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‎Workflows/downsample.md

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---
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label: Downsample
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description: Downsample data by barcode
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icon: fold-down
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order: 10
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---
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# :icon-fold-down: Downsample data by barcode
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=== :icon-checklist: You will need one of either
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- one alignment file [!badge variant="success" text=".bam"] [!badge variant="success" text=".sam"] [!badge variant="secondary" text="case insensitive"]
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- one set of paired-end reads in FASTQ format [!badge variant="success" text=".fq"] [!badge variant="success" text=".fastq"] [!badge variant="secondary" text="gzip recommended"] [!badge variant="secondary" text="case insensitive"]
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===
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While downsampling (subsampling) FASTQ and BAM files is relatively simple with tools such as `awk`, `samtools`, `seqtk`, `seqkit`, etc.,
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[!badge corners="pill" text="downsample"] allows you to downsample a BAM file (or paired-end FASTQ) _by barcodes_. That means you can
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keep all the reads associated with `d` number of barcodes. The `--invalid` proportion will determine what proportion of invalid barcodes appear in the barcode
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pool that gets subsampled, where `0` is none, `1` is all invalid barcodes, and a number in between is that proportion, e.g. `0.5` is half.
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Bear in mind that the barcode pool still gets subsampled, so the `--invalid` proportion doesn't necessarily reflect how many end up getting
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sampled, rather what proportion will be considered for sampling.
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!!! Barcode tag
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Barcodes must be in the `BX:Z` SAM tag for both BAM and FASTQ inputs. See [Section 1 of the SAM Spec here](https://samtools.github.io/hts-specs/SAMtags.pdf).
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!!!
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```bash usage
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harpy downsample OPTIONS... INPUT(S)...
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```
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```bash example
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# BAM file
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harpy downsample -d 1000 -i 0.3 -p sample1.sub1000 sample1.bam
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# FASTQ file
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harpy downsample -d 1000 -i 0 -p sample1.sub1000 sample1.F.fq.gz sample1.R.fq.gz
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```
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## :icon-terminal: Running Options
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In addition to the [!badge variant="info" corners="pill" text="common runtime options"](/commonoptions.md), the [!badge corners="pill" text="downsample"]
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module is configured using the command-line arguments below.
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{.compact}
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| argument | short name | default | description |
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| :-------------- | :--------: | :-----------: | :-------------------------------------------------------------------------------------------------------------------------------- |
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| `INPUT(S)` | | | [!badge variant="info" text="required"] One BAM file or both read files from a paired-end FASTQ pair |
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| `--downsample` | `-d` | | [!badge variant="info" text="required"] Number of barcodes to downsample to |
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| `--invalid` | `-i` | `1` | Proportion of barcodes to sample |
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| `--prefix` | `-p` | `downsampled` | Prefix for output files |
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| `--random-seed` | | | Random seed for sampling [!badge variant="secondary" text="optional"] |
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----
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## :icon-git-pull-request: Downsample Workflow
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```mermaid
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graph LR
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subgraph fastq
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R1([read 1]):::clean---R2([read 2]):::clean
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end
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subgraph bam
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bamfile([bam]):::clean
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end
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fastq-->|bam conversion|bam
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bam-->sub([extract and\n subsample barcodes]):::clean
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sub-->exreads([extract reads]):::clean
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bam-->exreads
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fastq-->exreads
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style fastq fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
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style bam fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
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classDef clean fill:#f5f6f9,stroke:#b7c9ef,stroke-width:2px
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```

‎Workflows/other.md

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{.compact}
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| module | description |
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| :------------- | :------------------------------------------------------------------------------- |
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| `downsample` | Downsample BAM or FASTQ files by barcode |
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| `imputeparams` | Create a template imputation parameter file |
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| `resume` | Continue a Harpy workflow from an existing output folder |
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| `popgroup` | Create generic sample-group file using existing sample file names (fq.gz or bam) |
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| `view` | View a workflow log, config, or snakefile |
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---
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### downsample
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While downsampling (subsampling) FASTQ and BAM files is relatively simple with tools such as `awk`, `samtools`, `seqtk`, `seqkit`, etc.,
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Harpy offers the `downsample` module, which allows you to downsample a BAM file (or paired-end FASTQ) _by barcodes_. That means you can
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keep all the reads associated with `d` number of barcodes. First, barcodes are extracted, then subsampled, then the reads associated
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with those barcodes are extracted. The `--invalid` proportion will determine what proportion of invalid barcodes appear in the barcode
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pool that gets subsampled, where `0` is none, `1` is all invalid barcodes, and a number in between is that proportion, e.g. `0.5` is half.
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Bear in mind that the barcode pool still gets subsampled, so the `--invalid` proportion doesn't necessarily reflect how many end up getting
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sampled, rather what proportion will be considered for sampling.
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!!! Barcode tag
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Barcodes must be in the `BX:Z` SAM tag for both BAM and FASTQ inputs. See [Section 1 of the SAM Spec here](https://samtools.github.io/hts-specs/SAMtags.pdf).
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!!!
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```bash usage
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harpy downsample OPTIONS... INPUT(S)...
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```
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```bash example
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# BAM file
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harpy downsample -d 1000 -i 0.3 -p sample1.sub1000 sample1.bam
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# FASTQ file
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harpy downsample -d 1000 -i 0 -p sample1.sub1000 sample1.F.fq.gz sample1.R.fq.gz
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```
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#### arguments
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{.compact}
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| argument | short name | default | description |
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| :-------------- | :--------: | :-----------: | :-------------------------------------------------------------------------------------------------------------------------------- |
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| `INPUT(S)` | | | [!badge variant="info" text="required"] One BAM file or both read files from a paired-end FASTQ pair |
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| `--downsample` | `-d` | | [!badge variant="info" text="required"] Number of barcodes to downsample to |
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| `--invalid` | `-i` | 1 | Proportion of barcodes to sample |
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| `--prefix` | `-p` | `downsampled` | Prefix for output files |
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| `--random-seed` | | | Random seed for sampling [!badge variant="secondary" text="optional"] |
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| `--snakemake` | | | Additional Snakemake arguments, in quotes |
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| `--threads` | `-t` | `4` | Number of threads to use |
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| `--quiet` | | | Don't show output text while running |
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---
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### imputeparams
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Create a template parameter file for the [!badge corners="pill" text="impute"](/Workflows/impute.md) module.
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The file is formatted correctly and serves as a starting point for using parameters that make sense for your study.

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