• hecatomb install - Install the databases (you should only need to do this once)
  • hecatomb run - Run the pipeline
  • hecatomb test - Run the test dataset
  • hecatomb config - Copy the default config file to the current directory (for use with --configfile)
  • hecatomb listHosts - List the currently-available host genomes
  • hecatomb addHost - Add your own host genome
  • hecatomb combine - Combine output from multiple Hecatomb runs


When working with paired-end reads, you can either specify a directory of reads, and Hecatomb will infer the sample names and forward/reverse files, or, you can specify a TSV file to explicitly assign sample names and point to the corresponding read files. In either case you just use --reads and Hecatomb will figure out if it's a file or directory.

When you specify a directory of reads, e.g. hecatomb run --reads readDir/, Hecatomb expects paired sequencing reads in the format sampleName_R1/R2.fastq(.gz), and uses the wildcards to match around the _R1 flag like so: {sampleName}_R1{fileExtension}. e.g.


This allows for some variation, such as sample1_R1.L001.fastq.gz or sample1_R1.fq.gz, but you might run into problems if you mix and match file extensions, or if you have _R1 as part of a sample's name.

When you specify a TSV file, e.g. hecatomb run --reads samples.tsv, Hecatomb expects a 3-column tab separated file with the first column specifying the sample name, and the other columns the relative or full paths to the forward and reverse read files. e.g.

sample1    /path/to/reads/sample1.1.fastq.gz    /path/to/reads/sample1.2.fastq.gz
sample2    /path/to/reads/sample2.1.fastq.gz    /path/to/reads/sample2.2.fastq.gz

For single-end sequencing (long or short reads), Hecatomb can accept either FASTA or FASTQ format files. When specifying a directory of reads, the file extensions must be either fasta.gz or fastq.gz, and they must be consistent across samples. Hecatomb matches with constrained wildcards like so: {sampleName}.{extension,fast[aq].gz}.

Alternatively you can pass a TSV file just like before, only it will be a 2-column tab separated file, but you can mix and match FASTAs and FASTQs to your heart's content.

Read annotation + assembly

By default, Hecatomb will annotate your reads and perform an assembly. If you have more than 32 threads available, you can increase the threads provided to the pipeline with --threads:

hecatomb run --reads fastq/ --threads 64

If you're running on a HPC cluster, you should first set up a Snakemake Profile. More info and example for Hecatomb here. Then you would specify your profile name when running Hecatomb. Assuming your profile is called slurm:

hecatomb run --reads fastq/ --profile slurm

Running Hecatomb on a HPC with a Snakemake profile is THE BEST WAY to run the pipeline. But if you're feeling lazy, just submit a single job with the max resources and use --threads.

Run specific stages

Optionally skip specific stages of Hecatomb by specifying the stages you want to run. For instance, skip assembly and run the read-annotations only:

hecatomb run --reads fastq/ --profile slurm preprocessing annotations

View the stages that are available to run:

hecatomb run print_stages

Quicker read annotation

The main pipeline bottleneck is the MMSeqs searches. Use the --search fast flag to run Hecatomb with less sensitive settings for MMSeqs. In limited testing, we find it performs almost as well but with considerable runtime improvements.

hecatomb run --reads fastq/ --profile slurm --search fast

Specifying a host genome

Hecatomb includes a thorough host read removal step which utilises a processed host genome. You can specify a host, or add your own.

By default, Hecatomb will use the human genome. If your sample is from a different source you will need to specify the host genome for your sample source.

To see what host genomes are available:

hecatomb listHosts

The following should be available by default: bat, mouse, camel, celegans, macaque, rat, dog, cat, tick, mosquito, cow, human

So if you are working with mouse samples you would run:

hecatomb run --reads fastq/ --host mouse

Add your own host genome

If the genome for the host you're working with isn't included in the available hosts, or you have a reference genome which you think is better, you can add it with addHost. This script will mask viral-like regions from your genome and add it to your Hecatomb host database.

You will need to specify the host genome FASTA file, as well as a name for this host. Assuming you want to add the llama genome and the FASTA genome file is called llama.fasta:

hecatomb addHost --host llama --hostfa llama.fasta

You will then be able to run Hecatomb with your new host genome:

hecatomb run --reads fastq/ --host llama

Combine multiple Hecatomb runs

You can now combine multiple Hecatomb runs. For some files such as the seqtable and the bigtable you can simply concatenate them (but removing duplicate headers). However, the assembly files need to be coalesced with FlyE, and the assembly-associated files need to be regenerated.

To run:

hecatomb combine --comb hecOutDir1/ --comb hecOutDir2/

and use --threads or --profile like you normally would.