Limitations and challenges of genetic barcode quantification

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1 Limitations and challenges of genetic barcode quantification Lars hielecke, im ranyossy, ndreas Dahl, Rajiv iwari, Ingo Roeder, Hartmut eiger, Boris Fehse, Ingmar lauche and Kerstin ornils SUPPLEMENRY D Figure S a 0.6 density B6 0 3 B6 0 4 B6 0 2 B B B b minimal HD 5 LR 3 LR Δ R U5 SFFV Fluorescence Marker wpre Δ R U5 B6 Illu - B32 - Illu st PR 2 nd PR mplification from gdn Library preparation PR mplification from gdn + Library preparation Illumina-Sequencing Fig. S: (a) Simulated average Hamming distance distributions for a randomly picked sample of a 6 and 32 nucleotides long barcode and for three different initial cell numbers (averaged over 00 simulations). (b) Optimization strategy for the B32 construct: additionally to the extension from 6 to 32 wobble bases, the construct was equipped with adaptors for Illumina-sequencing. hese modules allow the amplification and NS-library preparation in one PR step and with less cycles.

2 Figure S2 a read counts HDs read counts barcodes barcodes b read counts HDs read counts barcodes barcodes Fig. S2: Results of the error-correction method for one exemplary (a) B6 and (b) B32 minibulk. he remaining barcodes (shaded in grey) can not be reliably assigned to any of the original barcodes and are most likely contaminations (HD > 6). 2

3 Figure S3 a fraction of error corrected barcodes HD threshold b fraction of error corrected barcodes HD threshold Fig. S3: Sensitivity analysis of the effect of the chosen Hamming distance (HD) threshold for the error correction model. he dashed line indicates the chosen upper Hamming distance threshold for the definition of similarity during error correction for both barcode constructs. 3

4 Figure S4 5 LR 3 LR Δ R U5 SFFV Fluorescence Marker wpre B Δ R U5 ~kb p90 VIS-specific reverse primer lone enomic location Reverse primer Length of obtained fragment (bp) B6-B hr. q Bw-µL Kl B6- hr. 9q32 8 6Bw-µL Kl B6-2 hr. 5q35.3 6B-Klon2-hr 948 B6-D hr. 2q22.3 9a 6Bw-µL Kl.4 93 B6-E hr. 9q Bw-µL Kl B32- hr. 8q Bw-5µL Kl B32- hr. 0q24.4 a 32Bw-5µL Kl B32- hr. 7q Bw-5µL Kl B32-E hr. 9q Bw-5µL Kl Fig. S4 Viral integration site analysis and generation of kb fragments. he viral integration site (IS) was identified via LM-PR (26). o generate PR fragments with approximately kb in length, we used a forward-primer located on the provirus (p90) and a IS-specific reverse primer. he obtained PR-product contained the respective barcodesequence of each clone 4

5 Figure S5 a b

6 c Fig. S5: Detailed inspection of mutation patterns on the level of single nucleotide substitutions for only the most similar descendent barcodes (with a HD = and HD = 2 to the original barcodes) are displayed as ternary plots. Shown are the results for the B6 construct (a), for the B6 Q5 polymerase approach (b), and for the B32 construct (c). 6

7 Figure S6 a b read counts read counts B6 B B6 B6 2 B6 D B6 E B32 B32 B32 B32 E c B6 / B6 2 Fig. S6: Read count distribution of all identified descendent Bs for the (a) B6 and (b) B32 construct. (c) Read count ratio of the double integrated B6- and B6-2 Bs for all available minibulks is depicted as boxplot. 7

8 Figure S7 a b cumulative reads of descendents 3*0 4 2*0 4 *0 4 B6 B B6 B6 2 B6 D B6 E unknown cumulative reads of descendents 5*0 4 4*0 4 3*0 4 2*0 4 *0 4 B32 B32 B32 B32 E unknown 0*0 5 2*0 5 4* barcode reads 0*0 5 2*0 5 4*0 5 6*0 5 8*0 5 barcode reads Fig. S7: he ratio of read counts obtained from the original barcodes vs their related descendent barcodes, according to the chosen thresholds of (a) HD = 4 for the B6 and (b) HD = 8 for the B32 construct. Figure S8 ratio barcode reads barcodes original Bs descendent Bs B6 B32 Fig. S8: he distribution of the relative read count ratio between the smallest original barcode and the biggest descendent barcode (B descendent /B original ) for both of the studied barcode constructs is visualized as boxplots. 8

9 Figure S9 a b 00% 00% % % content % % content % % % % 0% B6 B B6 B6 2 B6 D B6 E 0% B32 B32 B32 E B32 Fig. S9: content within the barcode sequences of the chosen (a) B6 and (b) B32 barcodes. 9

10 Figure S0 343 plasmids (LeO-EFS-eFP-B) 69 Bs 69 Bs 69 Bs 68 Bs 68 Bs Stock Stock 2 Stock 3 Stock 4 Stock 5 Maxi Maxi 2 Maxi 3 Maxi 4 Maxi 5 B Library Virusproduction Fig. S0: 343 barcode equipped plasmids were selected and combined into five stocks, containing 68 or 69 different barcodes. fter transformation and plasmid preparation, the five maxi-preparations were merged to generate the final annotated barcode library. s a quality control of the different steps, NS of the selected plasmids for all stocks and the subsequently generated maxi preparations was performed. 0

11 able S Function Name Sequence (5'->3') Barcode Oligos eneration of B6 Barcode-FP-FW Barcode-FP-RV NNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNN eneration of B32 Poly-FP-barcode ggtgannn NNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNN 32B-Poly-rev PR and Sequencing primers Barcode identification B-PR-FW B-PR-RV_neu B-PR-Seq B6 barcode retrieval B-PR-FW B-PR-RV_neu Ill2_ail-complete Ill-ail2 B32 barcode retrieval MPLX-primer xxxxxx p43 DUL-Index-primer xxxxxxxx x LM-PR (5'LR) Identification of LV-5-LR- [biotin]- second Integration LV-5-LR-2 in clone B6- LV-5-LR-3 LV-5-LR-Seq O O2 digital droplet PR IS-specific ddpr LV-Seq Lenti-LR-BHQ FM--BHQ 7 6Bw-µL Kl.7 8 6Bw-µL Kl.8 6B-Klon2-hr 9a 6Bw-µL Kl.4 0 6Bw-µL Kl Bw-5µL Kl.3 a 32Bw-5µL Kl Bw-5µL Kl Bw-5µL Kl.7 B-specific ddpr 6B-dPR-FW 6B-dPR-P FM--BHQ 6B-KlonB 6B-Klon 6B-Klon2 6B-KlonD 6B-KlonE 32-dPR-FW 32-dPR-P FM--BHQ 32B-Klon-RV 32B-Klon-RV 32B-Klon-RV 32B-KlonE-RV VN ddpr FP-dPR-FW FP-dPR-BHQ FM--BHQ FP-dPR-RV Housekeeping mepo-fw mepo-probe HEX--BHQ mepo-rv kb fragments p90 7 6Bw-µL Kl.7 8 6Bw-µL Kl.8 6B-Klon2-hr 9a 6Bw-µL Kl.4 0 6Bw-µL Kl Bw-5µL Kl.3 a 32Bw-5µL Kl Bw-5µL Kl Bw-5µL Kl.7

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