Using Carrier DNA in Ultra-Low Input Library Preparations for Next-Generation Sequencing

Area of Science:

• Metagenomics
• Molecular Biology
• Bioinformatics

Background:

• Shotgun metagenome sequencing requires sufficient DNA input for robust library preparation.
• Ultra-low DNA concentrations (below 50 picograms) pose challenges for standard library preparation kits.
• Carrier DNA (exogenous DNA spike-in) is a potential method to increase DNA input for low-concentration samples.

Purpose of the Study:

• To evaluate the effectiveness of carrier DNA in improving shotgun metagenome library preparation for ultra-low DNA inputs.
• To assess the impact of carrier DNA on library robustness, sequencing output, and data quality.
• To determine if carrier DNA is beneficial for routine library preparation of samples with minimal DNA.

Main Methods:

• Utilized adaptive polymerase chain reaction (PCR) cycling for dynamic thermocycling until sufficient library amplification.
• Sequenced libraries prepared with and without carrier DNA from ultra-low input samples (down to 50 fg).
• Mapped sequencing reads to reference genomes (Lambda and mock community organisms) and analyzed outcome measures including read counts, on-target reads, coverage evenness, and PCR duplication rate.

Main Results:

• Libraries could be prepared from as little as 50 fg of input DNA.
• A strong correlation was observed between input DNA concentration and PCR duplication rate.
• Carrier DNA showed mild negative impacts on mock community distribution and resulted in a loss of sequencing output, though it partially offset data loss from PCR duplication.

Conclusions:

• The utility of carrier DNA for ultra-low input metagenomic samples is equivocal.
• Carrier DNA spike-in is not recommended for routine library preparation due to negative impacts on data quality and sequencing efficiency.
• Adaptive cycling PCR is an effective method for library preparation when input DNA concentrations are below detection limits.