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

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. Diverse pools of starting sequences used for selection are often flanked by fixed sequences used as primer binding sites. These low diversity regions often lead to data loss from complications with Illumina image processing algorithms. A common method to alleviate this problem is the addition of fragmented bacteriophage PhiX genome, which improves sequence quality but sacrifices a portion of usable sequencing reads. An alternative approach is to insert nucleotides of variable length and composition ("phased inserts") at the beginning of each molecule when adding sequencing adaptors. This approach preserves read depth but reduces the length of each read. Here, we test the ability of phased inserts to replace PhiX in a low-diversity sample generated for a high-throughput sequencing based ribozyme activity screen. We designed a pool of 4096 RNA sequence variants of the self-cleaving twister ribozyme from Oryza sativa. For each unique sequence, we determined the fraction of ribozyme cleaved during in vitro transcription via deep sequencing on an Illumina MiSeq. We found that libraries with the phased inserts produced high-quality sequence data without the addition of PhiX. We found good agreement between previously published data on twister ribozyme variants and our data produced with phased inserts even when PhiX was omitted. We conclude that phased inserts can be implemented following in vitro selection experiments to reduce or eliminate the use of PhiX and maximize read depth.

RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes

“…Reverse transcription was carried out using a 5′ RACE protocol using phased template switching oligo’s (TSO1-4, Supplementary file 1 ) as described previously ( Bendixsen et al, 2020 ). Briefly, reverse transcription reactions used 5 pmol RNA and 20 pmol of reverse transcription primer in a volume of 10 μL.…”

Dynamic RNA Fitness Landscapes of a Group I Ribozyme during Changes to the Experimental Environment

“…Reverse transcription (Protoscript II, NEB) reactions were incubated at 42 °C for 1 h, then 65 °C for 25 min. Second-strand synthesis was carried out by low cycle PCR (10 cycles, KAPA HiFi, Roche) using a mixture of four different forward primers ( table 1 : Tas2.1a-phased 1-4) to add sequence diversity and NGS Unique Dual Index compatible sequence to the second strand ( Bendixsen et al 2020 ). This second-strand synthesis PCR product was purified with silica-based columns (DCC, Zymo) and amplified with Illumina compatible NGS Unique Dual Index Primers (IDT).…”

Experimental Resurrection of Ancestral Mammalian CPEB3 Ribozymes Reveals Deep Functional Conservation

“…4 µl SMARTScribe 5× First-Strand Buffer (Clontech), 2 µl dNTP (10 mM), 2 µl DTT (20 mM), 2 µl phased template switching oligo mix (10 µM), 1 µl water and 1 µl SMARTScribe Reverse Transcriptase (10 units, Clontech) were added. The phased template switching oligo mix consisted of four oligonucleotides that were phased by the addition of 9, 12, 15, or 18 nucleotides ( Bendixsen et al. 2020 ).…”