We benchmarked the ability of novel base calling models to detect RNA modifications from native RNA reads generated on the Oxford Nanopore Technologies platform. The tested base calling models are listed below:
| Model (short) | Model (long) | Software used for training | Training data | Model type | Model size (MB) | Median accuracy (human) | Distribution | Basecaller Support |
|---|---|---|---|---|---|---|---|---|
| default | rna_r9.4.1_70bps_hac | tayaki | not disclosed | flip-flop | 1.99 | 91% | guppy v6.0.6 | all guppy versions |
| IVT | rna_r9.4.1_70bps_ivt_hac | tayaki | Nanopore-WGS-Consortium | flip-flop | 1.99 | 88% | this work | all guppy versions |
| SUP | rna_r9.4.1_70bps_sup | bonito | PRJEB40872 | CRF-CTC | 27 | 97% | this work | guppy v6.0.6 and upwards |
| IVT_SUP | rna_r9.4.1_70bps_ivt_sup | bonito | Nanopore-WGS-Consortium | CRF-CTC | 27 | 97% | this work | guppy v6.0.6 and upwards |
All files required to run guppy (v6.0.6) with these models can be
downloaded from OSF. Code required to reproduce
published results can be found in /scripts
When using guppy standalone make sure version 6.0.6 or higher is installed on your system. Next, download the files of each model from OSF. This should include two files per model:
rna_r9.4.1_70bps_<model>.cfg
template_rna_r9.4.1_70bps_<model>.jsnMake sure to place these files in the following folder within guppy
~/ont-guppy_<version>/data/In order to run them use the -c [ --config ] arg flag and specify the
desired config file
guppy_basecaller –i ./fast5 –s ./guppy_out -c rna_r9.4.1_70bps_<model>.cfg --num_callers 2 --cpu_threads_per_caller 1For use within the MasterofPores nextflow pipleline download the files
specified above into the mop_preprocess module as follows:
~/MOP2/mop_preprocess/bin/ont-guppy_<version>/data/specify them in the appropriate config file (either
drna_tool_splice_guppy6_opt.tsv or
drna_tool_unsplice_guppy6_opt.tsv) found in
~/mop_preprocess/tools_opts. An example file is shown below:
#step tool extrapars
demultiplexing deeplexicon ""
demultiplexing guppy ""
filtering nanofilt ""
basecalling guppy "-c rna_r9.4.1_70bps_<model>.cfg --disable_qscore_filtering"
filtering nanoq ""
mapping graphmap ""
mapping graphmap2 "-x rnaseq"
mapping minimap2 "-uf -ax splice -k14"
mapping bwa ""
counting htseq "-a 0"
counting nanocount ""
discovery bambu ""Running mop_preprocess from there as specified in the pipelines documentation will use the provided basecalling model.
| Software | Version |
|---|---|
| MasterOfPores | 2.0 |
| guppy | 6.0.6 |
| minimap2 | 2.17 |
| graphmap | 0.5.2 |
| EpiNano | 1.1 |
| singularity | 3.2.1 |
| nextflow | 21.04.3 |
| CUDA | 11 |
Packages required ro run each script are further specified in that script.
If you find this work useful, please cite:
Diensthuber G.*, Pryszcz L.*, Llovera L., Lucas MC., Delgado-Tejedor A, Cruciani S., Roignant JY., Begik 0. and Novoa EM.: Enhanced detection of RNA modifications and mappability with high-accuracy nanopore RNA basecalling models. bioRXiv 2023. doi: https://www.biorxiv.org/content/10.1101/2023.11.28.568965v1