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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