.. _generate:

Generating reads
================

InSilicoSeq comes with a set of pre-computed error models to allow the user to easily generate reads from the most popular Illumina instruments:

- HiSeq
- MiSeq
- NovaSeq

Per example generate 1 million MiSeq reads from a set of input genomes:

.. code-block:: bash

    iss generate --genomes genomes.fasta --model miseq --output miseq_reads

This will create 2 fastq files, `miseq_reads_R1.fastq` and `miseq_reads_R2.fastq` in your current directory, as well as `miseq_reads_abundance.txt`, a tab-delimited file containing the abundance of each genomes.

If you have created your custom model, change ``--model miseq`` to your custom model file:

.. code-block:: bash

    iss generate --genomes genomes.fasta --model model.npz --output model_reads


Required input files
--------------------

By default, InSilicoSeq only requires 1 file in order to start generating reads: 1 (multi-)fasta files containing your input genome(s).

If you don't want to use a multi-fasta file or don't have one at hand but are equipped with an Internet connection, you can download random genomes from the ncbi:

.. code-block:: bash

    iss generate --ncbi bacteria -u 10 --model miseq --output miseq_ncbi

or for bacteria and viruses:

.. code-block:: bash

    iss generate -k bacteria viruses -u 10 4 --model miseq --output miseq_ncbi

In addition the the 2 fastq files and the abundance file, the downloaded genomes will be saved in `miseq_ncbi_genomes.fasta` in your current directory.

*Note: If possible, I recommend using InSilicoSeq with a fasta file as input.*
*The eutils utilities from the ncbi can be slow and quirky.*


Abundance distribution
----------------------

With default settings, the abundance of the input genomes is drwan from a log-normal distribution.

Alternatively, you can use other distributions with the ``--abundance`` parameter: `uniform`, `halfnormal`, `exponential` or `zero-inflated-lognormal`

If you wish to fine-tune the distribution of your genomes, InSilicoSeq also accepts an abundance file:

.. code-block:: bash

    iss generate -g genomes.fasta --abundance_file abundance.txt -m HiSeq -o HiSeq_reads

Example abundance file for a multi-fasta containing 2 genomes: genome_A and genome_B.

.. code-block:: bash

    genome_A    0.2
    genome_B    0.8


For the abundance to make sense, the total abundance in your abundance file must equal 1.

.. figure:: distributions.png

    Histograms of the different distribution (drawn with 100 samples)


Full list of options
--------------------

--genomes
^^^^^^^^^

Input genome(s) from where the reads will originate

--ncbi
^^^^^^

Download input genomes from RefSeq instead of using --genomes.
Requires --n_genomes option.
Can be bacteria, viruses, archaea or a combination of the three (space-separated)

--n_genomes
^^^^^^^^^^^

How many genomes will be downloaded from the ncbi.
Required if --ncbi is set.
If more than one kingdom is set with --ncbi, multiple values are necessary (space-separated).

--abundance
^^^^^^^^^^^

Abundance distribution (default: lognormal).
Can be uniform, halfnormal, exponential, lognormal or zero_inflated_lognormal.

--abundance_file
^^^^^^^^^^^^^^^^

Abundance file for coverage calculations (default: None).

--n_reads
^^^^^^^^^

Number of reads to generate (default: 1000000).
Allows suffixes k, K, m, M, g and G (ex 0.5M for 500000).

--mode
^^^^^^^

Error model. If not specified, using kernel density estimation (default: kde).
Can be 'kde' or 'basic'

--model
^^^^^^^^

Error model file. (default: None).
Use HiSeq, NovaSeq or MiSeq for a pre-computed error model provided with the software, or a file generated with iss model.
If you do not wish to use a model, use --mode basic.
The name of the built-in models is case insensitive.

--gc_bias
^^^^^^^^^

If set, may fail to sequence reads with abnormal GC content.
Does not guarantee --n_reads (default: False)

--cpus
^^^^^^

Number of cpus to use. (default: 2).

--quiet
^^^^^^^

Disable info logging

--debug
^^^^^^^

Enable debug logging

--output
^^^^^^^^

Output file prefix (Required)