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Robust and scalable barcoding for massively parallel long-read sequencing.

Joaquín Ezpeleta1,2, Ignacio Garcia Labari3, Gabriela Vanina Villanova4,5

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NS-watermark barcodes enable massive parallel sequencing on long-read platforms, overcoming low raw accuracy. This technique successfully identified over 86% of 3840 distinct barcodes with minimal error, paving the way for advanced sequencing applications.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Nucleic-acid barcoding is crucial for many applications but limited by low accuracy in long-read sequencing.
  • NS-watermark barcodes offer validated in silico error correction capabilities.
  • Scalable synthesis of large oligonucleotide pools is essential for advanced barcoding.

Purpose of the Study:

  • To demonstrate the utility of NS-watermark barcodes in a proof-of-concept experiment using long-read sequencing.
  • To assess the performance of a large number of distinct, non-random barcodes in parallel sequencing.
  • To evaluate the feasibility of microarray-based synthesis for generating oligonucleotide pools for barcoding.

Main Methods:

  • Synthesized 3840 distinct NS-watermark barcodes using microarray-based methods.
  • Asymmetrically tagged amplicons from *Bordetella pertussis* and *Drosophila mojavensis*.
  • Sequenced tagged amplicons simultaneously on the Oxford Nanopore Technologies (ONT) MinION platform.

Main Results:

  • Successfully recovered the identity of over 86% of the synthesized NS-watermark barcodes.
  • Achieved a low crosstalk rate of 0.17% (one misassignment per 584 reads).
  • Demonstrated performance comparable to established high-accuracy Illumina sequencing despite low raw read accuracy.

Conclusions:

  • NS-watermark barcodes are robust and scalable for massive labeling applications in sequencing.
  • Microarray-based synthesis is a viable method for producing large oligonucleotide pools for barcoding.
  • This approach shows promise for current and future sequencing technologies, including long-read single-cell RNA sequencing.