DOI: 10.5061/dryad.zcrjdfn9v
title: Mitochondrial genome sequencing of marine leukemias reveals cancer contagion between clam species in the Seas of Southern Europe

description: Clonally transmissible cancers are tumour lineages that are transmitted
 between individuals via the transfer of living cancer cells. In marine
 bivalves, leukemia-like transmissible cancers, called hemic neoplasias,
 have demonstrated the ability to infect individuals from different
 species. We performed whole-genome sequencing in eight V. verrucosa clams
 that were diagnosed with hemic neoplasia, from two sampling points located
 more than 1,000 nautical miles away in the Atlantic Ocean and the
 Mediterranean Sea Coasts of Spain. Mitochondrial genome sequencing of
 tumour tissues from neoplastic animals revealed the coexistence of
 haplotypes from two different clam species. Phylogenies estimated from
 mitochondrial and nuclear markers confirmed this leukemia originated in C.
 gallina (or a closely related taxa) and was later transmitted to V.
 verrucosa, in which it survived as a contagious cancer. The analysis of
 mitochondrial and nuclear gene sequences supports all the studied tumours
 belonging to a single neoplastic C. gallina lineage that spread in the
 Seas of Southern Europe.
description: We performed whole-genome sequencing on 23 samples from 16 clam
 specimens, which includes eight neoplastic and eight non-neoplastic
 animals (Table 1), with Illumina paired-end libraries of 350 bp insert
 size and reads 150 bp long. Then, we run MITObim v1.9.1 (Hahn, et al.
 2013) to assemble the full mitochondrial genome of all sequenced samples,
 using gene baits from the following Cox1 and 16S reference genes to prime
 the assembly of clam mitochondrial genomes: V. verrucosa (Cox1, with
 GenBank accession number KC429139, 16S: C429301), C. gallina (Cox1:
 KY547757, 16S: KY547777) and C. striatula (Cox1: KY547747, 16S: KY547767).
 The draft sequences were polished twice with Pilon v1.23 (Walker, et al.
 2014), and conflictive repetitive fragments from the mitochondrial control
 region were resolved using long read sequencing with Oxford Nanopore
 technologies (ONT) on a set of representative samples from each species
 and tumours. ONT reads were assembled with Miniasm v0.3 (Li 2016) and
 corrected using Racon v1.3.1 (Vaser, et al. 2017). Protein-coding genes,
 rDNAs and tRNAs were annotated on the curated mitochondrial genomes using
 MITOS2 web server (Bernt, et al. 2013), and manually curated to fit ORFs
 as predicted by ORF-FINDER (Rombel, et al. 2002). The rebuilt sequences
 can be found here in <br> VVE_CGA_Mitochondrial_DNA.fasta and the
 annotations in VVE_CGA_Mitochondrial_DNA.gff. We then
 mapped the paired-end sequencing data from healthy and neoplastic tissues
 from all neoplastic samples onto the V. verrucosa and C. gallina reference
 mitochondrial genomes using BWA-mem v0.7.17-r1188 (Li and Durbin 2009)
 with default parameters. Duplicate reads were marked with Picard 2.18.14
 and removed from the analysis. The employed reference genome can be found
 in Ref_CGA_VVE.fa, and each bam file corresponds to a given animal
 (i.e. PLVV18_2249F-N0-alnCGA_VVE.sorted.detup.bam) in which the code
 represents the country (P, Portugal), and the locality (L, Lisbon) of
 procedence, the species (VV, Venus verrucosa), the year of collection (18,
 2018), an internal code of the animal (2249) and the sequenced tissue (F,
 Foot; Also H from Haemolymph and G from Gill). Some of them were obtained
 from Whole genome amplified samples (WGA). There is a reference to the
 intensity of the neoplasia detected in the animal (N0=Healthy; N1, N2, N3
 = different levels of neoplasia; N? = neoplasia but level
 unknown). COI Veneridae sequences were recovered from
 Genbank and Boldsystems and aligned with our mitochondria reconstructions
 and are in the COX_all_Venerids.phy file. This was later employed to
 reconstruct a molecular ML phylogeny shown as supplementary data in our
 manuscript. DEAH12 and TFIIH sequences, alignments and molecular
 phylogenies can be found here as well (alignments and trees).
 Satellite monomer clusters CL04 and CL17 reconstructions as
 recovered from repeatexplorer (Novak et al.
 2010) from C. gallina NGS data can be found at
 "Satellite_references.fasta" and coverage stats for each of the
 specimens with NGS data here is present
 in Satellite_coverage.txt.