De novo Genome Assembly of the Mediterranean Mussel

For sequencing, we extracted 4μg of DNA from muscle tissue from a single mussel extracted from the Ria of Vigo, Spain. Using this DNA, three sequencing libraries with insert sizes of 180, 500 and 800 bp were constructed and sequenced at BGI (Beijing Genomics Institute—China). These libraries were sequenced with the Illumina HiSeq2000 high-throughput platform using paired-end sequencing (100-bp reads). To clean the initial set of reads, we filtered out raw reads if they fulfilled any of these conditions: a) >5% ambiguous bases (represented by the letter N); b) poly-A structures; c) > = 20 bases with low quality scores; d) adapter contamination: reads with more than 10 bp aligned to the adapter sequence (no more than 3-bp mismatch allowed); or e) small insert-size reads in which paired reads overlapped more than or equal to 10 bp (10% mismatch allowed).
We used Jellyfish [26 (link)] for counting k-mers and obtaining their frequency distributions. With these data, we drew frequency plots using k-mer lengths of 15, 17, 19 and 21. To assign the “true” coverage peak, we compared these plots to identify the peak that changed in height (“heterozygous peak”) and the one that did not (“coverage peak”). The latter was then used to calculate the genome size as the total k-mer number divided by the coverage-peak depth [27 (link)]. Finally, we assembled de novo the reads resulting from the quality filtering step using SOAPdenovo v1.05 [27 (link)] with parameters -K 31 -d 1 -M 1 -F–R. Then, we ran the Assemblathon 2 script [28 (link)] to obtain assembly statistics. Using this script, we compared the genome assemblies of M. galloprovincialis with those of A. californica, L. gigantea, P. fucata, and C. gigas (S1 File). Genome surveys of other molluscs with scarce sequencing depth [22 (link)] were not included in these comparisons. We confirmed the identification of the studied mussel as M. galloprovincialis by scanning the assembled sequences with two Mytilus genetic markers, Glu-5’ [29 (link)] and EFbis [30 (link)], using BLASTN [31 (link)] and Geneious version 6.1.8 [32 (link)].