SUPPLEMENTARY INFORMATION. Intron retention is a widespread mechanism of tumor suppressor inactivation.

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1 SUPPLEMENTARY INFORMATION Intron retention is a widespread mechanism of tumor suppressor inactivation. Hyunchul Jung 1,2,3, Donghoon Lee 1,4, Jongkeun Lee 1,5, Donghyun Park 2,6, Yeon Jeong Kim 2,6, Woong-Yang Park 2,7, Dongwan Hong 1,5, Peter J. Park 8,9, Eunjung Lee 8 1 Research Institute, National Cancer Center, Gyeonggi-do, South Korea 2 Samsung Genome Institute, Samsung Medical Center, Seoul, South Korea 3 Bioinformatics and Systems Biology Graduate Program, University of California San Diego, San Diego, CA, USA 4 Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA 5 Cancer Immunology Branch, Division of Cancer Biology, National Cancer Center, Gyeonggi-do, South Korea 6 Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology, Samsung Electronics Company, Ltd., Seoul, South Korea 7 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, South Korea 8 Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, MA, USA 9 Center for Biomedical Informatics, Harvard Medical School, Boston, MA, USA Correspondence should be addressed to E.L. (elee20@partners.org), P.J.P. (peter_park@hms.harvard.edu), or D.H. (dwhong@ncc.re.kr)

2 Table of contents Supplementary Note... 3 Supplementary Figures Suppl. Figure 1 Flowchart with results of the identified SNVs associated with abnormal splicing... 8 Suppl. Figure 2 Examples of RNA-seq reads showing complex abnormal splicing... 9 Suppl. Figure 3 Comparison between the two splicing-altering SNV sets identified by AS and/or RS analysis Suppl. Figure 4 Fraction of SNVs showing reference allele expression only Suppl. Figure 5 Enrichment of splicing-altering SNVs in cancer gene sets Suppl. Figure 6 Experimentally confirmed intron retention-causing LBEMs in tumor suppressors Suppl. Figure 7 Splicing-disrupting LBEMs in TP Suppl. Figure 8 Splicing-disrupting LBEMs in SPSB Suppl. Figure 9 A diagram of TSG inactivation Suppl. Figure 10 TSGs with LBEM causing intron retention Suppl. Figures Characterization of genomic and transcriptomic patterns of LBEMs Suppl. Figure 11 Comparison of 5 splice site strength disrupted by LBEMs Suppl. Figure 12 Comparison of intronic size Suppl. Figure 13 Comparison of GC content Suppl. Figure 14 Comparison of density of intronic splicing regulatory elements Suppl. Figure 15 Comparison of expression level of adjacent up- and down-stream exons Suppl. Figure 16 Characterization of predicted LBEMs Suppl. Figure 17 Example of RNA-seq reads with a mutant allele supporting an intronic cryptic splice site Suppl. Figure 18 Exonic SNVs causing intron retention Suppl. Figure 19 Splice site SNVs causing intron retention References... 25

3 Supplementary Note Read pattern criteria for Ratio-based Spicing (RS) analysis Using the criteria described below, we classified somatic single-nucleotide variations (SNVs) into different types of abnormal splicing: intron retention, exon skipping, and intronic and exonic cryptic site activation. SNVs that showed multiple types of abnormal splicing were classified as complex abnormal splicing. As a negative control for the effect of an SNV on splicing in each sample, normally spliced transcripts were also required to be observed. Split reads supporting any alternative splicing transcripts from RefSeq annotation were not regarded as abnormal splicing. Using the original alignment data, we extracted reads mapped near SNVs and discarded PCR duplicates after marking them. Furthermore, reads with mapping quality scores less than 20 or those with indels were filtered out. For each target exon containing an SNV, we separately checked abnormal splicing (except for exon skipping) for its adjacent up- and down-stream exons. To identify an SNV significantly associated with abnormal splicing, we compared the ratio of abnormal reads to normal reads in a target mutated sample to two background ratio distributions (The definition of ratios for each category of abnormal splicing is described below). To create the background distributions, we first selected control samples from 741 normal samples or from 1,812 cancer samples that did not have mutations in the gene of interest. For each control sample, we calculated the same ratio of abnormal to normal reads; these were collected into two background ratio distributions; one for normal and one for cancer samples. When the target mutated sample s ratio was within both the top 1% and the top 5% of the normal and cancer background ratio distributions, respectively, then the SNV was considered significantly associated with the abnormal splicing. The criteria for each category of abnormal splicing are described below: Intron retention For an exon with an SNV (E*), its adjacent exon (E), and an intron between the two exons (I), 1) 2 abnormal reads that span each of the two exon-intron junctions (E*_I and I_E) were required for intron retention, covering 10 bp of the exon and 10 bp of the intron without split-read alignment. 2) 70% of bases in the intron (I) were required to be covered. 3) 2 normal reads spanning the exon-exon junction (E*_E) were required. 4) To ensure the whole intron was retained, both of the ratios of abnormal / normal reads, (E*_I) / (E*_E) and (E_I) / (E*_E), were required to be higher than those from the background distributions.

4 Exon skipping For an exon (E*) with an SNV, and its adjacent exons (E 1, E 2 ), 1) 2 abnormal reads spanning the junction of the two adjacent exons (E1_E2) were required. 2) The coverage of the two adjacent exons (E 1 and E 2 ) was required to be greater than that of exon E*. 3) 2 normal reads spanning the exon-exon junctions on each side (E1_E* and E*_E2) were required. 4) The ratio of abnormal / normal reads, (E1_E2) / (E1_E* + E*_E2), was required to be higher than that from the background distributions. Intronic cryptic splice site activation For an exon with an SNV (E*), its adjacent exon (E), and an intron between the two exons (I) which has a cryptic splice site (the parts before and after the cryptic site: I 1 and I 2 ), (when there are more than one cryptic splice sites, read pattern was examined for each splice site) 1) 2 abnormal reads that span the intronic cryptic splice site and the adjacent exon junction (I 1 _E). 2) 2 abnormal reads that span the exon and the first part of the intron junction (E*_I 1 ) covering both 10 bp of the exon and 10 bp of the intron. 3) 70% of bases of the intronic part before the cryptic splice site (I 1 ) were required to be covered. 4) 2 normal reads that span the exon-exon junction (E*_E). 5) To ensure the splice site activation, both of the ratios of abnormal / normal reads, (I 1 _E) / (E*_E) and (E*_I 1 ) / (E*_E), were required to be higher than those from the background distributions.

5 Exonic cryptic splice site activation For an exon with an SNV (E*) and an exonic cryptic splice site (the parts before and after the cryptic site: E* 1 and E* 2 ), its adjacent exon (E), and an intron between the exons (I), 1) 2 abnormal reads that span the exonic cryptic splice site and the adjacent exon junction (E* 1 _E). 2) The coverages of the exonic part before the cryptic splice site (E* 1 ) and the adjacent exon (E) should be greater than that of the exonic part after the cryptic splice site (E* 2 ). 3) 2 normal reads that span the exonic part after the cryptic splice site and the adjacent exon junction (E* 2 _E). 4) The ratio of abnormal / normal reads, (E* 1 _E) / (E* 2 _E), was required to be higher than that from the background distributions. Analysis of exonic splicing regulatory elements To test enrichment of SNVs associated with abnormal splicing in splicing regulatory elements, we first obtained splicing regulatory elements; 238 exonic splicing enhancers (ESEs) 1 and 176 exonic splicing silencers (ESS) 2. We utilized Burrows-Wheeler Aligner (BWA) 3 with default parameters, except for allowing exact match and all multiple alignment, to align the regulatory sequences to exons containing an SNV and annotated SNVs within the regulatory sequences in Supplementary Table 3. For each set of exonic SNVs causing different types of abnormal splicing (332 exon skipping, 74 exonic or 145 intronic cryptic site activation, 252 intron retention, and 45 complex splicing), we counted how often splicingaltering SNVs were within the splicing regulatory sequences. To evaluate whether the observed count was significantly different from the expected, we generated a null distribution based on randomly selecting the same number of SNVs out of 226,956 exonic somatic SNVs (from original total set; Supplementary Table 1) 100,000 times. The P value was calculated as the fraction of trials in which the number of SNVs in the splicing regulatory sequences is greater than or equal to the observed count. We found that SNVs associated with exon skipping were significantly enriched in ESEs (P = ), whereas those associated with other types of abnormal splicing were not. We did not find any enrichment of SNVs associated with

6 abnormal splicing in ESSs. Examination of relationship between abnormal splicing types Using splice-site SNVs associated with abnormal splicing, we found that exon skipping and intron retention occurred in a mutually exclusive manner (P = ). Intronic cryptic site activation and exon skipping were also mutually exclusive (P = ). The lack of co-occurrence of these aberrations suggests that mechanisms involved in exon-skipping act independently of those involving intronic elements. Enrichment of abnormal splicing in Tumor Suppressor Genes We also observed enrichment in the TSG sets when using an extended set of 453 intron retention-causing SNVs from AS analysis, including 22 non-lbems and 365 intronic splice site SNVs (P ; Fig. 4a and Supplementary Table 11; see Supplementary Fig. 18 and 19 for examples and Online Methods). In contrast, SNVs in the exon skipping or normal splicing groups were not enriched in the oncogene or tumor suppressor gene sets. Characteristics of genomic features We found that the genomic loci in the intron retention group had originally lower splicing strength than the loci in the other two groups (the scores calculated with a wild-type allele; P ; Supplementary Fig. 11b and 11c for MAXENT 4 and H-Bond scores 5, respectively). Both exons and introns in the intron retention group had higher GC content than those in the exon skipping group (P = ; Fig. 5b and Supplementary Fig. 13). However, we found that the density of intronic splicing regulatory elements (ISREs) was lower for the intron retention group than for the exon skipping group (P = ; Fig. 5b and Supplementary Fig. 14). Given that GC content dominates intrinsic nucleosome occupancy 6, the observed inverse correlation between GC content and ISRE density matches with a previous study reporting that ISREs were significantly enriched in nucleosome-disfavoring sequences 7. This suggests the possibility that in the intron retention group, shorter nucleosome-free regions limit regulatory protein access to ISREs. Although the density of intronic splicing regulatory elements (ISREs) was lower for the intron retention group than for the exon skipping group (Fig 5b and Supplementary Fig. 14), the density of exonic splicing regulatory elements (ESREs) did not show any differences between the groups (data not shown). Analysis of expression at the exon level revealed that the exon skipping group showed higher expression of adjacent exons relative to the exons with an LBEM (i.e., skipped exons) compared to the intron retention and normal splicing groups (Supplementary Fig. 15). Examination of excluded SNVs We excluded SNVs with additional SNVs or indels in the same gene, to unambiguously trace the causal SNV for each splicing event. For the excluded 2,797 SNVs near the exon-intron junctions (2,797 out of

7 19,540 SNVs with more than one SNVs or indels satisfied prerequisites of AS analysis), we performed AS analysis and found that 10 somatic SNVs caused intron retention as follows. Chr Position Gene Strand Transcript Variant type Reference Variant Sample chr COPE - NM_ Silent C T TCGA-B5-A0JY chr MBP - NM_ Missense C T TCGA-BG-A0M4 chrx KDM5C - NM_ Missense C A TCGA-B5-A11E chr SPG11 - NM_ Missense C A TCGA-D1-A103 chr NCOR2 - NM_ Missense C A TCGA-AP-A059 chr RALBP1 + NM_ Nonsense G T TCGA-AP-A051 chr JOSD2 - NM_ Missense C T TCGA-BS-A0UF chr LTBP4 + NM_ Silent C T TCGA-B5-A0JY chr NUP133 - NM_ Nonsense G A TCGA chr RALBP1 + NM_ Nonsense G T TCGA-AP-A051

8 Supplementary Figures Supplementary Figure 1. Flowchart with results of the identified SNVs associated with abnormal splicing. Allele-specific Splicing (AS) and Ratio-based Splicing (RS) analysis were performed to identify somatic SNVs associated with abnormal splicing. Given the difficulty of detecting exon skipping-causing SNVs (i.e., the absence of mutant allele in observed reads) in AS analysis, we first identified SNVs associated with exon skipping through RS analysis (n=707, outlined box). Since trasns-acting mutations are not likely to affect splicing of other genes in an allele-specific manner, we used the SNVs associated with exon skipping before removing SNVs from tumor samples with detects in splicing factors.

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10 Supplementary Figure 2. Examples of RNA-seq reads showing complex abnormal splicing. IGV browser 8 screenshots where RNA-seq reads showed complex abnormal splicing associated with an SNV in (a) CDKN2A and (b) TP53.

11 Supplementary Figure 3. Comparison between the two splicing-altering SNV sets identified by AS and/or RS analysis. (a) The number of identified exonic SNVs within 30 bp from exon-intron junction. The number in parenthesis in RS analysis indicates the number of exonic SNVs outside of 30 bp from exon-intron junction. (b) The number of identified intronic splice-site SNVs. Our AS analysis method cannot detect intronic splice-site SNVs causing exon skipping, because neither the wild-type nor mutant allele at intronic splice sites appear in RNA-seq reads unless the intron is retained (see Online Methods for AS analysis of intronic splice site SNVs). Therefore, the number of splice-site SNVs causing intron retention is only shown for AS analysis. For exon-skipping cases, the SNVs from AS analysis are a subset of those from RS analysis. AS analysis requires SNVs to pass RS analysis first (noted in the analysis flowchart in Supplementary Fig 1). For intron retention, both analyses provide high-confidence sets. For example, there are 66 LBEMs from AS, 44 from RS, and 29 are shared. When we examine each inconsistent case, we find that the discrepancy was mainly due to different criteria rather than false positive predictions. For example, RS analysis required many reads spanning exon-intron junctions of the exon with the SNV and the next exon and required 70% of bases in the intron to be covered, whereas AS analysis required reads spanning exon-intron junction of only the exon with the SNV in an allele-specific manner. Some SNVs from AS analysis, especially under the influence of NMD, were not able to meet those RS criteria.

12 Supplementary Figure 4. Fraction of SNVs showing reference allele expression only. (a) Fraction of synonymous SNVs that showed reference allele expression only. Gray and black solid lines represent the average fraction of nonsense and synonymous SNVs. Error bars represent standard errors. Synonymous SNVs at the last base of exons showed the highest fraction of reference allele expression only, comparable to that of nonsense SNVs demonstrating their strong mutational impact. (b) Fraction of missense SNVs that showed reference allele expression only is depicted in the same way as in (a).

13 Supplementary Figure 5. Enrichment of splicing-altering SNVs in cancer gene sets. Somatic synonymous and missense SNVs within 30 bp from exon-intron junctions identified by RS analysis were tested for their enrichment in two oncogene sets (upper panel) and four TSG sets (lower panel). As we categorized intronic cryptic site activation under intron retention in AS analysis, we followed the same strategy in this analysis. Due to relatively small number of SNVs associated with exonic cryptic site activation across all positions (n=80), we did not further analyze them. P value was assessed by random permutation test. The bars exceeding the y-axis value of 5 represent an empirical p-value of zero with 100,000 random permutation tests.

14 Supplementary Figure 6. Experimentally confirmed intron retention-causing LBEMs in tumor suppressors. Validation experiments for the LBEM in CDH1 (RNA-seq reads in Fig. 1c), PTCH1, and MLL2. The whole exon with an LBEM (green box) and flanking intronic (~ bp; green line) sequences of wild-type and mutant alleles were cloned and replaced an alternative cassette in a reporter plasmid. The percentages of abnormally spliced transcripts are shown above each band (mean ± SD % from triplicate).

15 Supplementary Figure 7. Splicing-disrupting LBEMs in TP53. (a) Positions of 11 TP53 LBEMs that caused intron retention were marked in TP53 gene structure. The LBEMs were clustered in three locations (the last base of exon 4, 6, and 9) rather than being distributed across the gene. (b) Screenshot examples of three different LBEMs causing intron retention. Reads with the reference allele preferentially showed normal splicing pattern whereas reads with the mutant allele spanned the exon-intron junction.

16 Supplementary Figure 8. Splicing-disrupting LBEMs in SPSB3. IGV browser 8 screenshots showing RNAseq reads spanning the exon-intron junction caused by the same missense LBEM of SPSB3 exon 6 (chr16: C>G) in two renal cancer samples.

17 Supplementary Figure 9. A diagram of TSG inactivation by somatic SNVs causing intron retention.

18 Supplementary Figure 10. TSGs with LBEM causing intron retention. (a) The upper panels are the same as the main figures 1a-d. The lower panel shows mrna level of TP53 (RPKM) across 502 breast cancer samples was plotted (vertical black dotted line for median expression; breast cancer sample BH-A0HL whose express data was not available was not used), and the expression levels of the two cases carrying the LBEM causing intron retention were marked by red lines. The average TP53 expression of 70 tumors with truncating (nonsense, frameshift indel, or splice-site) mutations in TP53 and the average TP53 expression of 111 tumors with missense mutations in TP53 were marked by orange and purple lines, respectively. P-values for differential expression due to truncating or missense mutations were calculated by Monte Carlo simulation of 100,000 permutations. (b) ARID1A. (c) CDH1. (d) TPR with LBEM that did not disrupt splicing.

19 Supplementary Figure 11. Comparison of 5 splice site strength disrupted by LBEMs. (a) H-bond 5 score was compared between the groups of SNVs causing different types of abnormal splicing. Splicing-disrupting SNVs decreased splicing strength more than those that did not disrupt splicing. (b-c) Splicing strength of 5 splice sites calculated using wild-type alleles. (b) MaxENT 4 and (c) H-bond 5 was calculated using a wildtype allele at the last base of exons. P-values were calculated using nonparametric Mann-Whitney test (P < 0.05).

20 Supplementary Figure 12. Comparison of intronic size. Comparison of down- (a) and up-stream (b) intronic length. Intronic length was significantly shorter for the intron retention group than for the exon skipping group, whereas exonic length did not show differences between the groups (data not shown). Down- (c) and up-stream (d) intronic size relative to exon. Intronic size relative to exon was significantly shorter for the intron retention group than for the exon skipping group.

21 Supplementary Figure 13. Comparison of GC content. Comparison of exonic (a) and up-stream intronic GC content (b). Down-stream GC content was shown in the main Fig 5b. Exons and Up-stream introns for the intron retention group had higher GC content than those for the exon skipping group. Supplementary Figure 14. Comparison of density of intronic splicing regulatory elements (ISREs) in upstream intronic regions. Density of them in down-stream intronic regions was shown in main Fig 5b. Density of ISREs was significantly lower for the intron retention group than for the exon skipping group. Density of exonic regulatory splicing elements did not show any differences between the groups (data not shown).

22 Supplementary Figure 15. Comparison of expression level of adjacent up- and down-stream exons. Expression level of adjacent (a) down- and (b) up-stream exons relative to that of exons with an LBEM was compared among different abnormal splicing groups. As expected, the exon skipping group showed higher expression of adjacent exons relative to the exons with an LBEM (i.e., skipped exons) compared to the intron retention and normal splicing groups.

23 Supplementary Figure 16. Characterization of predicted LBEMs. (a) Enrichment of LBEMs predicted to cause abnormal splicing in cancer-associated gene sets. Two oncogene- (upper panel) 9,10 and four tumor suppressor-like gene sets (lower panel) 9-11 were tested. P value was assessed by random permutation test (Online Methods). The predicted LBEMs showed a consistent enrichment pattern with the confirmed LBEMs through AS analysis (Note that many TSGs were manually confirmed and not used for the prediction). (b) Analysis of sequence motifs using LBEMs predicted to cause abnormal splicing. The motif pattern of LBEMs causing abnormal splicing mirrored left-handed (higher relative entropy scores in the exon) model like the LBEMs identified by AS analysis.

24 Supplementary Figure 17. Example of RNA-seq reads with a mutant allele supporting an intronic cryptic splice site. This abnormal splicing was caused by an LBEM of TP53 exon 6 in an ovarian cancer sample (TCGA ). The first two and last two reads with a mutant allele T spanned the exon-intron junction and the intronic cryptic splice site activation, respectively.

25 Supplementary Figure 18. Exonic SNVs causing intron retention. MLL2 and MLL3 had intron retention caused by missense and synonymous SNVs at the second last base of exon 13 and 24, respectively. The exonic SNVs in MLL2 and MLL3 caused intron retention and intronic cryptic site activation, respectively. Supplementary Figure 19. Splice site SNVs causing intron retention. The SNVs in BAP1 and TP53 were identified through AS analysis. IGV browser 8 screenshots showing RNA-seq reads spanning the exon-intron junction in BAP1 and TP53. The mutant allele was only observed in RNA-seq reads mapping to the retained part of the intron (Online Methods).

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