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CUPID-seq

CUPID-seq (Containerized Dual-Index Demultiplexing) is a containerized pipeline for demultiplexing dual-indexed 16S amplicon sequencing data. By packaging the analysis in Docker and Singularity/Apptainer images, CUPID-seq eliminates dependency installation headaches and ensures highly reproducible results across computing environments.

Key Features

  • Containerized — runs in Docker or Singularity/Apptainer with minimal dependency setup
  • Reproducible — identical environments across local machines and HPC clusters
  • Flexible — supports 13 pre-built 16S primer/index sets plus custom primers

Pipeline Overview

The CUPID-seq pipeline processes paired-end amplicon reads through the following stages:

  1. Input validation — checks file paths, naming conventions, and samplesheet/fastqlist consistency
  2. Demultiplexing — separates reads by dual inline indexes (round 1 + round 2 barcodes)
  3. Trimming — removes index, spacer, and primer sequences from demultiplexed reads
  4. Grouped output — organizes trimmed reads into user-defined group subdirectories

Read Structure

The dual-index primer design uses variable-length phased indexes on both read 1 and read 2:

Primer structure overview

Phased primer design

Supported 16S Regions

Pre-built index files are provided for the following regions. The default is V4.

Region Index File
V1 - V2 V1-V2_index_for_demux.txt
V1 - V3 V1-V3_index_for_demux.txt
V2 - V3 V2-V3_index_for_demux.txt
V3 V3_index_for_demux.txt
V3 - V4 V3-V4_index_for_demux.txt
V4 V4_index_for_demux.txt (default)
V4 - V5 V4-V5_index_for_demux.txt
V5 V5_index_for_demux.txt
V5 - V7 V5-V7_index_for_demux.txt
V6 V6_index_for_demux.txt
V6 - V7 V6-V7_index_for_demux.txt
V6 - V8 V6-V8_index_for_demux.txt
V7 - V9 V7-V9_index_for_demux.txt

These files are located in the other_index_for_demux/ directory of the repository.

Next Steps