6533b825fe1ef96bd1283391
RESEARCH PRODUCT
Directional high-throughput sequencing of RNAs without gene-specific primers.
Marja TiirolaAnita Mäkisubject
0301 basic medicineDNA Complementaryprimer biasComputational biologyBiologylevätGeneral Biochemistry Genetics and Molecular BiologyDNA sequencingWorkflow03 medical and health sciencesribosomitligationGeneDNA PrimersGene Library030102 biochemistry & molecular biologyplanktongeenitekniikkaHigh-Throughput Nucleotide SequencingRibosomal RNAmikrolevätrandom priming030104 developmental biologymikrobistoRNA RibosomalSpecific primersPhytoplanktonphytoplanktonRNAnext-generation sequencingribosomal RNABiotechnologydescription
Ribosomal RNA analysis is a useful tool for characterization of microbial communities. However, the lack of broad-range primers has hampered the simultaneous analysis of eukaryotic and prokaryotic members by amplicon sequencing. We present a complete workflow for directional, primer-independent sequencing of size-selected small subunit ribosomal RNA fragments. The library preparation protocol includes gel extraction of the target RNA, ligation of an RNA oligo to the 5′-end of the target, and cDNA synthesis with a tailed random-hexamer primer and further barcoding. The sequencing results of a phytoplankton mock community showed a highly similar profile to the biomass indicators. This method has universal potential for microbiome studies, and is compatible for the 5′-end sequencing of other RNA types with minimum library preparation costs.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2018-10-01 | BioTechniques |