Key Points
The IgNAR region >3.13 Mb was enriched 245.531-fold by CATCH.
Twenty holes of 3508 bp of the IgNAR region were filled using PacBio HiFi reads.
Five potential germline V alleles of IgNAR1 were found.
Abstract
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 255× 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.
Footnotes
This work was supported by Shenzhen-Hong Kong Collaboration Fund JCYJ20170412152916724 (20170331).
N.Y., J.S., and M.W. directed the project. H.D. performed shark sampling, laboratory experiments, data analysis, and figure creation and wrote the manuscript. S.Y., L.L., and J.C. raised the sharks. H.D., S.Y., and L.W. sampled sharks. H.D., Y.Z., J.W., H.X., and T.L. performed data analysis. L.W. provided the samples that were used for IgNAR1 germline sequence verification. B.R., X.L., and X.Z. revised the paper.
The online version of this article contains supplemental material.
Abbreviations used in this article
- BGI
- Beijing Genomics Institution
- CATCH
- Cas9-assisted targeting of chromosome segments
- CCS
- circular consensus sequence
- CISMR
- CRISPR-mediated isolation of specific megabase-sized regions of the genome
- IgNAR
- Ig new Ag receptor
- MDA
- multiple displacement amplification
- NDSK
- N-laurylsarcosine and proteinase K
- NGS
- next-generation sequencing
- PFGE
- pulsed-field gel electrophoresis
- sgRNA
- single-guide RNA
- TE
- Tris-EDTA
- vNAR
- variable domain of Ig new Ag receptor
- Received August 23, 2021.
- Accepted October 21, 2021.
- Copyright © 2021 by The American Association of Immunologists, Inc.
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