
0.262
0.254
0.250
0.248
0.239
0.235
0.235
0.232
0.222
0.219
0.217
0.215
0.209
0.205
0.204
0.204
0.202
0.201
0.201
0.198
0.195
1ng
Sequence
ARWEYYAMDY
ARLNYYGNLFV
ARHAYYDQTEVSFVY
ARHHRYAYYFDY
ASYRYGWYFDV
ARDVGYGNYFVY
ATYDGYYFDY
AAYYYGSSYGFAY
ATYDGYCFDY
ASYVYGYYFDY
ARGGISVDY
ARYRLGTYFDY
ARDINYDIYWYFDV
ANYYYGSSLFAY
ARLDLFAY
ARLIYGSSYIVDF
ARLGYYGYFAY
ARLGYYGGFAY
ARDKGRVFFDY
TTGFAY
ARLGYYGWFAY
ARSGKVRNAMDY
ARGGANVDY
AREARTTARFAY
ARKGAMITHYYAMDY
TIGGFDY
TREGTYDGCSGHFDY
ARGGHDFSLDY
ARWELGRAWFAY
ARHGITRLLDY
Freq.
1.187
0.776
0.739
0.565
0.532
0.440
0.392
0.338
0.316
0.311
0.302
0.287
0.279
0.278
0.273
0.268
0.253
0.250
0.239
0.236
0.228
0.224
0.222
0.219
0.217
0.215
0.214
0.214
0.197
0.196
Table S 3. List of all primers used for ligation, DA, and PE. fw: forward, rv: reverse, UAd: universal
adapter, Idx: index, RC: reverse complement.
Ligation 1
DA
IgH Universal Forward
TruSeq Universal Adapter + Diversity region + VH 5' specific region
IgH-UAd-fw1
IgH-UAd-fw2
IgH-UAd-fw3
IgH-UAd-fw4
IgH-UAd-fw5
IgH-UAd-fw6
IgH-UAd-fw7
IgH-UAd-fw8
IgH-UAd-fw9
IgH-UAd-fw10
IgH-UAd-fw11
IgH-UAd-fw12
IgH-UAd-fw13
IgH-UAd-fw14
IgH-UAd-fw15
IgH-UAd-fw16
IgH-UAd-fw17
IgH-UAd-fw18
IgH-UAd-fw19
Universal Adapter - NNNN GAKGTRMAGCTTCAGGAGTC
Universal Adapter - NNNN GAGGTBCAGCTBCAGCAGTC
Universal Adapter - NNNN CAGGTGCAGCTGAAGSASTC
Universal Adapter - NNNN GAGGTCCARCTGCAACARTC
Universal Adapter - NNNN CAGGTYCAGCTBCAGCARTC
Universal Adapter - NNNN CAGGTYCARCTGCAGCAGTC
Universal Adapter - NNNN CAGGTCCACGTGAAGCAGTC
Universal Adapter - NNNN GAGGTGAASSTGGTGGAATC
Universal Adapter - NNNN GAVGTGAWGYTGGTGGAGTC
Universal Adapter - NNNN GAGGTGCAGSKGGTGGAGTC
Universal Adapter - NNNN GAKGTGCAMCTGGTGGAGTC
Universal Adapter - NNNN GAGGTGAAGCTGATGGARTC
Universal Adapter - NNNN GAGGTGCARCTTGTTGAGTC
Universal Adapter - NNNN GARGTRAAGCTTCTCGAGTC
Universal Adapter - NNNN GAAGTGAARSTTGAGGAGTC
Universal Adapter - NNNN CAGGTTACTCTRAAAGWGTSTG
Universal Adapter - NNNN CAGGTCCAACTVCAGCARCC
Universal Adapter - NNNN GATGTGAACTTGGAAGTGTC
Universal Adapter - NNNN GAGGTGAAGGTCATCGAGTC
IgG Index Reverse
IgGall-IdxX-rv
TruSeq Adapter Index X (RC) + Diversity region + IgG constant specific
region (RC)
Adapter Index X - NNNN CARKGGATRRRCHGATGGGG
PE - PCR1
IgH Extension Forward
Extension + VH 5' specific region
IgH-extension-fw1
IgH-extension-fw2
IgH-extension-fw3
IgH-extension-fw4
IgH-extension-fw5
IgH-extension-fw6
IgH-extension-fw7
IgH-extension-fw8
IgH-extension-fw9
IgH-extension-fw10
IgH-extension-fw11
IgH-extension-fw12
IgH-extension-fw13
CCCTCCTTTAATTCCC GAKGTRMAGCTTCAGGAGTC
CCCTCCTTTAATTCCC GAGGTBCAGCTBCAGCAGTC
CCCTCCTTTAATTCCC CAGGTGCAGCTGAAGSASTC
CCCTCCTTTAATTCCC GAGGTCCARCTGCAACARTC
CCCTCCTTTAATTCCC CAGGTYCAGCTBCAGCARTC
CCCTCCTTTAATTCCC CAGGTYCARCTGCAGCAGTC
CCCTCCTTTAATTCCC CAGGTCCACGTGAAGCAGTC
CCCTCCTTTAATTCCC GAGGTGAASSTGGTGGAATC
CCCTCCTTTAATTCCC GAVGTGAWGYTGGTGGAGTC
CCCTCCTTTAATTCCC GAGGTGCAGSKGGTGGAGTC
CCCTCCTTTAATTCCC GAKGTGCAMCTGGTGGAGTC
CCCTCCTTTAATTCCC GAGGTGAAGCTGATGGARTC
CCCTCCTTTAATTCCC GAGGTGCARCTTGTTGAGTC
IgH-extension-fw14
IgH-extension-fw15
IgH-extension-fw16
IgH-extension-fw17
IgH-extension-fw18
IgH-extension-fw19
IgG Extension Reverse
IgGall-extension-rv
CCCTCCTTTAATTCCC GARGTRAAGCTTCTCGAGTC
CCCTCCTTTAATTCCC GAAGTGAARSTTGAGGAGTC
CCCTCCTTTAATTCCC CAGGTTACTCTRAAAGWGTSTG
CCCTCCTTTAATTCCC CAGGTCCAACTVCAGCARCC
CCCTCCTTTAATTCCC GATGTGAACTTGGAAGTGTC
CCCTCCTTTAATTCCC GAGGTGAAGGTCATCGAGTC
Extension (RC) + IgG constant specific region (RC)
GAGGAGAGAGAGAGAG CARKGGATRRRCHGATGGGG
PE - PCR2
IgALL Universal Forward
TruSeq Universal Adapter + Diversity region + Extension
IgALL-UAd-fw
Universal Adapter - NNNN CCCTCCTTTAATTCCC
IgALL Index Reverse
TruSeq Adapter Index (RC) + Diversity region + Extension (RC)
IgALL-IdxX-rv
Adapter Index X - NNNN GAGGAGAGAGAGAGAG
References
1.
Mazor, Y., Van Blarcom, T., Mabry, R., Iverson, B. L. & Georgiou, G. Isolation of engineered,
full-length antibodies from libraries expressed in Escherichia coli. Nat Biotechnol 25, 563–565
(2007).