# rnaSPAdes manual

1. About rnaSPAdes
2. rnaSPAdes specifics
2.1. Running rnaSPAdes
2.2. rnaSPAdes output
3. Assembly evaluation
4. Citation
5. Feedback and bug reports

## 1 About rnaSPAdes

rnaSPAdes is a tool for de novo transcriptome assembly from RNA-Seq data and is suitable for all kind of organisms. rnaSPAdes is a part of SPAdes package since version 3.9. Information about SPAdes download, requirements, installation and basic options can be found in SPAdes manual. Below you may find information about differences between SPAdes and rnaSPAdes.

## 2 rnaSPAdes specifics

### 2.1 Running rnaSPAdes

To run rnaSPAdes use

        rnaspades.py [options] -o <output_dir>

or

        spades.py --rna [options] -o <output_dir>

Note that we assume that SPAdes installation directory is added to the `PATH` variable (provide full path to rnaSPAdes executable otherwise: `<rnaspades installation dir>/rnaspades.py`).

Here are several notes regarding options :

- rnaSPAdes can take as an input only one paired-end library and multiple single-end libraries.
- rnaSPAdes does not support `--careful` and `--cov-cutoff` options.
- rnaSPAdes is not compatible with other pipeline options such as `--meta`, `--sc` and `--plasmid`.
- rnaSPAdes works using only a single k-mer size (55 by the default). We strongly recommend not to change this parameter. In case your RNA-Seq data set contains long Illumina reads (150 bp and longer) you may try to use longer k-mer size (approximately half of the read length). In case you have any doubts about your run, do not hesitate to contact us using e-mail given below.

### 2.2 rnaSPAdes output

rnaSPAdes outputs only one FASTA file named `transcripts.fasta`. The corresponding file with paths in the `assembly_graph.fastg` is `transcripts.paths`.

Contigs/scaffolds names in rnaSPAdes output FASTA files have the following format:
`>NODE_97_length_6237_cov_11.9819_g8_i2`
Similarly to SPAdes, `97` is the number of the transcript, `6237` is its sequence length in nucleotides and `11.9819` is the k-mer coverage. Note that the k-mer coverage is always lower than the read (per-base) coverage. `g8_i2` correspond to the gene number 8 and isoform number 2 within this gene. Transcripts with the same gene number are presumably received from same or somewhat similar (e.g. paralogous) genes. Note, that the prediction is based on the presence of shared sequences in the transcripts and is very approximate.

## 3 Assembly evaluation

rnaQUAST may be used for transcriptome assembly quality assessment for model organisms when reference genome and gene database are available. rnaQUAST also includes BUSCO and GeneMarkS-T tools for de novo evaluation.

## 4 Citation

If you use rnaSPAdes in your research, please include main SPAdes paper Bankevich, Nurk et al., 2012 in your reference list. Paper on rnaSPAdes is to be submitted.

## 5 Feedback and bug reports

Your comments, bug reports, and suggestions are very welcomed. They will help us to further improve rnaSPAdes.

If you have any troubles running rnaSPAdes, please send us `params.txt` and `spades.log` from the directory `<output_dir>`.

Address for communications: spades.support@cab.spbu.ru.