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High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes.

ABSTRACT: High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.

SUBMITTER: Singh M

PROVIDER: S-EPMC6635368 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes.

Singh Mandeep M Al-Eryani Ghamdan G Carswell Shaun S Ferguson James M JM Blackburn James J Barton Kirston K Roden Daniel D Luciani Fabio F Giang Phan Tri T Junankar Simon S Jackson Katherine K Goodnow Christopher C CC Smith Martin A MA Swarbrick Alexander A

Nature communications 20190716 1

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Reperto ...[more]

PMID: 31311926

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Project description:BackgroundAlternative splicing complexity plays a vital role in carcinogenesis and cancer progression. Improved understanding of novel splicing events and the underlying regulatory mechanisms may contribute new insights into developing new therapeutic strategies for colorectal cancer (CRC).MethodsHere, we combined long-read sequencing technology with short-read RNA-seq methods to investigate the transcriptome complexity in CRC. By using experiment assays, we explored the function of newly identified splicing isoform TIMP1 Δ4-5. Moreover, a CRISPR/dCasRx-based strategy to induce the TIMP1 exon 4-5 exclusion was introduced to inhibit neoplasm growth.ResultsA total of 90,703 transcripts were identified, of which > 62% were novel compared with current transcriptome annotations. These novel transcripts were more likely to be sample specific, expressed at relatively lower levels with more exons, and oncogenes displayed a characteristic to generate more transcripts in CRC. Clinical outcome data analysis showed that 1472 differentially expressed alternative splicing events (DEAS) were tightly associated with CRC patients' prognosis, and many novel isoforms were likely to be important determinants for patient survival. Among these, newly identified splicing isoform TIMP1 Δ4-5 was significantly downregulated in CRC. Further in vitro and in vivo assays demonstrated that ectopic expression of TIMP1 Δ4-5 significantly suppresses tumor cell growth and metastasis. Serine/arginine-rich splicing factor 1 (SRSF1) acts as a onco-splicing regulator through sustaining the inclusion of TIMP1 exon 4-5. Furthermore, CRISPR/dCasRx-based strategies designed to induce TIMP1 exon 4-5 exclusion have the potential to restrain the CRC growth.ConclusionsThis data provides a rich resource for deeper studies of gastrointestinal malignancies. Newly identified splicing isoform TIMP1 Δ4-5 plays an important role in mediating CRC progression and may be a potential therapy target in CRC.

Dataset Information

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes.

Publications

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes.

Similar Datasets

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