The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient...

Area of Science:

• Virology
• Molecular Biology
• Bioinformatics

Background:

• Sequence Independent Single Primer Amplification (SISPA) is a common method for preparing viral sequencing templates.
• SISPA utilizes random primers with a defined tag for amplification, but often results in biased sequence read distribution.
• This bias can affect downstream applications like viral metagenomics.

Purpose of the Study:

• To investigate the mechanisms causing biased sequence depth in random amplification methods.
• To identify factors contributing to sequence read distribution bias in SISPA.
• To optimize random nucleic acid amplification protocols for improved viral sequencing.

Main Methods:

• In silico analysis of sequence depth, GC-content, RNA secondary structure, and tag sequence complementarity using Avian paramyxovirus type 8.
• Experimental validation using fragmented and non-fragmented viral RNAs and primers with varying random oligomer lengths and tag sequences.
• Assessment of oligonucleotide annealing bias.

Main Results:

• The tag sequence was identified as the primary contributor to the observed bias in sequence depth.
• Extending the random oligomer length and using different tag sequences experimentally reduced the bias.
• In silico combination of data from SISPA experiments with different tags also mitigated the bias.

Conclusions:

• The tag sequence in SISPA plays a crucial role in sequence depth bias.
• Optimizing primer design (e.g., longer random oligomers) and data analysis strategies can reduce amplification bias.
• These findings enhance the reliability of SISPA for viral sequencing, metagenomics, and microarray analysis.