mansoni gen ome. SOAP was used to remove in silico all female and male reads that correspond to unique sequences, and velvet in combination with a commercial long read assembler was used to assemble the remaining sequences into 8,594 individual repeat contigs. The minimum length corresponds to the used velvet parameter. We then applied our earlier described whole genome inhibitor Cisplatin in silico subtractive hybridiza tion approach to identify female specific repeats. Thirty three new repeat sequences were iden tified to be specific for the female W chromosome, giving a total of 36 W specific repeats. Several in silico methods were used to classify the repeats and their specificity was confirmed by PCR on male and female individuals. The results are summarized in Table 2. Three repeats were already known, 33 repeats are new.
The size of the consensus sequence for each assembled repeat was confirmed by PCR on female and male individuals. EST data and RT PCR show that at least eight repeats are transcribed. For a subset, copy number was estimated by qPCR and is moderate, with the exception of SMAlphafem 1. The copy number was estimated using quantitative DNA with a unique W specific region on scaffold Smp scaff018821 as reference. We used SchistoDB to identify genes that could be located within the region that is spanned by the repeats. Eight putative genes were identified in the vicinity of the repeats. Manual inspection of all loci showed that female next generation sequencing hits can be found for four putative genes, and male hits. However, three genes are identical and the pre dicted coding regions are small.
No significant similarity to known proteins could be found with blastx. Blast against the genome shows that these putative genes are not unique and it remains to be answered whether these sequences are actually transcribed and code proteins. Female specific repeats are arranged as large satellite type blocks in the heterochromatic region of chromosome W To identify the localization of the most abundant female specific repeats, W1, W3 8 and W13, we used fluorescent in situ hybridization on late secondary sporocyst metaphases. All studied repeats are arranged as large satellite blocks and localized in the heterochro matic region of the W chromosome, either in the pericentromeric region or on the euchromatin/heterochromatin boundary of the long arm.
None of the tested repeats was found on the short arm of chromosome W. Repeats W6 and W7 are specific for the pericentromeric region of the q arm, and W1 and W4 are located on the frontier of the heterochromatic GSK-3 region. W1 was already known and we confirm the earlier FISH results that localized it to the distal part of the heterochromatic region of Wq. Hirai et al. described a euchromatic gap region in the vicinity of the W1 chromosome. We did not see this gap, which might be due to the lower resolution of our equipment or differences between the used S. mansoni strains.