High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing
Abstract Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular co...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-05-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-38324-9 |
| _version_ | 1797832019562987520 |
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| author | Zhuo-Xing Shi Zhi-Chao Chen Jia-Yong Zhong Kun-Hua Hu Ying-Feng Zheng Ying Chen Shang-Qian Xie Xiao-Chen Bo Feng Luo Chong Tang Chuan-Le Xiao Yi-Zhi Liu |
| author_facet | Zhuo-Xing Shi Zhi-Chao Chen Jia-Yong Zhong Kun-Hua Hu Ying-Feng Zheng Ying Chen Shang-Qian Xie Xiao-Chen Bo Feng Luo Chong Tang Chuan-Le Xiao Yi-Zhi Liu |
| author_sort | Zhuo-Xing Shi |
| collection | DOAJ |
| description | Abstract Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics. |
| first_indexed | 2024-04-09T14:01:02Z |
| format | Article |
| id | doaj.art-59b8c6b1b4d94ff0b107573a266dbe7f |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-04-09T14:01:02Z |
| publishDate | 2023-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-59b8c6b1b4d94ff0b107573a266dbe7f2023-05-07T11:18:17ZengNature PortfolioNature Communications2041-17232023-05-0114111310.1038/s41467-023-38324-9High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencingZhuo-Xing Shi0Zhi-Chao Chen1Jia-Yong Zhong2Kun-Hua Hu3Ying-Feng Zheng4Ying Chen5Shang-Qian Xie6Xiao-Chen Bo7Feng Luo8Chong Tang9Chuan-Le Xiao10Yi-Zhi Liu11State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceCollege of Life Sciences, University of Chinese Academy of SciencesState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangdong Key Laboratory of Liver Disease Research, the Third Affiliated Hospital of Sun Yat-sen UniversityState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceKey Laboratory of Genetics and Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants, Ministry of Education, College of Forestry, Hainan UniversityBeijing Institute of Radiation MedicineSchool of Computing, Clemson UniversityBGI Genomics, BGI ShenzhenState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceAbstract Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics.https://doi.org/10.1038/s41467-023-38324-9 |
| spellingShingle | Zhuo-Xing Shi Zhi-Chao Chen Jia-Yong Zhong Kun-Hua Hu Ying-Feng Zheng Ying Chen Shang-Qian Xie Xiao-Chen Bo Feng Luo Chong Tang Chuan-Le Xiao Yi-Zhi Liu High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing Nature Communications |
| title | High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing |
| title_full | High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing |
| title_fullStr | High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing |
| title_full_unstemmed | High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing |
| title_short | High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing |
| title_sort | high throughput and high accuracy single cell rna isoform analysis using pacbio circular consensus sequencing |
| url | https://doi.org/10.1038/s41467-023-38324-9 |
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