isONcorrect 0.0.3

De novo error-correction of long-read transcriptome reads.

isONcorrect

isONcorrect is a tool for error-correcting Oxford Nanopore cDNA reads. It is designed to handle highly variable coverage and exon variation within reads and achieves about a 0.5-1% median error rate after correction (see preprint for details). It leverages regions shared between reads from different isoforms achieve low error rates even for low abundant transcripts. See preprint for details.

Processing and error correction of full-length ONT cDNA reads is acheved by the pipeline of running pychopper --> isONclust --> isONcorrect

isONcorrect is distributed as a python package supported on Linux / OSX with python v>=3.4. .

Table of Contents

• INSTALLATION
  • Using conda
  • Using pip
  • Downloading source from GitHub
  • Dependencies
  • Testing installation
• USAGE
  • Running
  • Output
  • Parameters
• CREDITS
• LICENCE

INSTALLATION

Using conda

Conda is the preferred way to install isONcorrect.

1. Create and activate a new environment called isoncorrect

conda create -n isoncorrect python=3 pip 
source activate isoncorrect

2. Install isONcorrect and its dependency spoa.

pip install isONcorrect
conda install -c bioconda spoa

3. You should now have 'isONcorrect' installed; try it:

isONcorrect --help

Upon start/login to your server/computer you need to activate the conda environment "isonclust" to run isONcorrect as:

source activate isoncorrect

Using pip

pip is pythons official package installer and is included in most python versions. If you do not have pip, it can be easily installed from here and upgraded with pip install --upgrade pip.

To install isONcorrect, run:

pip install isONcorrect

Then install spoa.

Downloading source from GitHub

Dependencies

Make sure the below listed dependencies are installed (installation links below). Versions in parenthesis are suggested as isONcorrect has not been tested with earlier versions of these libraries. However, isONcorrect may also work with earliear versions of these libaries.

• spoa (1.1.5)
• edlib (1.1.2)
• NumPy (1.16.2)

In addition, please make sure you use python version >=3.

With these dependencies installed. Run

git clone https://github.com/ksahlin/isONcorrect.git
cd isONcorrect
./isONcorrect

Testing installation

You can verify successul installation by running isONcorrect on this small dataset. Simply download the test dataset and run:

isONcorrect --fastq [path to isONcorrect basefolder]/test_data/isoncorrect/0.fastq --outfolder [output path]

USAGE

Running

For a file with raw ONT cDNA reads the following pipeline is recommended (bash script provided below)

1. Get full-length ONT cDNA sequences produced by pychopper (a.k.a. cdna_classifier)
2. Cluster the full length reads where a cluster corresponds to a gene/gene-family
3. Make fastq files of each cluster
4. Correct individual clusters
5. Optional (join reads from separate clusters back to a single file)

Below shows specific pipeline script to go from raw reads raw_reads.fq to corrected full-length reads all_corrected_reads.fq (please modify/remove arguments as needed).

#!/bin/bash

# isonano pipeline to get high quality full length reads from transcriots

cdna_classifier.py  raw_reads.fq outfolder/reads_full_length.fq \
                      [-t cores]  [-w outfolder/rescued.fq  -u outfolder/unclassified.fq  -S outfolder/stats.txt] 

isONclust  --ont --fastq outfolder/reads_full_length.fq \
             --outfolder outfolder/clustering  [--t cores] 
isONclust write_fastq --N 1 --clusters outfolder/clustering/final_clusters.csv \
          --fastq reads_full_length.fq --outfolder  outfolder/clustering/fastq_files 
run_isoncorrect --fastq_folder outfolder/clustering/fastq_files  --outfolder /outfolder/correction/ 

# OPTIONAL BELOW 
touch all_corrected_reads.fq
for f in in outfolder/clustering/fastq_files/*/corrected_reads.fastq; 
do 
  cat {f} >> all_corrected_reads.fq
done

isONcorrect does not need ONT reads to be full-length (i.e., produced by pychopper), but unless you have specific other goals, it is advised to run pychopper for any kind of downstream analysis to guarantee full-length reads.

Output

The output of run_isoncorrect are one file per cluster with identical headers to the original reads.

CREDITS

Please cite [1] when using isONcorrect.

1. Kristoffer Sahlin, Botond Sipos, Phillip L James, Daniel Turner, Paul Medvedev (2020) Link.

Bib record:

LICENCE

GPL v3.0, see LICENSE.txt.