supplementary fig. 1. plasmids luciferase …...supplementary fig. 4. study flowchart. study...

1

Supplementary Figure 1

Supplementary Fig. 1. Plasmids luciferase constructs with AAV2 insertions. (a) pGL3-basic vector with firefly luciferase gene (luc+) is under the control of TERT promoter wild type, inserted with AAV2 from CHC985T, with scrambled sequence or with –124G>A and –146G>A mutations. (b) pmirGLO vector with PGK promoter controlling the expression of Firefly luciferase gene (luc2). Site of insertion for 3’UTR of TNFSF10 with CHC2557T and CHC1602T AAV2 insertion or with the scrambled sequence. Renilla luciferase gene (hRluc) is under the control of SV40 promoter. Plasmids representations are taken from Promega protocol. Multiple Cloning Site (MCS) are indicated.

Nature Genetics: doi:10.1038/ng.3389

2


Supplementary Fig. 2. TERT promoter luciferase construct with hot spot mutations and AAV2 insertion. The impact of AAV2 integration in TERT promoter similar to case CHC985T was evaluated using promoter luciferase assays in two different liver cell lines (Huh6 and Huh7) compared to the mutations –124 and –146 and to the wild type (WT). Error bars represent standard deviation of triplicate experiments corresponding to three independent transfections for each plasmid in each cell line. Results were normalized on the Renilla luciferase signal and on the WT construct. t-test was performed; ns, p > 0,05; *, 0,05 < p < 0,01; **, 0,01 < p < 0,001; ***, 0,001< p < 0,0001.


3


Supplementary Fig. 3. Positions of the primers used for screen AAV2 by PCR in 9 fragments. Coordinates and nucleotide sequences are provided in Supplementary Table 1.


4


Supplementary Fig. 4. Study flowchart. Study flowchart providing a framework of cases [samples] inclusion from patients series (Initial series, n = 150 and Exome series, n = 43). AAV2 status is defined according to molecular approaches: AAV2 PCR screening (+, positive; –, negative) and sequencing by viral capture or whole-exome (presence of AAV2 reads, positive; no AAV2 reads, negative). Presence of AAV2 sequences was defined as clonal or non-clonal insertion.


5



6

Supplementary Fig. 5. Integration sites in Human genome and in AAV2 DNA from viral capture sequencing. IGV (Integrative Genome Viewer) output of the alignment of the viral capture exome-seq reads to the region of integration of AAV2 into (a) CCNA2 intron 2 region, (b) 5’ UTR of TERT, (c) CCNE1 intron 4 region and (d) 3’ UTR of TNFSF10 genes, respectively. Blue reads represent sense DNAs, red reads represent antisense DNAs. Red lines define the boundaries region of the integration site and numbers refer to chimeric reads at this position. The consensus sequences of the integration site are represented above each representation. Viral sequences are denoted in red with nucleotides coordinate according to AF043303.1 reference AAV2 sequence. Human genome sequences are indicated in black, with coordinates that refer to the NCBI GRCh37..


7


Supplementary Fig. 6. IGV-Sashimi plot for RNA-Seq alignments from CCNE1-AAV2 inserted cases and controls. IGV-Sashimi plot for RNA-Seq alignments from the 2 CCNE1-AAV2 inserted cases (CHC1591T in red and CHC2208T in blue), a normal liver tissue in green and a non-inserted HCC in brown. RefSeq (NM_001238) canonical transcript for the gene is shown above. Alignments in exons are represented as read densities (numbers of reads are indicated in y-axis), and splice junction reads are drawn as arcs connecting a pair of exons, where arc width is drawn proportional to the number of reads aligning to the junction. The orange arrows define the AAV2 insertion site.


8


Supplementary Fig. 7: Insertion site of HBV and AAV2 in TERT, CCNE1 and KMT2B. We figured the recurrent HBV insertion in TERT, CCNE1 and KMT2B according to the results of deep sequencing published by Ding et al. 47 (orange arrows) and Sung et al. (yellow arrows)7. We also figured the AAV2 insertion (blue arrows) identified in the present study.


9


Supplementary Fig. 8. Phylogenetic tree constructed with the 11 inserted AAV2 in tumors together with reference AAV sequences reported in NCBI. Molecular Phylogenetic analysis was performed by Maximum Likelihood method. The integrated AAV sequences have highest similarity with AAV2 reference genome than any other strains of AAV.


10



11

Supplementary Fig. 9. Alignment of the AAV2 inserted sequences in tumors. Multiple nucleotide alignment was performed using MultAlin program 48 including 11 inserted AAV2 sequences with Genbank AAV2 reference sequence (AF043303.1). Similarities were indicated by dots, whereas differences were shown by nucleotide symbols. Recurrent substitutions are referred in Fig. 5.


12

Supplementary Table1. Primers used for identifying AAV2 by PCR, sequencing AAV2-Human chimeric regions and site directed mutagenesis

Primer name 5'- Primer sequence-3' Used For AAV2 ITR 5'F GTGGCCAACTCCATCACTAG AAV2 identification AAV2 ITR 5'R ACCACATGGTGTCGCAAA AAV2 identification AAV2 ORF 1F CGGCATTTCTGACAGCTTTG AAV2 identification AAV2 ORF 1R GGGTGCCTGCTCAATCA AAV2 identification AAV2 ORF 2F GGACAATGCGGGAAAGATTATG AAV2 identification AAV2 ORF 2R GGGATCGTACCCGTTTAGTT AAV2 identification AAV2 ORF2.2F AAAGGGTGGAGCCAAGAAA AAV2 identification AAV2 ORF2.2R TGCAGGGAAACAGCATCA AAV2 identification AAV2 ORF2.3F CAGGAGCGCCTTAAAGAAGATA AAV2 identification AAV2 ORF2.3R CCGTCTTAACAGGTTCCTCAA AAV2 identification

AAV2 ORF 3F 250bp GCACCAATGGCAGACAATAAC AAV2 identification AAV2 ORF 3R 250bp TGATGAGTCTTTGCCAGTCAC AAV2 identification

AAV2 ORF3.2F GCGTACCGGAAACAACTTTAC AAV2 identification AAV2 ORF3.2R GTACTGGTCGATGAGAGGATTC AAV2 identification AAV2 ORF3.3F CGGAGCAGTATGGTTCTGTATC AAV2 identification AAV2 ORF3.3R CCTGAAGGTACACATCTCTGTC AAV2 identification AAV2 ITR 3'F GGTCTCTGCGTATTTCTTTCTTATC AAV2 identification AAV2 ITR 3'R GTGGCCAACTCCATCACTA AAV2 identification TNFSF10_1F TCCTGACCCTATATTGTTGATGA Sanger Sequencing TNFSF10_1R TTTTGGTTGTGGCTGCTCTA Sanger Sequencing TNFSF10_2F ATGGACCATGAAGCCAGTTT Sanger Sequencing TNFSF10_2R AACCATTGCATTAATGTTTGGAA Sanger Sequencing TNFSF10_3F CAGCTTCCAAACATTAATG Sanger Sequencing TNFSF10_3R TGGCATGATCTCACCACACT Sanger Sequencing TNFSF10_6F TGGACCATGAAGCCAGTTTT Sanger Sequencing TNFSF10_6R TTCAGCAATTTCATTCTCTTGG Sanger Sequencing TNFSF10_7F TCTACAACACACACTGTTCTGAAA Sanger Sequencing TNFSF10_7R CTTGAGAGATGGATTGTTGCTC Sanger Sequencing TNFSF10_8F CAGCTTCCAAACATTAATGCAA Sanger Sequencing TNFSF10_8R TCTCACCACACTGCAACCTC Sanger Sequencing TNFSF10_9F TGTTGGCACATGCCTGTAGT Sanger Sequencing TNFSF10_9R GAAGTCTTTTCCCCCATTTTT Sanger Sequencing TNFSF10_10F TCATGTTTGCTGTAGTGCTCA Sanger Sequencing

TNFSF10_10R1 GCCTGGGTGACAGAGAGACT Sanger Sequencing TNFSF10_10R2 GTGGCTCATGCCTGTAATCC Sanger Sequencing

CCNA2_1F ATGAGCATGTCACCGTTCCT Sanger Sequencing CCNA2_1R TCACATCCTTTCTAAGCCTTG Sanger Sequencing CCNA2_2F CTGGACCCAGAAAACCATTG Sanger Sequencing CCNA2_2R TTCTCCCAACCAAGAACACA Sanger Sequencing


13

Primer name 5'- Primer sequence-3' Used For CCNA2_3F TCCAGTGCTCTTTCAGGTTC Sanger Sequencing CCNA2_3R CCCTCCTTCCTCACAATCAA Sanger Sequencing CCNA2_4F AGTTGTACCATGGCTGTTTACTC Sanger Sequencing CCNA2_4R GGTTTGTTATGGGCAAATGG Sanger Sequencing CCNA2_5F GCAGGAAAAGTCCTTTGGAA Sanger Sequencing CCNA2_5R GGCATGGTGGTTCTGTTAGG Sanger Sequencing CCNA2_6F GGGATCTGCAGTTTCTCTTGG Sanger Sequencing CCNA2_6R TGACTCTGGGACAATTCAAAAG Sanger Sequencing CCNA2_7F GCTTTGTTAATATGAGATTCCTTCC Sanger Sequencing CCNA2_7R CATTAATTACGAGAGGCTACATGCT Sanger Sequencing TERT_P_1F CCGGTGGGTGATTAACAGAT Sanger Sequencing TERT_P_1R GAGGCCCTGGGAACAGGT Sanger Sequencing TERT_P_2F CCTTCACGTCCGGCATTC Sanger Sequencing TERT_P_2R CAGCGCTGCCTGAAACTC Sanger Sequencing TERT_P_3F AGCCCCTCCCCTTCCTTT Sanger Sequencing TERT_P_3R TCCTTCAGGCAGGACACCT Sanger Sequencing TERT_P_4F CACAGCCTAGGCCGATTC Sanger Sequencing TERT_P_4R AAGGTGAAGGGGCAGGAC Sanger Sequencing

1591T_Hum1F CCGCCGCCGCCTGCCGGGA Sanger Sequencing 1591T_Chi_1F CTGGAGCGCGTCTGTGGA Sanger Sequencing 1591T_Chi_1R ACCAGGGTCGGAGTGGCGA Sanger Sequencing 1591T_Hum1R CTGTCCGCAGCCCGCGGCAG Sanger Sequencing

KMT2B_1F TGAGAAGAGACGGTCCATCC Sanger Sequencing KMT2B_1R CCCCAAGAGGAGGAGGAGTA Sanger Sequencing KMT2B_2F TCAGTTTACCCCAAGCGAAG Sanger Sequencing KMT2B_2R ACCTCGGCCTTAACAGGAGT Sanger Sequencing KMT2B_3F AGACACAGGCTCAGCTACTGC Sanger Sequencing KMT2B_3R TTAGGGGGTTGCAAAGAATG Sanger Sequencing KMT2B_4F GCCGTCACCACAGCAGAT Sanger Sequencing KMT2B_4R CAGAAAGGCAGGTTCTGAGG Sanger Sequencing KMT2B_5F TCAAGATCGATCAGCAGCAG Sanger Sequencing

KMT2B_5R1 AGGTCTGGGGAGGAACAAAG Sanger Sequencing KMT2B_5R2 AGACCAGCCTAGCCAATGTG Sanger Sequencing

2557T_Right1F AGCCTCAAGTTGCACCAGTT Sanger Sequencing 2557T_Right1R TGCAACAAAGCAGTGTCCTT Sanger Sequencing 2557T_Right2F AAACGGGGAAACAAAATTCA Sanger Sequencing 2557T_Right2R TTGCAAATCCCAACCCTTAG Sanger Sequencing 2557T_Right3F ACAGAGTCTCGCTGTGTTGC Sanger Sequencing 2557T_Right3R TGTTGGCATATGACAGAATCC Sanger Sequencing 2557T_Right4F CCGGAATACATCTCTTAAAGTTTG Sanger Sequencing


14

Primer name 5'- Primer sequence-3' Used For 2557T_Right4R GTTGGCAACTACCACCATTG Sanger Sequencing 2557T_Right5F CTTGGCAGTGCAAACCTGTA Sanger Sequencing 2557T_Right5R CATCTTGCCTGTGTCCAGAA Sanger Sequencing

CCNE1_1F GTCACATGGGGTTTCATGCT Sanger Sequencing CCNE1_1R CCCCAGTTCACCAGCTTTTA Sanger Sequencing CCNE1_2F GACTGGCACCGTCTGAGATT Sanger Sequencing CCNE1_2R CAGGAAATTTGGCAGGAACT Sanger Sequencing CCNE1_3F CAGCCTGACCTCAGCAAGA Sanger Sequencing CCNE1_3R GAGGGGCTTCTCTGCTAACC Sanger Sequencing

CCNE1_SD_1F ATGAATGGGTGAAGCTGTCC Sanger Sequencing CCNE1_SD_1R AAACACAAAATGTGTGGTGAGC Sanger Sequencing CCNE1_SD_2F TATGGCTTGTTGCACACTGG Sanger Sequencing CCNE1_SD_2R CCTTGCTCTCACTCCGTCTC Sanger Sequencing CCNE1_SD_3F GGGTAGGTGGTGGGAGAGAT Sanger Sequencing CCNE1_SD_3R GCGCTCCATATTTCACAGGT Sanger Sequencing CCNE1_SD_4F CTCCGACACTGCCTGCTC Sanger Sequencing CCNE1_SD_4R CCTTGGTATACCCCTGTTGA Sanger Sequencing CCNE1_SD_5F CCCTCCTGCACACATCTTTC Sanger Sequencing

CCNE1_SD_5R1 GGCTATTTGGTTGCCTTGAA Sanger Sequencing CCNE1_SD_5R2 CTCAGATGATCCACCCACCT Sanger Sequencing CCNE1_SD_5R3 ACCACCCCCAGCTAATTTTT Sanger Sequencing

L_CCNE1_1F ACTCAGGGCCCGGAACTC Sanger Sequencing L_CCNE1_1R GACGCGGGAGAAGTCTGG Sanger Sequencing L_CCNE1_2F GCAGGCCCTGTCACTTGG Sanger Sequencing L_CCNE1_2R GCTCAGAGCGGGACATTTA Sanger Sequencing L_CCNE1_3F CGCGCAGGGATTTTAAATGT Sanger Sequencing

L_CCNE1_3R1 GACTCACCAGGGTCTTGTCC Sanger Sequencing L_CCNE1_3R2 CCTCAGTGTCCCCTCCAC Sanger Sequencing L_CCNE1_4F AAAGGGGGAAGGGGTACTG Sanger Sequencing

L_CCNE1_4R1 ACGTTTGCCTTCCTCTTCCT Sanger Sequencing L_CCNE1_4R2 CAGGTTATGAGCTCCGTTCC Sanger Sequencing

TERT-124F GAGGGCCCGGAAGGGGCTGGGCC Site Directed Mutagenesis TERT-124R GGCCCAGCCCCTTCCGGGCCCTC Site Directed Mutagenesis TERT-146F GGACCCGGAAGGGGTCGGGACGG Site Directed Mutagenesis TERT-146R CCGTCCCGACCCCTTCCGGGTCC Site Directed Mutagenesis


15

Supplementary Table 2. Clinical and pathological features of the patients with AAV2 insertion


supplementary fig. 1. plasmids luciferase …...supplementary fig. 4. study flowchart. study...

Documents