Cas9-Based Local Enrichment and Genomics Sequence Revision of Megabase-Sized Shark IgNAR Loci

The 0.8-Mb Ig new Ag receptor (IgNAR) region of the whitespotted bamboo shark (Chiloscyllium plagiosum) is incompletely assembled in Chr_44 of the reference genome. Here we used Cas9-assisted targeting of chromosome segments (CATCH) to enrich the 2 Mb region of the Chr_44 IgNAR loci and sequenced it by PacBio and next-generation sequencing. A fragment >3.13 Mb was isolated intact from the RBCs of sharks. The target was enriched 245.531-fold, and sequences had up to 94% coverage with a 2553 mean depth. Compared with the previously published sequences, 20 holes were filled, with a total length of 3508 bp. In addition, we report five potential germline V alleles of IgNAR1 from six sharks that may belong to two clusters of the IgNAR. Our results provide a new method to research the germline of large Ig gene segments, as well as provide the enhanced bamboo shark IgNAR gene loci with fewer gaps.

Automated summary

In this study, we successfully enriched and sequenced a 2 Mb region of the IgNAR gene loci in whitespotted bamboo shark using Cas9-assisted targeting of chromosome segments (CATCH) technology. The results showed high levels of enrichment and coverage. We also reported partial sequences of the bamboo shark IgNAR gene and filled in some previous gaps.