ConFindr

https://olc-bioinformatics.github.io/ConFindr/
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ConFindr is a pipeline that detects contamination in bacterial next-generation sequencing (NGS)...
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### Manual pip installation with virtualenv

Source: https://olc-bioinformatics.github.io/ConFindr/install

Manual installation steps: create an empty directory, create a virtualenv with Python 3, activate it, and install ConFindr via pip. Using a virtual environment is highly recommended. After installation, you must install the listed dependencies (BBMap, Mash, KMA, Python, SAMtools, pysam) and add them to your $PATH.

```bash
mkdir ~/Virtual_Environments/ConFindr
```

```bash
virtualenv -p /usr/bin/python3 ~/Virtual_Environments/ConFindr
```

```bash
source ~/Virtual_Environments/ConFindr/bin/activate
```

```bash
pip install confindr
```

--------------------------------

### Download and extract example dataset

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Downloads the ConFindr example dataset from FigShare and extracts it into the current working directory.

```bash
wget https://figshare.com/ndownloader/files/41228577 -O test_samples.tar.gz && \
    tar -xzvf test_samples.tar.gz && \
    rm test_samples.tar.gz
```

--------------------------------

### ConFindr terminal output example

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Example of the terminal output generated when running ConFindr for the first time, showing database downloading and indexing.

```text
$ confindr -i test_samples -o output
  2023-05-16 15:25:51  Welcome to ConFindr 0.8.1! Beginning analysis of your samples... 
  2023-05-16 15:25:51  Could not find Escherichia_db_cgderived.fasta 
  2023-05-16 15:25:51  Could not find Listeria_db_cgderived.fasta 
  2023-05-16 15:25:51  Could not find Salmonella_db_cgderived.fasta 
  2023-05-16 15:25:51  Could not find refseq.msh 
  2023-05-16 15:25:51  Databases not present - downloading basic databases now... 
  2023-05-16 15:25:51  Downloading mash refseq sketch... 
  2023-05-16 15:25:53  Downloading cgMLST-derived data for Escherichia, Salmonella, and Listeria... 
  2023-05-16 15:25:55  Since this is the first time you are using this database, it needs to be indexed by KMA. This might take a while 
  2023-05-16 15:25:57  Since this is the first time you are using this database, it needs to be indexed by KMA. This might take a while 
  2023-05-16 15:25:58  Since this is the first time you are using this database, it needs to be indexed by KMA. This might take a while 
  2023-05-16 15:25:59  Did not find rMLST databases, if you want to use ConFindr on genera other than Listeria, Salmonella, and Escherichia, you'll need to download them. Instructions are available at https://olc-bioinformatics.github.io/ConFindr/install/#downloading-confindr-databases

  2023-05-16 15:25:59  Beginning analysis of sample SRX5084910_SRR8268082... 
  2023-05-16 15:25:59  Checking for cross-species contamination... 
  2023-05-16 15:26:00  Extracting conserved core genes... 
  2023-05-16 15:26:01  Quality trimming... 
  2023-05-16 15:26:01  Detecting contamination... 
  2023-05-16 15:26:05  Done! Number of contaminating SNVs found: 208

...

  2023-05-16 15:26:51  Contamination detection complete! 

```

--------------------------------

### Create conda environment and install ConFindr

Source: https://olc-bioinformatics.github.io/ConFindr/install

Creates a new conda environment named 'confindr' and installs ConFindr version 0.8.2 from the Bioconda channel. Requires the Bioconda channel to be added to your conda configuration first.

```bash
conda create -n confindr -c bioconda confindr=0.8.2
```

--------------------------------

### Install ConFindr in development mode

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Clones the repository and installs ConFindr in development mode within a Conda environment, including the necessary test data.

```bash
conda activate confindr
git clone https://github.com/OLC-Bioinformatics/ConFindr
cd ConFindr/
pip install -e .
wget https://figshare.com/ndownloader/files/41228577 -O tests/test_samples.tar.gz && \
  tar -xzvf tests/test_samples.tar.gz -C tests/ && \
  rm tests/test_samples.tar.gz
```

--------------------------------

### Example pytest output

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Shows the expected output when all automated tests pass.

```text
================================================ test session starts =================================================
platform linux -- Python 3.7.12, pytest-4.5.0, py-1.11.0, pluggy-0.11.0 -- /home/liam/miniconda3/envs/confindr-8.0/bin/python
cachedir: .pytest_cache
rootdir: /media/liam/Cerulean/projects/PRJ-LPB-00001/bin/confindr-8.0/ConFindr
collected 38 items
```

--------------------------------

### PubMLST consumer key and secret file

Source: https://olc-bioinformatics.github.io/ConFindr/install

Place the PubMLST consumer key on the first line and the consumer secret on the second line. This file is required by the `confindr_database_setup` command's `-s` argument.

```text
efKXmqp2D0EBlMBkZaGC2lPf
F$M+fQ2AFFB2YBDfF9fpHF^qSWJdmmN%L4Fxf5Gur3

```

--------------------------------

### Run ConFindr with core-gene databases

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Command to analyze samples in the test_samples directory with output directed to an output folder.

```bash
confindr -i test_samples -o output
```

--------------------------------

### Run ConFindr with rMLST databases

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Command to analyze samples using the --rmlst option after downloading the required databases.

```bash
confindr -i test_samples -o output --rmlst
```

--------------------------------

### Download Escherichia cgMLST FASTA

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Download the pre-clustered cgMLST FASTA file for Escherichia using wget.

```bash
wget 'https://scist01.blob.core.windows.net/olc/Escherichia_cgmlst.fasta?sp=r&st=2020-07-15T13:17:25Z&se=2029-07-15T21:17:25Z&spr=https&sv=2019-10-10&sr=b&sig=zF01kDCgmsmBWJ0cSpyfYi6CLldIjalwU2RgsswKmmI%3D' -O Escherichia_cgmlst.fasta
```

--------------------------------

### Run ConFindr with cgMLST

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Execute ConFindr using a custom cgMLST scheme file.

```bash
confindr.py -i folder-with-Escherichia-files -o cgmlst-output -cgmlst /path/to/Escherichia_cgmlst.fasta
```

--------------------------------

### Download Salmonella cgMLST FASTA

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Download the pre-clustered cgMLST FASTA file for Salmonella using wget.

```bash
wget 'https://scist01.blob.core.windows.net/olc/Salmonella_cgmlst.fasta?sp=r&st=2020-07-15T13:13:16Z&se=2029-07-15T21:13:16Z&spr=https&sv=2019-10-10&sr=b&sig=ncG%2F5MzKt57p1BUdyFtnhJUk9Yfi6x3rFhSQWPlT2Ek%3D' -O Salmonella_cgmlst.fasta
```

--------------------------------

### Activate conda environment

Source: https://olc-bioinformatics.github.io/ConFindr/install

Activates the conda environment named 'confindr' created in the previous step. After activation, typing `confindr -h` shows the help menu.

```bash
conda activate confindr
```

--------------------------------

### Integrate ConFindr into a Python script

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Use the confindr module to find paired reads and run contamination detection. Ensure databases are downloaded before execution.

```python
from confindr_src import confindr

# Find read files.
paired_reads = confindr.find_paired_reads('path_to_fastq_folder', forward_id='_R1', reverse_id='_R2')
# Run confindr. This assumes that you have already downloaded the databases. If you haven't,
# you can run confindr.check_for_databases_and_download(database_location='path/where/you/want/to/download, tmpdir='a/tmp/dir')
for pair in paired_reads:
    confindr.find_contamination(pair=pair,
                                forward_id='_R1', # change if yours is different
                                threads=4, 
                                output_folder='path/to/output',
                                databases_folder='path/to/databases')
```

--------------------------------

### Run automated tests with pytest

Source: https://olc-bioinformatics.github.io/ConFindr/usage

Executes the ConFindr test suite using pytest within the activated Conda environment.

```bash
conda activate confindr
cd ConFindr/
python -m pytest tests/ -vvv
```
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