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TCR Seq QC Report

Figure 1: a) Representative electrophoregram obtained from Tapestation from TCR a+b libraries generated using 100ng of Jurkat total RNA from the SMARTer Human TCR Profiling Kit ( Takara Bio USA Inc). Expected peak is 700-800bp shows enrichment of TCR a/b transcripts. b) Table 1 shows results from the quantification of the libraries.

Well Conc. [ng/ μl] Sample ID Alert Observations

C1 3.13

Sample Table

Size [bp] Calibrated Conc.

[ng/ μl]

Assigned Conc.

[ng/ μl]

Peak Molarity [nmol/l]

% Integrated Area

Peak Comment

Observations

25 5.54 - 341 - Lower Marker

709 3.13 - 6.80 100.00

1500 6.50 6.50 6.67 - Upper Marker

Peak Table

B

SMARTer Human TCR Profiling Kit libraryA

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Table 1: Representative example of overview of mapping metrics of TCR sequencing libraries generated using the SMARTer Human TCR a/b Profiling Kit from 100ng PBMC RNA and the MIXCR™ software. The sequencing files were shared by Takara and analysis was performed at MedGenome Inc.

Sample ID

Total sequencing reads (Paired End) 995932

Successfully aligned reads (Paired End) 624481 (62.7%)

Chimeras 8 (0%)

Paired-end alignment conflicts eliminated 75407 (7.57%)

Alignment failed, no hits (not TCR/IG?) 87456 (8.78%)

Alignment failed because of absence of V hits 3840 (0.39%)

Alignment failed because of absence of J hits 270706 (27.18%)

No target with both V and J alignments 2341 (0.24%)

Alignment failed because of low total score 7108 (0.71%)

Overlapped 805655 (80.89%)

Overlapped and aligned 459283 (46.12%)

Alignment-aided overlaps 41733 (9.09%)

Overlapped and not aligned 346372 (34.78%)

V gene chimeras 125 (0.01%)

J gene chimeras 33 (0%)

TRA chains 334164 (53.51%)

TRB chains 289127 (46.3%)

TRD chains 11 (0%)

TRG chains 10 (0%)

IGH chains 13 (0%)

IGK chains 23 (0%)

IGL chains 1124 (0.18%)

Sample ID ControlRNA-alpha

Final clonotype count 182432

Average number of reads per clonotype 3.21

Reads used in clonotypes, percent of total 586109 (58.85%)

Reads used in clonotypes before clustering, percent of total 590008 (59.24%)

Number of reads used as a core, percent of used 581771 (98.6%)

Mapped low quality reads, percent of used 8237 (1.4%)

Reads clustered in PCR error correction, percent of used 3899 (0.66%)

Reads pre-clustered due to the similar VJC-lists, percent of used 0 (0%)

Reads dropped due to the lack of a clone sequence 26338 (2.64%)

Reads dropped due to low quality 324 (0.03%)

Reads dropped due to failed mapping 7811 (0.78%)

Reads dropped with low quality clones 0 (0%)

Clonotypes eliminated by PCR error correction 2543

Clonotypes dropped as low quality 0

Clonotypes pre-clustered due to the similar VJC-lists 0

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Table 2: Representative example of top clonotypes identified using the TCR-sequencing libraries and CDR3 sequences for the top clones. The report shared with the customer will have the information on all clonotypes that were identified in the form of an excel document.

Figure 2: a) Representative TCR b library profile obtained from 300ng of gDNA using immunoSEQ ™ Assay from Adaptive Technologies. B) Table shows quantification of the library generated.

Adaptive immunoSEQ AssayA

Well Conc. [ng/ μl] Sample ID Alert Observations

B1 17.6

Sample Table

Size [bp] Calibrated Conc.

[ng/ μl]

Assigned Conc.

[ng/ μl]

Peak Molarity [nmol/l]

% Integrated Area

Peak Comment

Observations

25 5.73 - 353 - Lower Marker

371 17.1 - 70.3 96.77

866 0.571 - 1.01 3.23

1500 6.50 6.50 6.67 - Upper Marker

Peak Table

B

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Table 3: Screenshot of the immunoSEQ browser output showing frequency of TCRB clones identified upon sequencing the TCRB libraries using the Adaptive immunoSEQ kit.

Table 4: CDR3 sequences identified by immunoSEQ software from the libraries sequenced previously.

Amino Acid Sum (Productive Frequency) Present In Sample 1 Sample 2 Sample 3 Sample 4

CASGENRGKNIQYF 72.16% 2.00 0.019% 0.00% 0.00% 0.00%

CASSLTVGGNTIYF 54.62% 1.00 0.000% 0.00% 0.00% 54.62%

CSVEDLGQLGDTQYF 53.29% 15.00 0.009% 2.54% 4.26% 0.68%

CASSKVNRSPDEQYF 52.60% 1.00 0.000% 0.00% 0.00% 0.00%

CASRNSTNYGYTF 37.52% 2.00 0.009% 0.00% 0.00% 0.00%

CASSLRAPYEQYF 32.50% 1.00 0.000% 0.00% 0.00% 0.00%

CATSDPTGGAKTGELFF 26.18% 1.00 0.000% 0.00% 0.00% 0.00%

CASSLDRSKNTEAFF 24.08% 1.00 0.000% 24.08% 0.00% 0.00%

CSARTVSYNEQFF 20.98% 1.00 0.000% 0.00% 20.98% 0.00%

CASSRASTSYSYEQYF 19.11% 1.00 0.000% 0.00% 0.00% 0.00%

CAARAGEYYGELFF 18.24% 2.00 0.000% 0.00% 0.00% 0.00%

CAIRTAMNTEAFF 16.39% 15.00 0.019% 0.55% 7.17% 0.11%

CASSRLRNTIYF 15.33% 1.00 0.000% 0.00% 0.00% 0.00%

CATSGYNEQFF 14.83% 1.00 0.000% 0.00% 0.00% 0.00%

CASSQDYGRGTHTDTQYF 14.49% 1.00 0.000% 0.00% 0.00% 0.00%

CASSHTGGRAFF 14.33% 7.00 0.000% 0.00% 0.00% 0.00%

CASSPPTDKVVYEQYF 13.79% 14.00 0.000% 5.73% 0.04% 0.54%

CASSVEQGPANTEAFF 13.16% 1.00 0.000% 0.00% 0.00% 0.00%

CAWTPRWDTEAFF 13.05% 2.00 0.000% 0.00% 0.00% 0.00%

CASSLRVYEQYF 12.68% 14.00 0.000% 1.92% 0.22% 0.61%

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10X VDJ Single-cell solution A

B

Figure 3: a) Representative chromatogram of single-cell TCR libraries generated using the 10X V(D)J TCR Profiling Kit . b) Table shows quantification of the libraries.

Table 5: Representative sequencing metrics obtained from the 10X Loupe browser of libraries generated by sequenc-ing purified T cells using the 10X V(D)J solution for single-cell TCR sequencing.

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From [bp] To [bp] Average Size [bp]

Conc.[ng/ μl]

Region Molarity[nmol/l]

% of Total Region Comment

Color

240 1510 657 9.96 27.8 82.91

Region Table