# Demultiplex Raw Sequences

at least 2 cores/threads available
paired-end reads from an Illumina sequencer in FASTQ format gzip recommended

When pooling samples and sequencing them in parallel on an Illumina sequencer, you will be given large multiplexed FASTQ files in return. These files contain sequences for all of your samples and need to be demultiplexed using barcodes to separate the sequences for each sample into their own files (a forward and reverse file for each sample). These barcodes should have been added during the sample DNA preparation in a laboratory. The demultiplexing strategy will vary based on the haplotag technology you are using (read Haplotag Types).

usage
harpy demultiplex METHOD OPTIONS... R1_FQ R2_FQ I1_FQ I2_FQ

example using wildcards
harpy demultiplex gen1 --threads 20 --schema demux.schema Plate_1_S001_R*.fastq.gz Plate_1_S001_I*.fastq.gz

# Running Options

In addition to the common runtime options , the demultiplex gen1 module is configured using these command-line arguments:

argument	short name	description
`R1_FQ`		required The forward multiplexed FASTQ file
`R2_FQ`		required The reverse multiplexed FASTQ file
`I1_FQ`		required The forward FASTQ index file provided by the sequencing facility
`I2_FQ`		required The reverse FASTQ index file provided by the sequencing facility
`--keep-unknown`	`-u`	Keep reads that could not be demultiplexed
`--qxrx`	`-q`	Include the `QX:Z` and `RX:Z` tags in the read header
`--schema`	`-s`	required Tab-delimited file of sample<tab>barcode

# Keeping Unknown Samples

It's not uncommon that some sequences cannot be demultiplexed due to sequencing errors at the ID location. Use --keep-unknown to have Harpy still separate those reads from the original multiplex. Those reads will be labelled _unknown_sample.R*.fq.gz

# Keep QX and RX tags

Using --qx-rx, you can opt-in to retain the QX:Z (barcode PHRED scores) and RX:Z (nucleotide barcode) tags in the sequence headers. These tags aren't used by any subsequent analyses, but may be useful for your own diagnostics.

# Haplotag Types gzip recommended

Barcode configuration: 13 + 13
sequencing mask: 151+13+13+151
Sample identifier: Cxx barcode
Facility should not demultiplex

These are the original 13 + 13 barcodes described in Meier et al. 2021. You should request that the sequencing facility you used do not demultiplex the sequences. Requires the use of bcl2fastq without sample-sheet and with the settings --use-bases-mask=Y151,I13,I13,Y151 and --create-fastq-for-index-reads. With Generation I beadtags, the C barcode is sample-specific, meaning a single sample should have the same C barcode for all of its sequences.

# Demultiplexing Schema

Generation I haplotags typically use a unique Cxx barcode per sample-- that's the barcode segment that will be used to identify sequences by sample. However, any of the 4 segments (A,B,C,D) are valid, so long as the schema only features a single segment. You will need to provide a simple text file to --schema (-s) with two columns, the first being the sample name, the second being the identifying segment barcode (e.g., C19). This file is to be tab or space delimited and must have no column names.

example sample sheet
Sample01    C01
Sample02    C02
Sample03    C03
Sample04    C04

This will result in splitting the multiplexed reads into individual file pairs Sample01.F.fq.gz, Sample01.R.fq.gz, Sample02.F.fq.gz, etc. A sample can have multiple barcodes, but a barcode cannot have multiple samples:

Sample01    D01
Sample02    D02
Sample03    D03
Sample03    D21

Sample01    C01
Sample02    C02
Sample03    C02

Sample01    C01
Sample02    D02
Sample03    C03

# Gen I Demultiplex Workflow

Barcode correction and migration into the read headers is performed using demult_fastq (Harpy renames it to demuxGen1), which is distributed by the team behind haplotagging. Demultiplexing the pooled FASTQ files into individual samples is performed in parallel and using the beloved workhorse grep.

graph LR
    subgraph Inputs
        direction TB
        A[multiplexed FASTQ]:::clean---BX
        BX[index reads FASTQ]:::clean---SCH
        SCH[Sample Schema]:::clean
    end
    Inputs-->B([demultiplex samples]):::clean
    B-->D([quality metrics]):::clean
    style Inputs fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
    classDef clean fill:#f5f6f9,stroke:#b7c9ef,stroke-width:2px

The default output directory is Demultiplex with the folder structure below. Sample1 and Sample2 are generic sample names for demonstration purposes. The resulting folder also includes a workflow directory (not shown) with workflow-relevant runtime files and information.

Demultiplex/
├── Sample1.F.fq.gz
├── Sample1.R.fq.gz
├── Sample2.F.fq.gz
├── Sample2.R.fq.gz
└── reports
    └── demultiplex.QC.html

item	description
`*.F.fq.gz`	Forward-reads from multiplexed input `--file` belonging to samples from the `samplesheet`
`*.R.fq.gz`	Reverse-reads from multiplexed input `--file` belonging to samples from the `samplesheet`
`reports/demultiplex.QC.html`	phased vcf annotated with phased blocks

FASTQC metrics

This is the summary report Harpy generates for this workflow. You may right-click the image and open it in a new tab if you wish to see the example in better detail.