Supplementary Information for White shark genome: ancient elasmobranch adaptations associated with wound healing and the maintenance of genome stability. Nicholas J. Marraa,b,1,2, Michael J. Stanhopeb,1,3,4, Nathaniel Juec, Minghui Wangd, Qi Sund, Paulina Pavinski Bitarb, Vincent P. Richardse, Aleksey Komissarovf, Mike Raykog, Sergey Kliverf, Bryce J. Stanhopeb, Chuck Winklerh, Stephen J. O’Brienf,i, Agostinho Antunes,j,k, Salvador Jorgensenl, Mahmood S. Shivjia,3,4. authors to whom correspondence should be addressed: Michael J. Stanhope: [email protected] Mahmood S. Shivji: [email protected] Stephen J. O’Brien: [email protected] This PDF file includes: Figs. S1 to S3: pgs. 2-4 Table S1: pgs. 5-14 RESULTS; Shark Vision: pg. 15 Additional Methods; Transcriptome and Genome Annotation: pgs. 15-17 Captions for databases S1 to S8: pgs. 17-18. References for SI reference citations: pgs. 18-26 1 www.pnas.org/cgi/doi/10.1073/pnas.1819778116 Supplementary figures Fig. S1. K-mer plot of white shark genome sequence reads. Includes 23 bp lengths for all 2x150 bp reads (with the exclusion of the SWIFT kit sequences) included in the SOAPdenovo genome assembly, versus their frequency in the read pool. Several k-mer analyses were conducted to obtain an average estimate of genome size, of which this figure is representative. Genome size was calculated using the frequency of the k- mers present in the largest local maximum peak of the plot, using the following formula: G = Nk-mer/Ck-mer where G is genome size, N is the total number of error free k-mers expected from the sequence data, and C is the frequency of k-mers at the main peak. The plot also indicates a low level of heterozygosity (genomes with high heterozygosity have a secondary peak to the left of the local maximum), and a high repeat content, as shown by the large tail on the bottom right of the figure. There are also several areas of possible duplicated sequence, evidenced by the presence of several small secondary peaks to the right of the main genome size peak (magnified in the inset figure). The axes are plotted on a log-log scale to illustrate better the repeat tail. 2 Fig S2. Benchmarking Universal Single-Copy Orthologs (BUSCO). Assessment of the presence and completeness of Metazoan and Vertebrate BUSCO in the independently, de novo constructed genomes, of C. milli, R. typus, and C. carcharias as well as a multi- tissue transcriptome of C. carcharias. 3 Fig. S3. Break-down of the composition of the white shark genome assembly. The percent of the genome assembly belonging to introns and exons came from the MAKER output which provides an average intron and exon size along with the predicted number of genes. Segmental duplications were identified as sections of the genome that had >90% similarity to another portion of the genome over a continuous 1 kbp region as identified by BLASTN or that had 6 of 7 consecutive sliding windows of 1 kbp with a mean read depth of > mean + 3.5 SD, when the reads of the single end 150 bp library were mapped back to the genome. All repeat categories were determined by using the program RepeatMasker by using vertebrate + custom white shark repeats. The remaining percentage of the genome assembly not covered by these categories was defined as general 'Non-coding Regions'. 4 Table S1. Annotated list of positively selected genome stability related genes presented in Table 1 of the manuscript. Gene Protein name Role in genome Genome stability stability relevant GO terms CHEK2* Serine/threonine- DNA repair, apoptosis, DNA damage checkpoint; protein kinase tumor suppressor (1-8) double-strand break repair; Chk2 regulation of apoptotic process; regulation of signal transduction by p53 class mediator RFC5* Replication factor DNA repair; translesion DNA damage response, C subunit 5 synthesis; nucleotide detection of DNA damage; excision repair (9) DNA repair; translesion synthesis; nucleotide- excision repair FBXO45*! F-box/SPRY Regulates/degrades cellular response to DNA domain- tumor suppressor TP73 damage stimulus containing protein (10) 1 DICER1* Endoribonuclease siRNA and miRNA apoptotic DNA Dicer biogenesis; DNA repair fragmentation; miRNA (11-15) loading onto RISC involved in gene silencing by miRNA; conversion of ds siRNA to ss siRNA involved in RNA interference INO80B* INO80 complex Chromatin remodelling DNA repair; chromatin subunit B and DNA repair (16-18) remodelling; DNA recombination; cellular response to DNA damage stimulus DTL* Denticleless DNA damage response cellular response to DNA protein and translesion DNA damage stimulus; signal synthesis (19-25) transduction involved in G2 DNA damage checkpoint; POLD3* DNA polymerase DNA repair (26-30) nucleotide-excision repair, delta subunit 3 DNA incision, 5'-to lesion; telomere maintenance; translesion synthesis FEM1B* Protein fem-1 Apoptosis and DNA regulation of extrinsic homolog B repair (31,32) apoptotic signaling pathway via death domain receptors; regulation of 5 DNA damage checkpoint SIRT7* NAD-dependent DNA repair; chromatin chromatin organization; protein remodelling; apoptosis; NAD-dependent histone deacetylase regulates p53 (33-37) deacetylase activity (H3- sirtuin-7 K18 specific) PLK2* Serine/threonine- Cell cycle control; DNA damage response, protein kinase regulates tumor growth signal transduction by p53 PLK2 and apoptosis (38-42) class mediator resulting in cell cycle arrest; negative regulation of apoptotic process; CENPS* Centromere DNA repair (43-45) DNA repair; interstrand protein S cross-link repair; chromatin binding; cellular response to DNA damage stimulus; replication fork processing; CASS4* Cas scaffolding Cell adhesion and cell Cell adhesion protein family spreading; apoptosis member 4 (46-48) UFD1* Ubiquitin Protein negative regulation of recognition factor deubiquitination; core telomerase activity; in ER-associated component of p97- ubiquitin-dependent degradation UFD1-NPL4 complex ERAD pathway protein 1 involved in protein extraction from chromatin (49,50) AGT* Angiotensinogen Apotosis; cell regulation of apototic proliferation (51-54) process; regulation of cell proliferation; ERK1 and ERK2 cascade RPS6* 40S ribosomal Apoptosis (55,56) regulation of apoptotic protein S6 process; TOR signalling MYOG* Myogenin Regulation of cell positive regulation of cell proliferation and cell cycle arrest; regulation of cycle arrest (57,58) cell proliferation; chromatin DNA binding USP13* Ubiquitin Controls autophagy and regulation of autophagy; carboxyl-terminal p53 levels; cell cell proliferation; protein hydrolase 13 proliferation (59) deubiquitination PRIM1* DNA primase Okazaki fragment DNA replication, synthesis small subunit synthesis (60) of RNA primer; telomere maintenance via semi- conservative replication; G1/S transition of mitotic 6 cell cycle ALKBH7* Alpha- Necrosis (61) cellular response to DNA ketoglutarate damage stimulus; dependent programmed cell death; dioxygenase alkB regulation of homolog7 mitochondrial membrane permeability involved in programmed necrotic cell death BUD23* 18S rRNA Chromatin remodeling chromatin organization; (guanine-N(7))- (62) methyltransferase FIGL1# Fidgetin-like-1 Double strand break regulation of double-strand repair; regulation of break repair via meiotic recombination homologous (63,64) recombination; negative regulation of reciprocal meiotic recombination CTNNBL1# Beta-catenin-like Transcription coupled positive regulation of protein 1 repair; apoptosis (65-67) apoptotic process; mRNA splicing, via spliceosome CMTM7# CKLF-like Tumor suppressor (68) cytokine activity MARVEL transmembrane domain- containing protein 7 MDM4# Protein MDM4 p53 regulator (69-71) DNA damage response; signal transduction by p53 class mediator resulting in cell cycle arrest ARL6IP5# PRA1 family Apoptosis (72-79) intrinsic apoptotic protein 3 signaling pathway in response to oxidative stress; positive regulation of stress-activated MAPK cascade KIAA1324# UPF0577 protein Autophagy; tumor positive regulation of KIAA1324 suppressor (80-84) autophagosome assembly SALL4# Sal-like protein 4 DNA damage response; somatic stem cell stem cell maintenance population maintenance; (85-87) regulation of transcription, DNA-templated 7 PDCD2@! Programmed cell Apoptosis; regulation of positive regulation of death protein 2 stem cell proliferation apoptotic process; positive (88-90) regulation of hematopoietic stem cell proliferation PDCD4! Programmed cell Apoptosis; tumor positive regulation of death protein 4 suppressor (91-94) vascular associated smooth muscle cell apoptotic process; positive regulation of endothelial cell apoptotic process; cell aging; negative regulation of apoptotic process NHP2! H/ACA Telomere maintenance telomere maintenance via ribonucleoprotein (95-97) telomerase complex subunit 2 RRS1! Ribosome p53 regulator (98-102) regulation of signal biogenesis transduction by p53 class regulatory protein mediator homolog *= white shark # = whale shark @ = elephant shark ! = elasmobranchs Annotation and references: Genome stability PS genes CHK2 Activated when DNA undergoes a double-strand break (1); more specifically, phosphatidylinositol kinase family protein (PIKK) ATM, phosphorylates site Thr68 and activates CHK2 (1,2). CHK2 then in turn, phosphorylates targets such as CDC25 (cell division control protein 25), responsible for removing phosphate from active site residues and activating the cyclin-dependent kinases (CDKs). Thus, CHK2’s inhibition of CDC25 phosphatases prevents entry of the cell into mitosis. The CHK2 protein also interacts with and stabilizes