Mycobacterium tuberculosis complex sample processing by mechanical lysis, an essential step for reliable whole genome...

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Whole genome sequencing (WGS) for Mycobacterium tuberculosis complex (MTBC) relies heavily on DNA extraction efficiency.
• Current DNA extraction protocols can limit turnaround time and success rates for MTBC WGS.
• Effective mycobacterial lysis is essential for sufficient DNA yield for drug susceptibility prediction and genotyping.

Purpose of the Study:

• To compare four DNA extraction protocols from MGIT cultures for MTBC WGS.
• To evaluate the impact of mechanical lysis (bead beating) on DNA yield and WGS success.
• To assess the feasibility of early WGS from pre-positive cultures and optimize protocols for Nanopore sequencing.

Main Methods:

• Comparison of four DNA extraction protocols from BD BACTEC™ MGIT tubes.
• Inclusion of a bead beating (mechanical lysis) step in selected protocols.
• Evaluation of DNA yield, WGS success rates, and read lengths (N50).
• Assessment of DNA extraction from primary and pre-positive enriched cultures.
• Optimization of bead beating duration for Nanopore sequencing.

Main Results:

• Bead beating (Protocol B) increased DNA yield approximately 60-fold compared to non-mechanical lysis.
• Significantly improved WGS success rates were observed with the bead beating protocol.
• High success rates were achieved for DNA extraction from primary cultures using bead beating (Protocol C).
• Reliable WGS was possible up to five days earlier from pre-positive cultures using bead beating (Protocol D).
• Shortened bead beating (15s) for Nanopore sequencing increased read length (N50 from 1.4kb to 2.6kb) while maintaining lysis efficiency.
• Bead beating-based DNA capture/purification was as effective as column-based methods.

Conclusions:

• Incorporating a mechanical lysis step, specifically bead beating, dramatically enhances DNA yield and WGS success rates for MTBC.
• This optimized protocol allows for earlier detection and genotyping of MTBC through WGS, reducing overall turnaround time.
• The bead beating method simplifies and accelerates DNA extraction, making WGS more accessible and efficient.