NanoMetaPipe Options

The NanoMetaPipe script contains the main pipeline to take basecalled metagenomic nanopore reads through preprocessing to final assembled bins for microbiome analysis. The user will provide either raw reads or basecalled reads with the path to the output folder to place generated outputs. Other options include different thresholds necessary to trimming, filtering and classification steps.

Required Arguments

• -b or --basecalled: Folder of basecalled reads in FASTQ format. Reads must be basecalled previously!. Example: path/to/exp_folder/pass. This option does not work with raw reads.
• -c or --barcodes: List of barcodes used e.g. -c barcode01 barcode02
• -e or --isolates: List of isolates used. Must give sequence type separated by underscore e.g. -e isolate1_dna isolate2_cdna. Must correspond to the order of barcodes given i.e. barcode01=isolate1_dna.
• -kr or --kraken: Full path to the install kraken package. If kraken is in the $PATH, simply write kraken2
• -kb or kraken_DB: Full path to the installed kraken database
• -r or --reference: Full path to the reference genome to align against
• -s or --sequence_type: The type of sequence you want to be analysed. DNA and cDNA are supported individually and together in a multiplexed dataset. Ensure to identify the sequence types in the isolate names given i.e. Sequence files should be given as isolate1_DNA or isolate2_cDNA or isolate2_dscDNA.
• -br or --bracken: Full path to the installed bracken. If in the $PATH, simply write bracken
• -nd or --ncbi_db: The full path to the local NCBI nt database

Optional Arguments

• -bt or --bracken-hit-threshold: A minimum number of kmers that must be matched to place a contig into a taxonomic group by bracken re-estimation. Default is 20
• -cr or --cdna_ref: If using both DNA and CDNA sequence types, please provide the path to the transcriptome assembly here. If only using cDNA sequence files, this option is not necessary!
• -mcr or --make_cdna_ref: Generate a genome-guided transcriptome to remove host sequences from input cDNA sequences. Only works with with the detection of indicated cDNA input isolates. This option does not work with the -cr option. Default is Off. Activate this option with just -mcr or --make_cdna_ref. To be used in conjunction with the -cd option
• -cd or --ref_cdna_reads: Path to cDNA reads to generate a host transcriptome. Must be used with -mcr option
• -ca or --cdna_adapter: Full path to a FASTA file containing the adapters used for your cDNA sequencing reads. Must be used with the -mcr option
• -d or --demultiplexer: Choice of demultiplexer to use. Guppy or Qcat. If you wish to use guppy, you must have access to a GPU set up with tensorflow and other requirements set by Oxford Nanopore. Default is qcat
• -dfl or --dna_filter_length: Filter length threshold for DNA input sequences Default is 500
• -cfl or --cdna_filter_length: Filter length threshold for cDNA input sequences Default is 100
• -flw or --flowcell: The flowcell used for the sequencing experiment. Default is FLO-MIN106
• -fq or --filter_quality: Phred quality threshold to filter demultiplexed reads. Default is 10
• -ft or --filter: Carries out filtering of reads after demultiplexing. Default is On. Turn off with just -ft
• -g or --gff: The GFF file associated with the reference genome provided with the -r option
• -k or --kit: The sequencing kit used for the experiment. The SQK prefix is not necessary i.e. for SQK-LSK109, only LSK109 should be stated
• -kt or --kraken_Hit_Threshold: A minimum number of groups that must be matched to place a contig into a taxonomic group
• -km or --kraken_mode: Choose the method to use with kraken taxonomic classification. Classification can be done with just the host-free reads, the metagenome assembly or both. Default is assembly
• -n or --name: Give a name for the experiment. This will be used to name files and folders where appropriate
• -o or --out_dir: Full path to the output directory. Default will create the output file in the current working directory
• -p or --expansion: The expansion kit used for barcoding isolates in this experiment. The 'EXP' prefix is not necessary. i.e. EXP-NBD104 should be given as NBD104. Default is NBD104
• -rd or --redo_demulp: Carry out demultiplexing alone. To be used to re-generate demultiplexed reads if previously deleted. Default is Off, turn on with -rd
• -t or --threads: Number of threads to use. Default is 24
• -w or --cleanup: To clean up temporary files and other generated files as the pipeline progresses. Large generated files and folders would be deleted e.g. Basecalled outputs, demultiplexed reads and others will be deleted after being used. Log files will detail which files and folders were deleted. Default is Off, turn on with -w
• -mxm or --max_memory: Maximum memory to usefor the pipeline. Large input files and large classification databases may require a large amount of memory. Default is based on size of complete Refseq Kraken database which is ~132G. Default is 150G