PMID- 31341290 OWN - NLM STAT- MEDLINE DCOM- 20191126 LR - 20210827 IS - 1750-2799 (Electronic) IS - 1754-2189 (Print) IS - 1750-2799 (Linking) VI - 14 IP - 8 DP - 2019 Aug TI - RNase H-dependent PCR-enabled T-cell receptor sequencing for highly specific and efficient targeted sequencing of T-cell receptor mRNA for single-cell and repertoire analysis. PG - 2571-2594 LID - 10.1038/s41596-019-0195-x [doi] AB - RNase H-dependent PCR-enabled T-cell receptor sequencing (rhTCRseq) can be used to determine paired alpha/beta T-cell receptor (TCR) clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples. With the enhanced specificity of RNase H-dependent PCR (rhPCR), it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step. For single cells, the protocol includes sorting of single cells into plates, generation of cDNA libraries, a TCR-specific amplification step, a second PCR on pooled sample to generate a sequencing library, and sequencing. In the bulk method, sorting and cDNA library steps are replaced with a reverse-transcriptase (RT) reaction that adds a unique molecular identifier (UMI) to each cDNA molecule to improve the accuracy of repertoire-frequency measurements. Compared to other methods for TCR sequencing, rhTCRseq has a streamlined workflow and the ability to analyze single cells in 384-well plates. Compared to TCR reconstruction from single-cell transcriptome sequencing data, it improves the success rate for obtaining paired alpha/beta information and ensures recovery of complete complementarity-determining region 3 (CDR3) sequences, a prerequisite for cloning/expression of discovered TCRs. Although it has lower throughput than droplet-based methods, rhTCRseq is well-suited to analysis of small sorted populations, especially when analysis of 96 or 384 single cells is sufficient to identify predominant T-cell clones. For single cells, sorting typically requires 2-4 h and can be performed days, or even months, before library construction and data processing, which takes ~4 d; the bulk RNA protocol takes ~3 d. FAU - Li, Shuqiang AU - Li S AD - Broad Institute of MIT and Harvard, Cambridge, MA, USA. AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. AD - Translational Immunogenomics Lab, Dana-Farber Cancer Institute, Boston, MA, USA. FAU - Sun, Jing AU - Sun J AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. FAU - Allesoe, Rosa AU - Allesoe R AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. AD - Department of Bio and Health Informatics, Technical University of Denmark, Kongens Lyngby, Denmark. FAU - Datta, Krishnalekha AU - Datta K AD - Integrated DNA Technologies, Redwood City, CA, USA. FAU - Bao, Yun AU - Bao Y AD - Integrated DNA Technologies, Redwood City, CA, USA. FAU - Oliveira, Giacomo AU - Oliveira G AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. FAU - Forman, Juliet AU - Forman J AD - Broad Institute of MIT and Harvard, Cambridge, MA, USA. AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. AD - Translational Immunogenomics Lab, Dana-Farber Cancer Institute, Boston, MA, USA. FAU - Jin, Roger AU - Jin R AD - Massachusetts Institute of Technology, Cambridge, MA, USA. FAU - Olsen, Lars Ronn AU - Olsen LR AD - Department of Bio and Health Informatics, Technical University of Denmark, Kongens Lyngby, Denmark. FAU - Keskin, Derin B AU - Keskin DB AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. AD - Translational Immunogenomics Lab, Dana-Farber Cancer Institute, Boston, MA, USA. FAU - Shukla, Sachet A AU - Shukla SA AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. AD - Translational Immunogenomics Lab, Dana-Farber Cancer Institute, Boston, MA, USA. FAU - Wu, Catherine J AU - Wu CJ AD - Broad Institute of MIT and Harvard, Cambridge, MA, USA. AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. FAU - Livak, Kenneth J AU - Livak KJ AUID- ORCID: 0000-0001-9105-5856 AD - Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. kennethj_livak@dfci.harvard.edu. AD - Translational Immunogenomics Lab, Dana-Farber Cancer Institute, Boston, MA, USA. kennethj_livak@dfci.harvard.edu. LA - eng GR - P01 CA229092/CA/NCI NIH HHS/United States GR - P50 CA101942/CA/NCI NIH HHS/United States GR - R21 CA216772/CA/NCI NIH HHS/United States GR - R01 CA155010/CA/NCI NIH HHS/United States GR - U24 CA224331/CA/NCI NIH HHS/United States GR - R50 CA211482/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20190724 PL - England TA - Nat Protoc JT - Nature protocols JID - 101284307 RN - 0 (RNA, Messenger) RN - 0 (Receptors, Antigen, T-Cell) RN - EC 3.1.26.4 (Ribonuclease H) SB - IM MH - Cells, Cultured MH - Cloning, Molecular MH - Humans MH - Polymerase Chain Reaction/*methods MH - RNA, Messenger/*genetics/metabolism MH - Receptors, Antigen, T-Cell/*genetics/metabolism MH - Ribonuclease H/metabolism MH - Sequence Analysis, RNA/*methods MH - Single-Cell Analysis/*methods MH - T-Lymphocytes/chemistry/cytology PMC - PMC7189368 MID - NIHMS1563655 COIS- Competing interests J.S. is currently an employee of Moderna Therapeutics. K.D. is an employee of IDT; Y.B. was an employee of IDT during the time of this work; K.J.L. was a paid consultant of IDT during a portion of this work; D.B.K. has previously advised Neon Therapeutics, and owns equity in Aduro Biotech, Agenus Inc., Ampliphi BioSciences Corp., Biomarin Pharmaceutical Inc., Bristol Myers Squibb Com., Celldex Therapeutics Inc., Editas Medicine Inc., Exelixis Inc., Gilead Sciences Inc., IMV Inc., Lexicon Pharmaceuticals Inc., Sangamo Therapeutics, and Stemline Therapeutics Inc.; C.J.W. is a founder of Neon Therapeutics and a member of its scientific advisory board. The remaining authors declare no competing financial interests. C.J.W. is subject to a conflict of interest management plan for the reported studies because of her competing financial interests in Neon Therapeutics. Under this plan, C.J.W. may not access identifiable human subjects data nor otherwise participate directly in the IRB-approved protocol reported herein. C.J.W.'s contributions to the overall program strategy and data analyses occurred on a de-identified basis. EDAT- 2019/07/26 06:00 MHDA- 2019/11/27 06:00 PMCR- 2020/04/29 CRDT- 2019/07/26 06:00 PHST- 2018/11/27 00:00 [received] PHST- 2019/05/07 00:00 [accepted] PHST- 2019/07/26 06:00 [pubmed] PHST- 2019/11/27 06:00 [medline] PHST- 2019/07/26 06:00 [entrez] PHST- 2020/04/29 00:00 [pmc-release] AID - 10.1038/s41596-019-0195-x [pii] AID - 10.1038/s41596-019-0195-x [doi] PST - ppublish SO - Nat Protoc. 2019 Aug;14(8):2571-2594. doi: 10.1038/s41596-019-0195-x. Epub 2019 Jul 24.