fqtk

A toolkit for working with FASTQ files, written in Rust.

Visit us at Fulcrum Genomics to learn more about how we can power your Bioinformatics with fqtk and beyond.

Currently fqtk contains a single tool, demux for demultiplexing FASTQ files based on sample barcodes. fqtk demux can be used to demultiplex one or more FASTQ files (e.g. a set of R1, R2 and I1 FASTQ files) with any number of sample barcodes at fixed locations within the reads. It is highly efficient and multi-threaded for high performance.

Usage for fqtk demux follows:

Performs sample demultiplexing on FASTQs.

The sample barcode for each sample in the metadata TSV will be compared against the sample
barcode bases extracted from the FASTQs, to assign each read to a sample.  Reads that do not
match any sample within the given error tolerance will be placed in the ``unmatched_prefix``
file.

FASTQs and associated read structures for each sub-read should be given:

- a single fragment read (with inline index) should have one FASTQ and one read structure
- paired end reads should have two FASTQs and two read structures
- a dual-index sample with paired end reads should have four FASTQs and four read structures
  given: two for the two index reads, and two for the template reads.

If multiple FASTQs are present for each sub-read, then the FASTQs for each sub-read should be
concatenated together prior to running this tool
(e.g. `zcat s_R1_L001.fq.gz s_R1_L002.fq.gz | bgzip -c > s_R1.fq.gz`).

(Read structures)[<https://github.com/fulcrumgenomics/fgbio/wiki/Read-Structures>] are made up of
`<number><operator>` pairs much like the `CIGAR` string in BAM files.
Four kinds of operators are recognized:

1. `T` identifies a template read
2. `B` identifies a sample barcode read
3. `M` identifies a unique molecular index read
4. `S` identifies a set of bases that should be skipped or ignored

The last `<number><operator>` pair may be specified using a `+` sign instead of number to
denote "all remaining bases". This is useful if, e.g., fastqs have been trimmed and contain
reads of varying length. Both reads must have template bases.  Any molecular identifiers will
be concatenated using the `-` delimiter and placed in the given SAM record tag (`RX` by
default).  Similarly, the sample barcode bases from the given read will be placed in the `BC`
tag.

Metadata about the samples should be given as a headered metadata TSV file with at least the
following two columns present:

1. `sample_id` - the id of the sample or library.
2. `barcode` - the expected barcode sequence associated with the `sample_id`.

For reads containing multiple barcodes (such as dual-indexed reads), all barcodes should be
concatenated together in the order they are read and stored in the `barcode` field.

IUPAC bases are supported in the (expected) `barcode` column.  An observed IUPAC base must be
at least as specific as the corresponding base in the expected sample barcode.  E.g. If the
observed base is an N, it will only match expected sample barcrods with an N.  And if the
observed base is an R, it will match R, V, D, and N, since the latter IUPAC codes allow both
A and G (R/V/D/N are a superset of the bases compare to R).

The read structures will be used to extract the observed sample barcode, template bases, and
molecular identifiers from each read.  The observed sample barcode will be matched to the
sample barcodes extracted from the bases in the sample metadata and associated read structures.

An observed barcode matches an expected barcode if all the following are true:
1. The number of mismatches (edits/substitutions) is less than or equal to the maximum
   mismatches (see `--max-mismatches`).
2. The difference between number of mismatches in the best and second best barcodes is greater
   than or equal to the minimum mismatch delta (`--min-mismatch-delta`).

The expected barcode sequence may contains Ns, which are not counted as mismatches regardless
of the observed base (e.g. the expected barcode `AAN` will have zero mismatches relative to
both the observed barcodes `AAA` and `AAN`).

## Outputs

All outputs are generated in the provided `--output` directory.  For each sample plus the
unmatched reads, FASTQ files are written for each read segment (specified in the read
structures) of one of the types supplied to `--output-types`.  FASTQ files have names
of the format:

```bash
{sample_id}.{segment_type}{read_num}.fq.gz

where segment_type is one of R, I, and U (for template, barcode/index and molecular barcode/UMI reads respectively) and read_num is a number starting at 1 for each segment type.

In addition a demux-metrics.txt file is written that is a tab-delimited file with counts of how many reads were assigned to each sample and derived metrics.

Example Command Line

As an example, if the sequencing run was 2x100bp (paired end) with two 8bp index reads both reading a sample barcode, as well as an in-line 8bp sample barcode in read one, the command line would be:

fqtk demux \
    --inputs r1.fq.gz i1.fq.gz i2.fq.gz r2.fq.gz \
    --read-structures 8B92T 8B 8B 100T \
    --sample-metadata metadata.tsv \
    --output output_folder

Usage: fqtk demux [OPTIONS] --inputs ... --read-structures <READ_STRUCTURES>... --sample-metadata <SAMPLE_METADATA> --output

Options: -i, --inputs ... One or more input FASTQ files each corresponding to a sequencing read (e.g. R1, I1)

-r, --read-structures <READ_STRUCTURES>... The read structures, one per input FASTQ in the same order

-b, --output-types <OUTPUT_TYPES>... The read structure types to write to their own files (Must be one of T, B, or M for template reads, sample barcode reads, and molecular barcode reads).

      Multiple output types may be specified as a space-delimited list.

      [default: T]

-s, --sample-metadata <SAMPLE_METADATA> A file containing the metadata about the samples

-o, --output The output directory into which to write per-sample FASTQs

-u, --unmatched-prefix <UNMATCHED_PREFIX> Output prefix for FASTQ file(s) for reads that cannot be matched to a sample

      [default: unmatched]

  --max-mismatches <MAX_MISMATCHES>
      Maximum mismatches for a barcode to be considered a match

      [default: 1]

-d, --min-mismatch-delta <MIN_MISMATCH_DELTA> Minimum difference between number of mismatches in the best and second best barcodes for a barcode to be considered a match

      [default: 2]

-t, --threads The number of threads to use. Cannot be less than 3

      [default: 8]

-c, --compression-level <COMPRESSION_LEVEL> The level of compression to use to compress outputs

      [default: 5]

-S, --skip-reasons <SKIP_REASONS> Skip demultiplexing reads for any of the following reasons, otherwise panic.

      1. `too-few-bases`: there are too few bases or qualities to extract given the read structures.  For example, if a read is 8bp long but the read structure is `10B`, or if a read is empty and the read structure is `+T`.

-h, --help Print help information (use -h for a summary)

-V, --version Print version information

<!-- end usage -->

## Installing

### Installing with `conda`
To install with conda you must first [install conda](https://conda.io/projects/conda/en/latest/user-guide/install/index.html#installation).
Then, in your command line (and with the environment you wish to install fqtk into active) run:

```console
conda install -c bioconda fqtk

Installing with cargo

To install with cargo you must first install rust. Which (On Mac OS and Linux) can be done with the command:

curl https://sh.rustup.rs -sSf | sh

Then, to install fqtk run:

cargo install fqtk

Building From Source

First, clone the git repo:

git clone https://github.com/fulcrumgenomics/fqtk.git

Secondly, if you do not already have rust development tools installed, install via rustup:

curl --proto '=https' --tlsv1.2 -sSf https://sh.rustup.rs | sh

Then build the toolkit in release mode:

cd fqtk
cargo build --release
./target/release/fqtk --help

Developing

fqtk is developed in Rust and follows the conventions of using rustfmt and clippy to ensure both code quality and standardized formatting. When working on fqtk, before pushing any commits, please first run ./ci/check.sh and resolve any issues that are reported.

Releasing a New Version

Pre-requisites

Install cargo-release

cargo install cargo-release

Prior to Any Release

Create a release that will not try to push to crates.io and verify the command:

cargo release [major,minor,patch,release,rc...] --no-publish

Note: "dry-run" is the default for cargo release.

See the cargo-release reference documentation for more information

Semantic Versioning

This tool follows Semantic Versioning. In brief:

• MAJOR version when you make incompatible API changes,
• MINOR version when you add functionality in a backwards compatible manner, and
• PATCH version when you make backwards compatible bug fixes.

Major Release

To create a major release:

cargo release major --execute

This will remove any pre-release extension, create a new tag and push it to github, and push the release to creates.io.

Upon success, move the version to the next candidate release.

Finally, make sure to create a new release on GitHub.

Minor and Patch Release

To create a minor (patch) release, follow the Major Release instructions substituting major with minor (patch):

cargo release minor --execute

Release Candidate

To move to the next release candidate:

cargo release rc --no-tag --no-publish --execute

This will create or bump the pre-release version and push the changes to the main branch on github. This will not tag and publish the release candidate. If you would like to tag the release candidate on github, remove --no-tag to create a new tag and push it to github.