CIDER-Seq Data Analysis Software v.2.0

This repository contains data analysis software for CIDER-Seq (Circular DNA Enrichment Sequencing) including an implementation of the DeConcat algorithm for sequence de-concatenation and a new eccDNA detection module.

Update:

CIDER-Seq 2.0 comes with an additional eccDNA detection script. This script filters the output of the CIDER-Seq software (either cider-seq.py or cs-tools.py) and provides a list of likely eccDNA candidates and their source loci on the host genome. To run the script:

python3 cs-eccDNA.py [LIST-FILE] [GENOME-FILE]

The list file is a text file containing paths to the set of outputs generated by CIDER-Seq. These are a fasta file and a stat file, per sample. Fasta and stat files for multiple samples can include separate rows in the list files. These will be treated as biological replicates by the software. The genome file is a simple Fasta file for the relevant host genome.

Please cite:

Mehta D, Cornet L, Hirsch-Hoffmann M, Zaidi, SSA, Vanderschuren H (2020) Full-length sequencing of circular DNA viruses and extra-chromosomal circular DNA using CIDER-Seq. Nature Protocols, Volume 15, pages 1673–1689; https://doi.org/10.1038/s41596-020-0301-0

Mehta D, Hirsch-Hoffmann M, Were M, Patrignani A, Were H, Gruissem W, Vanderschuren H (2019) A new full-length circular DNA sequencing method for viral-sized genomes reveals that RNAi transgenic plants provoke a shift in geminivirus populations in the field. Nucleic Acids Research, Volume 47, Issue 2; https://doi.org/10.1093/nar/gky914

CODE:

Pseudocode / Algorithm Description:

Please refer to the Figures and Methods sections of Mehta et al., 2019 Nucleic Acids Research: https://doi.org/10.1093/nar/gky914

Table of contents

• Prerequisites
  • Python Modules
  • Standard Modules:
• Installation
• Structure
• Usage
  • For small datasets, or manual job handling
    • Options
  • For large datasets
    • COMMAND
    • Options
    • Typical Usage
• Input Files
• Output Files
• Config File
  • Explanation of name/value pairs
• License
• Disclamer
• References

Prerequisites

(All of the following are essential to run CIDER-Seq)

See howto python macOS or howto_python_LINUX.md for a brief guide to installing the following:

• Python 3
• BLAST
• MUSCLE

Python Modules

• NumPy
• Biopython **Note:**Please use Biopython version 1.77 or previous
• Click
• Matplotlib

Standard Modules:

(Should come pre-installed with Python)

• sys
• os
• logging
• tempfile
• uuid
• json

CIDER-Seq Installation

The following are minimal steps to install the package after prerequisites are fulfilled:

1. Clone/Download this Git repository onto your machine.
2. Create a copy of configuration file examples/ciderseq_config.json.
3. Edit the configuration file and set all necessary parameters (see Config File)
4. Run ciderseq example (see Usage)

Structure

The primary ciderseq.py script runs a pipeline on input sequence data consisting of the following process modules in order:

• Separation (cider/separate.py): Performs a BLASTn of input sequences against a user-defined dataset to bin reads into categories based on closed hit in the dataset. Non-hits are binned separately. This is useful in order to only process reads related to certain target sequences such as Virus A, Virus B and to filter out reads that do not match a target sequence(s). If switched off all the reads will be processed.

• Align (cider/align.py): An optional step which aligns reads against a reference sequence in order to trim sequence ends. Switch off if no reference sequence is available or if you wish to process all the reads in your input file (rather than just reads belonging to a specific target).

• DeConcat (cider/deconcat.py): The main de-contenation module. For a detailed description of the algorithm refer to Mehta et al., 2017, bioRxiv.

• Annotation (cider/annotate.py): This module annotates reads with gene/ORFs based on a reference dataset of proteins. It performs a tBLASTn to define the start and end sites of annotated genes/ORFs. It is capable of annotating circular sequences with genes crossing the sequence breakpoints.

• Phasing (cider/phase.py): Phasing ensures that all output sequences start at similar sites. This is essential for proper phylogenetic analysis. phase.py uses annotated sequences produced by annotate.py to phase genomes based on a user-defined phaseto gene. Hence, only annotated reads can be phased by this module.

Note that annotate.py and phase.py are designed to deal with circular DNA sequences. The modules cannot be executed directly but only via ciderseq.py script.

Usage

For regular users we recommend you ask your SMRT sequencing service provider to install CIDER-Seq on their computing cluster. If you have access to your own cluster or wish to process only a small dataset read on:

For small datasets, or manual job handling

The primary run command is:

python3 ciderseq.py [options] CONFIGFILE INPUTFILE

Options

The options allow the user to skip certain processes in the ciderseq.py pipeline:

--no-separation
--no-alignment
--no-deconcatenation
--no-annotation
--no-phasing

Additionally, the option --format allows you to choose the format of your input file from either fasta, fastq, tab or gb. The default option is fasta.

For example, for processing the files in examples/ we run:

python3 ciderseq.py --format fastq examples/ciderseq_config.json examples/example1.fastq

If we run:

python3 ciderseq.py --format fastq --no-separation --no-alignment examples/ciderseq_config.json examples/example1.fastq

the Separation and Alignment steps described above will be skipped.

Run python3 ciderseq.py --help for a brief description of usage and options.

For large datasets

We provide cstools.py in order to process large sequence datasets on a computing cluster (similar to the one your SMRT Analysis software is installed on). cstools.py is basically a file-handling program which will allow you to split your input sequences into batches and then join the outputs into a single results directory. Command plot will create a set of different plots based on the processed results, separation- and phase-results are required.

To run:

python3 cstools.py COMMAND [options] CONFIGFILE INPUTFILE

The CONFIGFILE and INPUTFILE are the same as the ones used by ciderseq.py. See below for details.

COMMAND

cstools.py has two primary actions: split and join which split input files into several jobs and then join the split jobs once processed. A third action called plot can be run after processing to produce DeConcat statistics. See examples/plots for examples of each chart.

Options

--format
--numjobs
--cluster
--clean

format: is the format of the input file, default FASTA

numjobs: is the number of processing jobs to create. the sequences equally distributed to the provides number of jobs. The default is 1.

cluster: contains cluster submission parameters, e.g. "bsub -n 4" in a LSF environment.

clean: if absent, the folders with the split data will not be deleted. Useful for debugging.

Typical Usage

The typical run command is:

python3 cstools.py split --format fastq --numjobs 8 --cluster "bsub -n 1" examples/ciderseq_config.json examples/example1.fastq

The split command outputs all necessary execution commands and ask confirmation before executing them.

followed by (for joining):

python3 cstools.py join --clean examples/ciderseq_config.json examples/example1.fastq

The clean option will remove the folder and files crated during split.

And (for plotting):

python3 cstools.py plot examples/ciderseq_config.json examples/example1.fastq

Input Files

There are only two input files required:

• CONFIGFILE: file containing editable parameters to customise your CIDER-Seq run. (see below for details)

• INPUTFILE: file containing your raw sequence data in a Seq.IO compatible format (fasta, fastq, tab, gb)

Output Files

All output files are saved in the directory specified in the CONFIGFILE. We recommend you create separate folders for each step defined in the CONFIGFILE.

See our example file examples/ciderseq_config.json for details.

Config File

ciderseq.py reads configuration parameters from the provided CONFIGFILE. see examples/ciderseq_config.json for an example.

Please read on for a detailed explanation of how to edit the CONFIGFILE file:

We recommend using examples/ciderseq_config.json and editing the values for your analysis.

There are several options available to modify in the CONFIGFILE.

Essential changes that you will almost certainly need to make are highlighted in green in the images below.

Overall Settings

Configuring separate.py:

Configuring align.py:

Configuring deconcat.py:

Configuring annotate.py:

Configuring phase.py:

Configuring Phasing:

A more detailed explanation of name/value pairs

• loglevel : "DEBUG", will write log information into output dir

• outputdir : "destination of log information"

• separate (settings used in cider/separate.py)

  • outputdir : "output directory of results"
  • blastinit : "python code to initialize blast environment, if necessary"
  • blastexe : "blast executable"
  • blastndb : "location of the indexed blast database"
  • evalue : "used evalue for blastn", suggested="1"

• align (settings used in cider/align.py)

  • outputdir : "output directory of results"
  • muscleinit : "python code to initialize muscle environment, if necessary"
  • muscleexe : "muscle executable"
  • targets : (array of align target DNA fasta files)
    • "name of genome" : "path to DNA fasta file"
  • windowsize : "size of the sliding window", suggested="10"

• deconcat (settings used in cider/deconcat.py)

  • outputdir : "output directory of results"
  • muscleinit : "python code to initialize muscle environment, if necessary"
  • muscleexe : "muscle executable"
  • fragmentsize : "size of de-concatenation fragment", recommended="30"
  • statistics : (write statistic output) ("1"=on, "0"=off)

• annotate (settings used in cider/annotate.py)

  • outputdir : "output directory of results"
  • blastinit : "python code to initialize blast environment, if necessary"
  • blastexe : "blast executable"
  • tblastndb : "location of the indexed blast database"
  • evalue : "used evalue for tblastn", suggested="0.01"

• phase (settings used in cider/phase.py)

  • outputdir : "output directory of results"
  • outputformat : (format of results as array, use SeqIO valid formats) e.g. ["genbank","fasta"]=outputs both .gb and .fasta formats
  • phasegenomes : (array of genomes for phasing)
    • "name of genome" : (array of phasing parameters)
      • proteins : array of proteins in genome
        • "name of protein" : (protein strand information)
          • "strand" : "1"=forward strand, "-1"=reverse strand"
      • phaseto : "name of protein to set sequence start position"
      • offset : (offset to start before protein position) if "10", phaseto protein will start at position 10

License

Copyright © 2019, Matthias Hirsch-Hoffmann and Devang Mehta. CIDER-Seq is licensed under the GNU General Public License, version 3 (https://www.gnu.org/licenses/gpl-3.0.en.html) or see LICENSE.txt.

Disclamer

This software is supplied 'as is' without any warranty or guarantee of support. The developers are not responsible for its use, misuse, or functionality. In no event shall the authors or copyright holders be liable for any claim, damages, or other liability arising from, out of, or in connection with this software.

References

Hunter JD (2007) Matplotlib: A 2D graphics environment. Computing in Science & Engineering 9:3 90-95 doi:10.5281/zenodo/573577