NextSV3: automated structrual variation detection from long-read sequencing using state-of-the-art tools

NextSV3 uses Minimap2/Winnowmap/NGMLR to do read mapping and uses two state-of-the-art SV callers (Sniffles2 and cuteSV2) to do SV calling.

Table of Contents

• Table of Contents
• Installation
• Usage
  • Aligners
  • Quick Example
  • Output files
  • Full Usage
• Contact
• Citation
• Included tools

Installation

Prerequisites

• Operating System: Linux
• Python 3.8 or later
• samtools (v1.9 or later)
• sniffles (v2.0.7 or later)
• cuteSV (v2.0.1 or later)

Conda environment is not required but we highly recommend you install the prerequisites in a new conda environment to avoid potential dependency issues.

conda create -n nextsv3 python=3.8
conda activate nextsv3
pip install "setuptools<58.0"
pip install sniffles
pip install cuteSV

Installation of NextSV3

If your OS is Linux, you can acquire precompiled binaries from the release page with:

wget https://github.com/Nextomics/nextsv/releases/download/v3.2.0/NextSV-3.2.0-linux-x86_64.tar.gz
tar xzf NextSV-3.2.0-linux-x86_64.tar.gz

If you want to compile from the source code, you need to have the following tools/libraries installed:

• GCC (v7.0 or later)
• GNU make
• CMake
• zlib development files

You may encounter problems during the compilation process if your operating system is missing any of the above tools/libraries, and we strongly recommend using our pre-compiled binaries if your operating system is Linux.

To compile NextSV from source:

git clone https://github.com/Nextomics/nextsv.git
cd nextsv
make

Usage

Aligners

The default aligner is minimap2 as it is faster. But you can use the -a/--aligners argument to specifiy aligners. You can use any combination of the following 3 aligners: minimap2, winnowmap, ngmlr (separated by the + sign):

# use minimap2 only (default) 
-a minimap2

# use minimap2 and winnowmap
-a minimap2+winnowmap

# use minimap2, winnowmap and ngmlr
-a minimap2+winnowmap+ngmlr

# use winnowmap and ngmlr
-a winnowmap+ngmlr

# use minimap2 and ngmlr
-a minimap2+ngmlr

# use ngmlr only
-a ngmlr

# use minimap2 only
-a minimap2

Please note that if you use more aligners, it would take more time to finish the pipeline.

Quick Example

# PacBio HiFi reads
python path/to/nextsv3.py -a minimap2+winnowmap+ngmlr -i path/to/fastq/folder/ -o ./nextsv_output -s unique_sample_name -r path/to/hg38.fasta -t 8 -p hifi -e nextsv3

# PacBio CLR reads
python path/to/nextsv3.py -a minimap2+winnowmap+ngmlr -i path/to/fastq/folder/ -o ./nextsv_output -s unique_sample_name -r path/to/hg38.fasta -t 8 -p clr -e nextsv3

# Oxford Nanopore reads
python path/to/nextsv3.py -a minimap2+winnowmap+ngmlr -i path/to/fastq/folder/ -o ./nextsv_output -s unique_sample_name -r path/to/hg38.fasta -t 8 -p ont -e nextsv3

Memory consumption of the pipeline depends on number of threads and the size of the reference genome. For human genomes (3Gb), we recommend 4GB memory per thread.

Output files

NextSV will generate a work.sh in the output directory. Run this work.sh and you will get output files. SV calls of sniffles and cuteSV will be generated in the out_dir/3_SV_calls folder.

Full Usage

usage: nextsv3.py [-h] -i path/to/input_dir -o path/to/output_dir -s sample_name -r ref.fasta -p sequencing_platform -a aligners_to_use [-t INT]
                  [-e conda_env] [--samtools path/to/samtools] [--sniffles path/to/sniffles] [--cuteSV path/to/cuteSV]
                  [--minimap2 path/to/minimap2] [-v]

nextsv3: an automated pipeline for structrual variation detection from long-read sequencing. Contact: Li Fang([email protected])

options:
  -h, --help            show this help message and exit
  -i path/to/input_dir, --in_dir path/to/input_dir
                        (required) path to input FASTQ/FASTA directory (all fastq/fasta files in this directory will be used as input files)
  -o path/to/output_dir, --out_dir path/to/output_dir
                        (required) path to output fastq directory
  -s sample_name, --sample_name sample_name
                        (required) a unique name or id for the input sample
  -r ref.fasta, --ref_fasta ref.fasta
                        (required) path to reference genome sequence in FASTA format
  -p sequencing_platform, --platform sequencing_platform
                        (required) sequencing platform. Three valid values: ont/clr/hifi ont: oxford nanopore; clr: PacBio CLR; hifi: PacBio
                        HiFi/CCS
  -a aligners_to_use, --aligners aligners_to_use
                        (optional) which aligner(s) to use. Three supported aligners: minimap2, ngmlr, winnowmap. Use "+" to combine multiple
                        aligners. Examples: minimap2+ngmlr, winnowmap+minimap2, minimap2+ngmlr+winnowmap, minimap2, ngmlr, winnowmap
  -t INT, --threads INT
                        (optional) number of threads (default: 4)
  -e conda_env, --conda_env conda_env
                        (optional) conda environment name (default: NULL)
  --samtools path/to/samtools
                        (optional) path to samtools (default: using environment default)
  --sniffles path/to/sniffles
                        (optional) path to sniffles (default: using environment default)
  --cuteSV path/to/cuteSV
                        (optional) path to cuteSV (default: using environment default)
  --minimap2 path/to/minimap2
                        this parameter is deprecated as minimap2 is supplied in NextSV now
  -v, --version         show version number and exit

Contact

If you have any questions/suggestions, please feel free to post it on the Issue page. You may also email me at [email protected].

Citation

NextSV

Fang, L., Hu, J., Wang, D. et al. NextSV: a meta-caller for structural variants from low-coverage long-read sequencing data. BMC Bioinformatics 19, 180 (2018). https://doi.org/10.1186/s12859-018-2207-1

Minimap2

Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics, 34:3094-3100 (2018). https://doi.org/10.1093/bioinformatics/bty191

Winnowmap

Jain, C., Rhie, A., Hansen, N.F., Koren, S., and Phillippy, A.M. (2022). Long-read mapping to repetitive reference sequences using Winnowmap2. Nat Methods 19, 705-710. https://doi.org/10.1038/s41592-022-01457-8

Jain, C., Rhie, A., Zhang, H., Chu, C., Walenz, B.P., Koren, S., and Phillippy, A.M. (2020). Weighted minimizer sampling improves long read mapping. Bioinformatics 36, i111-i118. https://doi.org/10.1093/bioinformatics/btaa435

NGMLR and Sniffles

Sedlazeck, F.J., Rescheneder, P., Smolka, M. et al. Accurate detection of complex structural variations using single-molecule sequencing. Nat Methods 15, 461–468 (2018). https://doi.org/10.1038/s41592-018-0001-7

Smolka, M., Paulin, L.F., Grochowski, C.M., Mahmoud, M., Behera, S., Gandhi, M., Hong, K., Pehlivan, D., Scholz, S.W., Carvalho, C.M.B., et al. (2022). Comprehensive Structural Variant Detection: From Mosaic to Population-Level. bioRxiv. https://doi.org/10.1101/2022.04.04.487055

CuteSV

Jiang, T., Liu, Y., Jiang, Y. et al. Long-read-based human genomic structural variation detection with cuteSV. Genome Biol 21, 189 (2020). https://doi.org/10.1186/s13059-020-02107-y

Cao, S., Jiang, T., Liu, Y., Liu, S., and Wang, Y. (2022). Re-genotyping structural variants through an accurate force-calling method. bioRxiv. https://doi.org/10.1101/2022.04.04.487055

Samtools

Petr Danecek, James K Bonfield, Jennifer Liddle, John Marshall, Valeriu Ohan, Martin O Pollard, Andrew Whitwham, Thomas Keane, Shane A McCarthy, Robert M Davies, Heng Li, Twelve years of SAMtools and BCFtools, GigaScience, Volume 10, Issue 2, February 2021, giab008, https://doi.org/10.1093/gigascience/giab008

Open source software tools included in this repository

To provide consistent output and better user experience, we included some open source software tools.

LongReadQC https://github.com/fangli80/longreadqc

Seqtk https://github.com/lh3/seqtk

Minimap2 https://github.com/lh3/minimap2

NGMLR https://github.com/philres/ngmlr

pigz https://github.com/madler/pigz

Winnowmap https://github.com/marbl/Winnowmap