DOI: 10.5061/dryad.tht76hf10
title: Deep sequencing data for document titled: Rolling circle RNA synthesis catalysed by RNA

description: RNA-catalysed RNA replication is widely considered a key step in the
 emergence of life’s first genetic system. However, RNA replication can be
 impeded by the extraordinary stability of duplex RNA products, which must
 be dissociated for re-initiation of the next replication cycle. Here we
 have explored rolling circle synthesis (RCS) as a potential solution to
 this strand separation problem. RCS on small circular RNAs - as indicated
 by molecular dynamics simulations - induces a progressive build-up of
 conformational strain with destabilisation of nascent strand 5’ and 3’
 ends. At the same time, we observe sustained RCS by a triplet polymerase
 ribozyme on small circular RNAs over multiple orbits with strand
 displacement yielding concatemeric RNA products. Furthermore, we show RCS
 of a circular Hammerhead ribozyme capable of self-cleavage and
 re-circularisation. Thus, all steps of a viroid-like RNA replication
 pathway can be catalysed by RNA alone. Our results have implications for
 the emergence of RNA replication and for understanding the potential of
 RNA to support complex genetic processes.
description: RNA from primer extension reactions catalysed by the triplet
 polymerase ribozyme were PAGE purified, adaptor ligated and prepared for
 illumina sequencing as described in the main text.
 Illumina Sequencing data were acquired and processed as FASTQ
 files using Terminal (and available software packages such as
 FASTX-toolkit). Prior to analysis the whole output file form illumina
 sequencing runs (containing also unrelated sequences) was split based on
 barcodes identifying the individual samples and trimmed starting with the
 original (P9<sub>1</sub>) primer sequence (GAAGAACTG). After
 the primer sequence, the triplets at positions 1, 2 ,3 etc. would be
 identifiable representing extension products made by the ribozyme. The
 presence for the 3’ adapter sequence (GTCGAATAT…) in the aligned sequences
 marked the end of the original RNA extension product.
 File 1 include the split and trimmed sequence data for circular
 and linear one-pot analysis (Circular_onepot and Linear_onepot,
 respectively), File 2 include the split and trimmed sequence data for
 branched RCS analysis (B3)).
description: File_1_2_ReadMe.txt (uploaded to Zenodo) describes the simple
 code executed to analyze the deep-sequencing datasets by counting the
 number of times the sequences in the List-files was present.
 File_1 includes deep-sequencing datasets from sequencing of the
 circular and linear onepot reactions (Circular_onepot.fastq and
 Linear_onepot.fastq), the list (List.txt) containing the sequences that
 was counted in the deep-sequencing datasets to allow analysis, and the
 results (results_circular and results_linear) containing the number of
 times (count) each sequence in the list appered in the deep-sequencing
 datasets (arranged in the same order as the corresponding sequence in
 List.) File_2 includes deep-sequencing dataset from
 sequencing of the branched RCS reaction (B3.fastq), the list (List.txt)
 containing the sequences that was counted in the deep-sequencing datasets
 to allow analysis, and the results (result) containing the number of times
 (count) each sequence in the list appered in the deep-sequencing datasets
 (arranged in the same order as the corresponding sequence in
 List.)