ena-STUDY-BOISE STATE UNIVERSITY-24-06-2019-13:25:13:331-51
Phased nucleotide inserts for sequencing low-diversity RNA samples from in vitro selection experiments
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 at the beginning of the sequence causes reduced sequence output and poor sequence quality on Illumina platforms due to complications with fluorescence imaging algorithms. A common solution to 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 sacrificing several bases in the 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 self-cleaving ribozyme activity. Phased nucleotides were inserted during reverse-transcription using four different template-switching oligos. The resulting cDNA was sequenced with and without the addition of PhiX DNA on a MiSeq platform. We found that libraries with the phased inserts produced equally high-quality sequence data whether or not PhiX DNA was added. In this experiment, the increase in read depth achieved without PhiX improved the consistency of activity measurements as compared to previously reported data. We conclude that phased inserts can be implemented following in vitro selection experiments to eliminate the use of PhiX when read length is not critical.
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 at the beginning of the sequence causes reduced sequence output and poor sequence quality on Illumina platforms due to complications with fluorescence imaging algorithms. A common solution to 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 sacrificing several bases in the 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 self-cleaving ribozyme activity. Phased nucleotides were inserted during reverse-transcription using four different template-switching oligos. The resulting cDNA was sequenced with and without the addition of PhiX DNA on a MiSeq platform. We found that libraries with the phased inserts produced equally high-quality sequence data whether or not PhiX DNA was added. In this experiment, the increase in read depth achieved without PhiX improved the consistency of activity measurements as compared to previously reported data. We conclude that phased inserts can be implemented following in vitro selection experiments to eliminate the use of PhiX when read length is not critical.