Optimized whole-genome amplification strategy for extremely AT-biased template.

Area of Science:

• Genomics
• Molecular Biology
• Parasitology

Background:

• Direct pathogen genome sequencing from clinical samples is crucial for genetic and medical research.
• Low DNA yield, particularly from AT-rich genomes like Plasmodium falciparum, limits sequencing success.
• Existing whole-genome amplification (WGA) methods can introduce bias and chimeras.

Purpose of the Study:

• To develop an improved WGA strategy for low-yield Plasmodium falciparum DNA.
• To enhance amplification and coverage of AT-rich genomic regions.
• To minimize amplification bias and chimera formation for short-read sequencing.

Main Methods:

• Developed novel WGA conditions incorporating tetramethylammonium chloride (TMAC).
• Applied the WGA method to low-input Plasmodium falciparum DNA samples (as low as 10 pg).
• Evaluated amplification efficiency, coverage uniformity, bias, and chimera formation.

Main Results:

• The TMAC-inclusive WGA method significantly improved amplification and coverage of AT-rich regions.
• Reduced amplification bias and chimera formation compared to standard WGA protocols.
• Demonstrated successful WGA from as little as 10 pg of input DNA.

Conclusions:

• The developed WGA strategy effectively addresses DNA yield limitations for Plasmodium falciparum sequencing.
• This method enables reliable genome sequencing from small blood volumes, facilitating parasite genetic studies.
• Improved WGA protocols are essential for advancing pathogen genomics from clinical samples.