An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing

Methods and Protocols

article

Daniela Cordeiro¹  Alexandra Camelo²  Ana Carolina Pedrosa¹  Inês Brandão¹  Jorge Canhoto¹  Christophe Espírito Santo¹  Sandra Correia¹ 

[1]Centre for Functional Ecology, TERRA Associate Laboratory, Department of Life Sciences, University of Coimbra

[2]Centro de Apoio Tecnológico Agro-Alimentar

[3]InnovPlantProtect CoLab

关键词: amplification-free protocol;    direct cDNA sequencing;    high-throughput sequencing;    MinION;    Oxford Nanopore Technologies®;    poly(A) RNA;    Solanaceae;    Solanum betaceum;    transcriptome;    woody plant;   

DOI  :  10.3390/mps6020031

学科分类：电力

来源: mdpi

【 摘 要 】

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

【 授权许可】

CC BY