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This is an explanation of how to use NANOME pipeline on some specific scenarios. For general usage, please check Usage.

1. How to use other reference genome in NANOME?

NANOME support other reference genome. Below is an example of running NANOME for any other reference genomes, please make sure put reference genome file .fasta and the indexed file into directory [reference-genome-dir], the --chrSet is the chromosomes params for the specific genome.

nextflow run LabShengLi/nanome\
    -profile singularity \
    --dsname [dataset-name]\
    --input [input-file]\
    --genome [reference-genome-dir]\
    --chrSet '[chromosomes separated by a space]'

The reference genome folder other_ref will be like below:

tree other_ref/

other_ref
├── Ecoli_k12_mg1655.fasta
├── Ecoli_k12_mg1655.fasta.fai

Note:

  • NANOME support the default behaviour of each tool's running, if the tool performed on the specified genome that is same as human/E. coli data.
  • please ensure there is only one .fasta file in genome reference folder, the index file is same name with suffix as .fasta.fai
  • please set param --chrSet with the interested chromosomes names, seperated by a space, such as --chrSet 'chr1 chr2 chr3' (the single quote character is important)
  • for human or ecoli genome, NANOME will automatically set default chromosomes chr1-22, X, Y for human, NC_000913.3 for ecoli. Users can change the default setting by specifying --chrSet param

2. How to input basecalled data into NANOME?

NANOME support the basecalled input data. You can use --skipBasecall param to inform NANOME to skip redo the basecalling step. We suggest keeping the Guppy basecalled data/directory structure as input. The .fastq.gz file, sequencing_summary.txt file, and the workspace/*.fast5 files are need to be located in the default directory.

Example:

nextflow run main.nf -profile test,singularity\
    --skipBasecall\
    --input test_data/ecoli_ci_basecalled

Basecalled input data directory structure like below:

tree test_data/ecoli_ci_basecalled

test_data/ecoli_ci_basecalled
├── abc.fastq.gz
├── fail
├── pass
├── sequencing_summary.txt
└── workspace
    ├── kelvin_20160617_FN_MN17519_sequencing_run_sample_id_74930_ch138_read698_strand.fast5
    └── kelvin_20160617_FN_MN17519_sequencing_run_sample_id_74930_ch139_read4507_strand.fast5

Note:

  • please use .fastq.gz, not .fastq, keep their locations in base folder, or pass, fail folders
  • please keep sequencing_summary.txt file for QC analysis
  • please keep all fast5 files in workspace/ directory
  • NANOME input can be a directory/tar/tar.gz file, user can compress the basecalled folder using tar -czf ecoli_ci_basecalled.tar.gz ecoli_ci_basecalled/
  • by default, NANOME run top four performers. If user wants to run Nanopolish tool only, just specify not run other three tools using params --runMegalodon false --runDeepSignal false --runGuppy false

3. Update latest version of NANOME.

Below is the command for updating the latest version of NANOME, if users have executed NANOME old version previously. -r [branch-name] is used to get a specific version from a branch in the GitHub for Nextflow.

nextflow pull LabShengLi/nanome

nextflow pull LabShengLi/nanome -r [branch-name]

4. Perform only basecall and QC.

Using option --runMethcall false will not run methylation calling, it will only perform basecall and QC.

nextflow run LabShengLi/nanome\
    -profile singularity,winter \
    --dsname APL\
    --input '/fastscratch/liuya/nanome/APL_ont_out/APL_sept/sept_dir/*'\
    --genome hg38 \
    --runMethcall false

5. Support T2T-CHM13 genome

Example as below:

nextflow run LabShengLi/nanome \
    -profile test_human,singularity \
    --genome chm13

6. Support R10.4.1 flow cells

Example as below:

nextflow run LabShengLi/nanome \
    -profile test_human,singularity \
    --input https://storage.googleapis.com/jax-nanopore-01-project-data/nanome-input/testdata_r10_4_1.tar.gz \
    --runGuppy \
    --GUPPY_BASECALL_MODEL dna_r10.4.1_e8.2_400bps_hac.cfg \
    --GUPPY_METHCALL_MODEL dna_r10.4.1_e8.2_400bps_modbases_5mc_cg_hac.cfg \
    --runNanopolish false --runDeepSignal false --runMegalodon false