tools-taggi

tools-taggi enables construction of Adapterama-style fusion primers and generation of dual- and quadruple-indexed Illumina amplicon libraries for multiplexed sequencing and demultiplexing.

Key Features:

• Universal Stub and Primer System: Uses universal stubs and primers based on the Adapterama system (Glenn et al., 2016) to produce thousands of unique index combinations compatible with iTru primers.
• Fusion Primers for Locus-Specific Amplification: Fuses partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences.
• Combinatorial Amplification: Supports combinatorial indexing in a 96-well format (e.g., 8 forward primers × 12 reverse primers = 96 unique combinations) to multiplex many samples.
• Dual-Indexed Library Preparation: Employs a second PCR with dual-indexed iTru or iNext primers to generate full-length, quadruple-indexed libraries that provide base diversity compatible with pooling with standard Illumina libraries.
• Adjustable Sequencing Depth for Cost Efficiency: Enables adjustment of reads per amplicon pool to allow deep sequencing when required or reduced per-sample sequencing cost when lower coverage suffices.
• Primer Design and Demultiplexing Software: Includes primer-design compatibility with iTru primers and references the demultiplexing program Mr_Demuxy for handling internal indexes.

Scientific Applications:

• Large-scale genomic projects: Facilitates scalable, cost-effective construction of amplicon libraries for Illumina sequencing in high-throughput studies.
• Microbiome studies: Enables multiplexed amplicon sequencing for microbial community profiling.
• Genetic diversity analysis: Supports genotyping and population genetics via multiplexed targeted sequencing.
• Pathogen detection: Enables targeted amplicon sequencing for identification and surveillance of pathogens.

Methodology:

Uses a two-step PCR: first PCR with locus-specific fusion primers containing partial Adapterama adapters and variable-length tags, then a second PCR with dual-indexed iTru or iNext primers to produce quadruple-indexed libraries; internal indexes can be demultiplexed with Mr_Demuxy.