nano-rave

Nextflow pipeline designed for rapid onsite QC and variant calling of Oxford Nanopore data (following basecalling and demultiplexing with Guppy).

Pipeline summary

Getting started

Running on a personal computer

1. Install nextflow.

2. Install Docker.

3. Run pipeline direct from Github repository.

   Example:

   nextflow run github.com/sanger-pathogens/nano-rave --sequencing_manifest ./test_data/pipeline/inputs/test_manifest.csv --reference_manifest ./test_data/pipeline/inputs/reference_manifest.csv --variant_caller medaka_haploid --min_barcode_dir_size 5 --results_dir my_output

   See usage for all available pipeline options.

4. Once your run has finished, check output in the results_dir and clean up any intermediate files. To do this (assuming no other pipelines are running from the current working directory) run:

   rm -rf work .nextflow*

Running on the farm (Sanger HPC clusters)

1. Add the following line to ~/.bashrc (if not already present):

   [[ -f  /software/pathogen/farm5 ]] && source /software/pathogen/etc/pathogen.profile

2. Source the updated .bashrc file

   source ~/.bashrc

3. Load the module

   module load nano-rave/<version>

4. The pipeline should now be directly available with the command nano-rave

   nano-rave --help

5. before excuting nano-rave, it is recommended to set the $SINGULARITY_CACHEDIR and $NXF_SINGULARITY_CACHEDIR environment variables so that they both point to a folder with enough space. This location is that one where singularity images supporting the pipeline dependencies will be downloaded; by default it is downloaded inside your home directory (spcifically in ${HOME}/.singularity/cache), which has space limitations and will rapidly fill up, causing the pipeline to fail. On the Sanger HPC, it is recommended to point to a location on your lustre scratch space.

6. Start your analysis

   To use the appropriate Sanger configuration, please run with -profile sanger_local option. Here is an example command:

   bsub -o nano-rave.o -e nano-rave.e -q long -n 4 -R "select[mem>16000] rusage[mem=16000]" -M16000 \
   nano-rave -profile sanger_local --sequencing_manifest ./test_data/pipeline/inputs/test_manifest.csv --reference_manifest ./test_data/pipeline/inputs/reference_manifest.csv --variant_caller medaka_haploid --min_barcode_dir_size 5 --results_dir my_output

   This will run the whole pipeline i.e. all per-sample processes within a single siubmitted job, so please tailor your resource request accordingly.

   NB: we are working on providing a sanger_lsf profile that will enable th e proper use of LSF cluster integration, meaning that each process is executed by submitting as a separate job on the HPC; under such configuration, you would be advised to submitted the main job (workflow head process) to the oversubscribed queue.

   See usage for all available pipeline options.

7. Once your run has finished, check output in the results_dir and clean up any intermediate files. To do this (assuming no other pipelines are running from the current working directory) run:

   rm -rf work .nextflow*

Usage

nextflow run main.nf

Options:
    --sequencing_manifest        Manifest containing paths to sequencing directories and sequencing summary files (mandatory)
    --reference_manifest         Manifest containing reference identifiers and paths to fastq reference files (mandatory)
    --results_dir                Specify results directory [default: ./nextflow_results] (optional)
    --variant_caller             Specify a variant caller to use [medaka (default), medaka_haploid, freebayes, clair3] (optional)
    --clair3_args                Specify clair3 variant calling parameters - must include model e.g. --clair3_args "--model_path /opt/models/r941_prom_sup_g5014" (optional)
    --min_barcode_dir_size       Specify the expected minimum size of the barcode directories, in MB. Must be > 0. [default: 10] (optional)
    --keep_bam_files             Save BAM files in results directory [default: false] (optional)
    --help                       Print this help message (optional)

Note:

• Please refer to https://github.com/HKU-BAL/Clair3#usage for a comprehesive list of options that can be used with --clair3_args. Currently, by default, the software will assume you will want to variant call human chromosome contigs. If this is not the case, or you wish to use a custom set of contigs, please see clair3 options --include_all_ctgs or --ctg_name. You may also want to skip phasing with clair3 option --no_phasing_for_fa if this is not required or useful for you.

Sequencing manifest format

The sequencing manifest is in a csv format and contains two columns

• sequencing_dir : folder containing all the Oxford Nanopore sequencing data
• sequence_summary_file : required for QC - usually found in the sequencing directory. In this file, the paths to the fast5 read files (first column) must be full/absolute paths.

The pipeline assumes that sequencing_dir contains Guppy output for a particular sample. In particular, the parent and child folders of the given sequencing_dir assume the following structure:

<sample>/<sequencing_dir>/fastq_pass/barcode*

Where each barcode* directory contains fastq.gz files. Only barcode* directories whose total size on disk exceeds the threshold set with --min_barcode_dir_size are considered.

Example manifest:

sequencing_dir,sequence_summary_file
./test_data/PIPELINE/inputs/sample/sequencing_dir,./test_data/PIPELINE/inputs/sample/sequencing_dir/sequencing_summary.txt

Note: When using relative paths in the manifest, they are relative to the current working directory (from which nextflow is run).

Reference manifest format

The reference manifest is in csv format and contains two columns

• reference_id : identifier for the reference (e.g. gene name or reference genome name)
• reference_path : path to the reference file (fasta format)

Example for amplicon data:

reference_id,reference_path
ama1,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_ama1.fasta
crt,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_crt.fasta
csp,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_csp.fasta
dhfr,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_dhfr.fasta
dhps,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_dhps.fasta
eba175_3d7,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_eba175_3d7.fasta
k13,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_k13.fasta
mdr1,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_mdr1.fasta
msp1,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_msp1.fasta
msp2,./test_data/PIPELINE/inputs/references/ref_target_gene_cds_seq_msp2.fasta

Note: When using relative paths in the manifest, they are relative to the current working directory (from which nextflow is run).

Variant callers

Three variant callers are currently supported:

• medaka : See medaka_variant usage
• medaka_haploid : See medaka_haploid_variant usage
• freebayes : See freebayes usage
• clair3 : See run_clair3.sh usage

Software versions

The pipeline makes use of docker images to ensure reproducibility. This version of the pipeline uses the following software dependencies:

Software	Version	Image URL
bedtools	2.29.2	quay.io/biocontainers/bedtools:2.29.2--hc088bd4_0
clair3	1.0.0	docker.io/hkubal/clair3@sha256:3c4c6db3bb6118e3156630ee62de8f6afef7f7acc9215199f9b6c1b2e1926cf8
freebayes	1.3.5	docker.io/gfanz/freebayes@sha256:d32bbce0216754bfc7e01ad6af18e74df3950fb900de69253107dc7bcf4e1351
medaka	1.4.4	quay.io/biocontainers/medaka:1.4.4--py38h130def0_0
minimap2	2.17	quay.io-biocontainers-minimap2:2.17--hed695b0_3
nanoplot	1.38.0	quay.io/biocontainers/nanoplot:1.38.0--pyhdfd78af_0
pycoqc	2.5.2	quay.io/biocontainers/pycoqc:2.5.2--py_0
samtools	1.15.1	quay.io/biocontainers/samtools:1.15.1--h1170115_0
tabix	1.11	quay.io/biocontainers/tabix:1.11--hdfd78af_0

Contributions and testing

Developer contributions to this pipeline will only be accepted if all pipeline tests pass. To check:

1. Make your changes.

2. Download the test data. A utility script is provided:

   python3 scripts/download_test_data.py
   

3. Install nf-test (>=0.7.0) and run the tests:

   nf-test test tests/*.nf.test
   

   If running on Sanger HPC cluster, add the option --profile sanger_local.

4. Submit a PR.

Citations

If you use this pipeline for your analysis, please cite our paper:

Drug resistance and vaccine target surveillance of Plasmodium falciparum using nanopore sequencing in Ghana

Sophia T. Girgis, Edem Adika, Felix E. Nenyewodey, Dodzi K. Senoo Jnr, Joyce M. Ngoi, Kukua Bandoh, Oliver Lorenz, Guus van de Steeg, Alexandria J. R. Harrott, Sebastian Nsoh, Kim Judge, Richard D. Pearson, Jacob Almagro-Garcia, Samirah Saiid, Solomon Atampah, Enock K. Amoako, Collins M. Morang’a, Victor Asoala, Elrmion S. Adjei, William Burden, William Roberts-Sengier, Eleanor Drury, Megan L. Pierce, Sónia Gonçalves, Gordon A. Awandare, Dominic P. Kwiatkowski, Lucas N. Amenga-Etego & William L. Hamilton

Nature Microbiology 8:2365–2377 (2023); doi: 10.1038/s41564-023-01516-6.

Which was initially released as a pre-print:

Nanopore sequencing for real-time genomic surveillance of Plasmodium falciparum

Sophia T. Girgis, Edem Adika, Felix E. Nenyewodey, Dodzi K. Senoo Jnr, Joyce M. Ngoi, Kukua Bandoh, Oliver Lorenz, Guus van de Steeg, Sebastian Nsoh, Kim Judge, Richard D. Pearson, Jacob Almagro-Garcia, Samirah Saiid, Solomon Atampah, Enock K. Amoako, Collins M. Morang’a, Victor Asoala, Elrmion S. Adjei, William Burden, William Roberts-Sengier, Eleanor Drury, Sónia Gonçalves, Gordon A. Awandare, Dominic P. Kwiatkowski, Lucas N. Amenga-Etego, William L. Hamilton

bioRxiv 2022.12.20.521122; doi: 10.1101/2022.12.20.521122

This pipeline was adapted from the nf-core/nanoseq pipeline.

A systematic benchmark of Nanopore long read RNA sequencing for transcript level analysis in human cell lines

Ying Chen, Nadia M. Davidson, Yuk Kei Wan, Harshil Patel, Fei Yao, Hwee Meng Low, Christopher Hendra, Laura Watten, Andre Sim, Chelsea Sawyer, Viktoriia Iakovleva, Puay Leng Lee, Lixia Xin, Hui En Vanessa Ng, Jia Min Loo, Xuewen Ong, Hui Qi Amanda Ng, Jiaxu Wang, Wei Qian Casslynn Koh, Suk Yeah Polly Poon, Dominik Stanojevic, Hoang-Dai Tran, Kok Hao Edwin Lim, Shen Yon Toh, Philip Andrew Ewels, Huck-Hui Ng, N.Gopalakrishna Iyer, Alexandre Thiery, Wee Joo Chng, Leilei Chen, Ramanuj DasGupta, Mile Sikic, Yun-Shen Chan, Boon Ooi Patrick Tan, Yue Wan, Wai Leong Tam, Qiang Yu, Chiea Chuan Khor, Torsten Wüstefeld, Ploy N. Pratanwanich, Michael I. Love, Wee Siong Sho Goh, Sarah B. Ng, Alicia Oshlack, Jonathan Göke, SG-NEx consortium

bioRxiv 610741; doi: 10.1101/610741

A full list of citations for tools used in the pipeline is given in CITATIONS.md

Copyright

Copyright (C) 2022,2023 Genome Research Ltd.