Bovreproseq

Bioinformatics pipeline for targeted syndromic next-generation sequencing panel for simultaneous detection of pathogens associated with bovine reproductive failure
For more info please see https://doi.org/10.1128/jcm.01433-24
Outputs html files with results, consensus sequences, metagenomics reports (Kraken2/Bracken/Blast), Krona plots, IGV reports and MultiQC quality reports.

Usage

nextflow run main.nf \
    --input <path_to_fastq_directory> \
    --out_dir <output_directory> \
    --kraken_db <path_to_kraken2_db> \
    --blastdb_path <path_to_blast_db> \
    --blastdb_name <blast_db_name>

Parameters

Required

• --input: Path to input directory containing FASTQ files (can be gzipped).
• --out_dir: Directory where results will be saved.
• --kraken_db: Path to the Kraken2 database directory.
• --blastdb_path: Path to the directory containing BLAST databases.
• --blastdb_name: Name of the BLAST database to use.

Optional

• --dehost: Enable host read filtering (default: true).
• --host_db: Path to kraken2 database with Bos taurus reference genome FASTA (required if --dehost is true).
• --qscore: Minimum mean Q-score filter for reads (default: 10).
• --trim_barcodes: Enable barcode and adapter trimming using Porechop (default: false).
• --read_count_threshold: Threshold for minimum read count for consensus generation amplicons (default: 10).
• --consensus_mode: Mode for consensus generation with samtools('simple' or 'bayesian').

Workflow Description

1. QC & Preprocessing: Merges FASTQ files, filters by Q-score (Nanoq), and optionally trims barcodes (Porechop). Generates QC metrics (NanoPlot).
2. Dehosting (Optional): Uses kraken2 filter out Bos taurus reads
3. Targeted Amplicon Analysis :
   • Aligns reads to the Bovreproseq_reference.fasta.
   • Generates consensus sequences using minimap2, samtools and medaka.
   • Identifies pathogens in the sample using Abricate.
   • Performs MLST typing to get subspecies ID of Campylobacter fetus.
   • Generates IGV HTML reports for visual inspection of alignments.
4. Metagenomics:
   • Classifies reads using Kraken2 and estimates abundance with Bracken.
   • Visualizes taxonomy with Krona.
   • Validation of hits using BLAST against specified databases.
5. Reporting:
   • Consolidates results into an interactive HTML report.
   • Generates a separate Metagenomics report.
   • MultiQC report for run statistics.

Dependencies

• nextflow
• docker
• wsl2 (if on Windows)

Software Used

• nanoq: Steinig, E., & Coin, L. (2022). Nanoq: ultra-fast quality control for nanopore reads. Journal of Open Source Software, 7(69), 2991. https://doi.org/10.21105/joss.02991
• NanoPlot: De Coster, W., D'Hert, S., & Schultz, D. T. (2018). NanoPack: visualizing and processing long-read sequencing data. Bioinformatics, 34(12), 2066–2068. https://doi.org/10.1093/bioinformatics/bty149
• porechop: Wick, R. R. (2017). Porechop: adapter trimmer for Oxford Nanopore reads. https://github.com/rrwick/Porechop
• minimap2: Li, H. (2018). Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics, 34(18), 3094–3100. https://doi.org/10.1093/bioinformatics/bty191
• kraken2: Wood, D. E., Lu, J., & Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biology, 20, 257. https://doi.org/10.1186/s13059-019-1891-0
• bracken: Lu, J., Breitwieser, F. P., Thielen, P., & Salzberg, S. L. (2017). Bracken: estimating species abundance in metagenomics data. PeerJ Computer Science, 3, e104. https://doi.org/10.7717/peerj-cs.104
• KrakenTools: Lu, J. (2020). KrakenTools. https://github.com/jenniferlu717/KrakenTools
• krona: Ondov, B. D., Bergman, N. H., & Phillippy, A. M. (2011). Interactive metagenomic visualization in a Web browser. BMC Bioinformatics, 12(1), 385. https://doi.org/10.1186/1471-2105-12-385
• megahit: Li, D., Liu, C.-M., Luo, R., Sadakane, K., & Lam, T.-W. (2015). MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics, 31(10), 1674–1676. https://doi.org/10.1093/bioinformatics/btv033
• samtools: Danecek, P., Bonfield, J. K., Liddle, J., Marshall, J., Ohan, V., Pollard, M. O., ... & Li, H. (2021). Twelve years of SAMtools and BCFtools. GigaScience, 10(2), giab008. https://doi.org/10.1093/gigascience/giab008
• bedtools: Quinlan, A. R., & Hall, I. M. (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 26(6), 841–842. https://doi.org/10.1093/bioinformatics/btq033
• abricate: Seemann, T. (2016). Abricate: mass screening of contigs for antimicrobial resistance or virulence genes. https://github.com/tseemann/abricate
• medaka: Oxford Nanopore Technologies. Medaka: Sequence correction provided by Oxford Nanopore Technologies. https://github.com/nanoporetech/medaka
• mlst: Seemann, T. (2016). mlst: scan contig files against PubMLST typing schemes. https://github.com/tseemann/mlst (PubMLST: Jolley, K. A., & Maiden, M. C. J. (2010). BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics, 11, 595. https://doi.org/10.1186/1471-2105-11-595)
• blast+: Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., & Madden, T. L. (2009). BLAST+: architecture and applications. BMC Bioinformatics, 10, 421. https://doi.org/10.1186/1471-2105-10-421
• igv-reports: Robinson, J. T., Thorvaldsdóttir, H., Winckler, W., Guttman, M., Lander, E. S., & Getz, G. (2011). Integrative genomics viewer. Nature Biotechnology, 29(1), 24–26. https://doi.org/10.1038/nbt.1754
• multiqc: Ewels, P., Magnusson, M., Lundin, S., & Käller, M. (2016). MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics, 32(19), 3047–3048. https://doi.org/10.1093/bioinformatics/btw354
• rmarkdown: Allaire, J., Xie, Y., McPherson, J., Luraschi, J., Ushey, K., Atkins, A., Wickham, Cheng, J., Chang, W., & Iannone, R. (2021). rmarkdown: Dynamic Documents for R. R package version 2.7. https://rmarkdown.rstudio.com/