🚀 seqdemu: A No-Nonsense Nanopore Demultiplexer

Tired of sifting through your nanopore sequences manually? Meet seqdemu, your no-nonsense, super-efficient demultiplexer for sequences with custom barcodes! 🎉

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✨ Features

✅ Demultiplex sequences with custom barcodes—on both ends!

✅ Got barcodes in the middle of the sequence? No problem!

✅ Allows for mismatches—because we all make mistakes. 😏

✅ Detects barcode issues (like multiple forward/reverse primers) and automatically excludes problematic sequences.

✅ Supports multiple CPUs for faster processing.

⚡ Installation

Clone the Repository & Set Up the Environment

# Clone the repo
git clone https://github.com/hsgweon/seqdemu.git
cd seqdemu

# Create the seqdemu environment (ensure that you have conda installed)
mamba create -n seqdemu_env -y -c conda-forge -c bioconda conda-forge::biopython progressbar2

🎬 Running seqdemu

1️⃣ What You Need:

1. A gzipped basecalled FASTQ file (your sequence data).
2. A barcode file (check out the example format below).

Barcode File Format Your barcodes should be listed like this:

Forward Primer	Reverse Primer	Sample ID
GCCTCAGGCTTA	CGCATAAGGCAA	S01
CTTATGCAATGC	ATGGACTCGCAA	S02
TCCACCAGAGGT	CAATGCGTCCAA	S03
GGAGAAGAAGAA	TGTGGTTGCTAA	S04
AAGCGGAGAGAA	CCTCATGGAAGA	S05
CTCTCTCCAGAA	TGGAGACCAAGA	S06
TCTCTCCAGGAA	TACGAGAAGAGA	S07
GCTACGTTACAA	GTAGTCCGCAGA	S08
ATGTCGGTTAGA	TAGCCTGCGTCA	S09
CATGTATCTGGA	CTCCGGTATCTA	S10
AGACCTAACCGA	CGAGCGCTGTTA	S11
GTTCGAATGTGA	GTCACCGAGAAG	S12
AGCCTCTTAACA	AGTTAGCGGAAG	S13
GCATGTTAGACA	CCGGTTAACAAG	S14
ACAATGGCCGCA	TCACCATGTAAG	S15
GAGAACCTTGCA	TAAGTGCCACAG	S16

2️⃣ Activate the seqdemu environment

mamba activate seqdemu_env

3️⃣ Run seqdemu

Make sure seqdemu is in your PATH, then run:

seqdemu.py -i GZIPPED_DORADO_FASTQ_FILE -b BARCODE_FILE -m NUMBER_OF_MISMATCH -o OUTPUT_FILENAME -t NUMBER_OF_CPUs -f both

🧪 Running a Test Example

cd test
../seqdemu.py -i testseqs.fastq.gz -b barcodes.csv -m 0 -o testseqs -t 10 -f both

📂 Understanding the Output Files

You'll get different output files, each with a specific purpose:

🟢 _barc → Sequences with barcodes attached.

🟡 _full → Sequences without trimming.

🔵 _noba → Barcodes removed—use this for downstream analysis!

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That's it! 🚀 Now go forth and demultiplex with confidence! 🎉