Phased nucleotide inserts for sequencing low-diversity RNA samples from in vitro selection experiments.

Phased nucleotide inserts for sequencing low-diversity RNA samples from in vitro selection experiments. RNA. 2020 Apr 16;: Authors: Bendixsen DP, Roberts J, Townshend B, Hayden EJ Abstract In vitro selection combined with high-throughput sequencing is a powerful experimental approach with broad application in the engineering and characterization of RNA molecules. The diverse pools of starting sequences used for selection are often flanked by fixed sequences used as primer binding sites. The low nucleotide diversity of these sequences causes reduced output and poor read quality on Illumina platforms due to complications with fluorescence imaging algorithms. A common method to alleviate this problem is the addition of fragmented bacteriophage PhiX genome. The increased diversity allows for sequencing, but sacrifices a portion of the usable sequencing reads. An alternative approach to improving nucleotide balance is to insert nucleotides of variable length and composition at the beginning of each molecule when adding adaptors prior to sequencing. This approach preserves read depth by slightly reducing the usable length of each read. Here, we test the ability of inserted nucleotides to replace PhiX in a low-diversity sample generated from a high-throughput sequencing based ribozyme activity screen. We designed a pool of RNA sequences based on the twister ribozyme from Oryza sativa and screened the resulting 4,096 sequence variants for se...
Source: RNA - Category: Genetics & Stem Cells Authors: Tags: RNA Source Type: research