Precise Characterization of <i>Bombyx mori Fibroin Heavy Chain</i> Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies
To study the evolution of gene function and a species, it is essential to characterize the tandem repetitive sequences distributed across the genome. Cas9-based enrichment combined with nanopore sequencing is an important technique for targeting repetitive sequences. Cpf1 has low molecular weight, l...
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| Format: | Article |
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MDPI AG
2021-09-01
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| Series: | Insects |
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| Online Access: | https://www.mdpi.com/2075-4450/12/9/832 |
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| author | Wei Lu Xinhui Lan Tong Zhang Hao Sun Sanyuan Ma Qingyou Xia |
| author_facet | Wei Lu Xinhui Lan Tong Zhang Hao Sun Sanyuan Ma Qingyou Xia |
| author_sort | Wei Lu |
| collection | DOAJ |
| description | To study the evolution of gene function and a species, it is essential to characterize the tandem repetitive sequences distributed across the genome. Cas9-based enrichment combined with nanopore sequencing is an important technique for targeting repetitive sequences. Cpf1 has low molecular weight, low off-target efficiency, and the same editing efficiency as Cas9. There are numerous studies on enrichment sequencing using Cas9 combined with nanopore, while there are only a few studies on the enrichment sequencing of long and highly repetitive genes using Cpf1. We developed Cpf1-based enrichment combined with ONT sequencing (CEO) to characterize the <i>B. mori FibH</i> gene, which is composed of many repeat units with a long and GC-rich sequence up to 17 kb and is not easily amplified by means of a polymerase chain reaction (PCR). CEO has four steps: the dephosphorylation of genomic DNA, the Cpf1 targeted cleavage of <i>FibH</i>, adapter ligation, and ONT sequencing. Using CEO, we determined the fine structure of <i>B. mori</i><i>FibH</i>, which is 16,845 bp long and includes 12 repetitive domains separated by amorphous regions. Except for the difference of three bases in the intron from the reference gene, the other sequences are identical. Surprisingly, many methylated CG sites were found and distributed unevenly on the <i>FibH</i> repeat unit. The CEO we established is an available means to depict highly repetitive genes, but also a supplement to the enrichment method based on Cas9. |
| first_indexed | 2024-03-10T07:34:15Z |
| format | Article |
| id | doaj.art-d12f2c7c062441fc828de7c3f1baa3a2 |
| institution | Directory Open Access Journal |
| issn | 2075-4450 |
| language | English |
| last_indexed | 2024-03-10T07:34:15Z |
| publishDate | 2021-09-01 |
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| series | Insects |
| spelling | doaj.art-d12f2c7c062441fc828de7c3f1baa3a22023-11-22T13:37:15ZengMDPI AGInsects2075-44502021-09-0112983210.3390/insects12090832Precise Characterization of <i>Bombyx mori Fibroin Heavy Chain</i> Gene Using Cpf1-Based Enrichment and Oxford Nanopore TechnologiesWei Lu0Xinhui Lan1Tong Zhang2Hao Sun3Sanyuan Ma4Qingyou Xia5State Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing 400715, ChinaState Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing 400715, ChinaState Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing 400715, ChinaState Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing 400715, ChinaState Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing 400715, ChinaState Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing 400715, ChinaTo study the evolution of gene function and a species, it is essential to characterize the tandem repetitive sequences distributed across the genome. Cas9-based enrichment combined with nanopore sequencing is an important technique for targeting repetitive sequences. Cpf1 has low molecular weight, low off-target efficiency, and the same editing efficiency as Cas9. There are numerous studies on enrichment sequencing using Cas9 combined with nanopore, while there are only a few studies on the enrichment sequencing of long and highly repetitive genes using Cpf1. We developed Cpf1-based enrichment combined with ONT sequencing (CEO) to characterize the <i>B. mori FibH</i> gene, which is composed of many repeat units with a long and GC-rich sequence up to 17 kb and is not easily amplified by means of a polymerase chain reaction (PCR). CEO has four steps: the dephosphorylation of genomic DNA, the Cpf1 targeted cleavage of <i>FibH</i>, adapter ligation, and ONT sequencing. Using CEO, we determined the fine structure of <i>B. mori</i><i>FibH</i>, which is 16,845 bp long and includes 12 repetitive domains separated by amorphous regions. Except for the difference of three bases in the intron from the reference gene, the other sequences are identical. Surprisingly, many methylated CG sites were found and distributed unevenly on the <i>FibH</i> repeat unit. The CEO we established is an available means to depict highly repetitive genes, but also a supplement to the enrichment method based on Cas9.https://www.mdpi.com/2075-4450/12/9/832Cpf1ONT<i>FibH</i>methylation |
| spellingShingle | Wei Lu Xinhui Lan Tong Zhang Hao Sun Sanyuan Ma Qingyou Xia Precise Characterization of <i>Bombyx mori Fibroin Heavy Chain</i> Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies Insects Cpf1 ONT <i>FibH</i> methylation |
| title | Precise Characterization of <i>Bombyx mori Fibroin Heavy Chain</i> Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies |
| title_full | Precise Characterization of <i>Bombyx mori Fibroin Heavy Chain</i> Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies |
| title_fullStr | Precise Characterization of <i>Bombyx mori Fibroin Heavy Chain</i> Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies |
| title_full_unstemmed | Precise Characterization of <i>Bombyx mori Fibroin Heavy Chain</i> Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies |
| title_short | Precise Characterization of <i>Bombyx mori Fibroin Heavy Chain</i> Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies |
| title_sort | precise characterization of i bombyx mori fibroin heavy chain i gene using cpf1 based enrichment and oxford nanopore technologies |
| topic | Cpf1 ONT <i>FibH</i> methylation |
| url | https://www.mdpi.com/2075-4450/12/9/832 |
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