PacBio sequencing output increased through uniform and directional fivefold concatenation

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• DNA sequencing technologies have advanced, focusing on short or long reads.
• Sequencing medium-length DNA fragments (600-5,000 bp) remains less efficient.
• Existing methods like PacBio Sequel I offer variable accuracy (90-99%) for such fragments.

Purpose of the Study:

• To develop an efficient and accurate method for sequencing medium-length DNA fragments (~870 bp).
• To achieve high sequencing depth and 99% accuracy for DNA populations.
• To reduce the cost and complexity of medium-length DNA sequencing.

Main Methods:

• Optimized a robust method to concatenate ~870 bp genes five times, creating ~5,000 bp DNA molecules.
• Utilized PacBio Single Molecule, Real-Time (SMRT) long-read sequencing for the concatenated DNA.
• Developed a user-friendly analysis pipeline, DeCatCounter, for efficient data processing.

Main Results:

• Achieved greater sequencing depth and high-quality reads compared to previous concatenation methods.
• Successfully sequenced nine DNA populations from a protein engineering study.
• Demonstrated a significant cost reduction, achieving sequencing at one-fifth of the previous cost.

Conclusions:

• The optimized concatenation and PacBio SMRT sequencing method provides an efficient solution for medium-length DNA sequencing.
• The DeCatCounter pipeline simplifies data analysis, making the method accessible.
• This approach offers a cost-effective alternative for DNA sequencing in research, particularly in protein engineering.