XSTK

XSTK performs multiplexed barcode primer design and sequence deconvolution to support genomic and metagenomic analyses using high-throughput DNA sequencing.

Key Features:

• Implementation: A collection of C and C++ libraries with command-line programs for DNA sequence analysis.
• Multiplexing support: Enables covalent linkage of short, unique barcode DNA segments to genomic DNA and incorporation of barcodes into PCR primers for multiplexed sequencing.
• Barcode primer design (barcrawl): Designs barcoded primers ensuring barcode sequence uniqueness and robustness to sequencing errors.
• Sequence deconvolution (bartab): Deconvolutes mixed DNA sequence datasets by assigning reads to samples based on barcode sequences.
• Bias mitigation: Integrates barcode uniqueness and error robustness into primer design to reduce PCR amplification and sequencing biases.

Scientific Applications:

• Metagenomic studies: Design and validation of barcoded PCR primers and deconvolution of multiplexed high-throughput sequencing datasets for community profiling.
• Genomic multiplexing: Sample multiplexing workflows that require accurate read assignment and mitigation of amplification/sequencing bias.

Methodology:

Uses a systematic computational approach to primer design and sequence deconvolution: barcrawl generates barcodes that are unique and resilient to errors, and bartab separates multiplexed sequence datasets into constituent samples based on those barcodes; this workflow was exemplified by a proof-of-concept using barcoded rRNA primers.