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Related Experiment Video

Updated: May 7, 2026

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE

Published on: June 26, 2019

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

Ping Li¹, Jeremy Kahsen¹, Karen Olsson-Francis²

¹Genomics and Microbiome Core Facility Rush University Medical Center.

Journal of Biomolecular Techniques : JBT

|May 6, 2026

Summary

This summary is machine-generated.

Adding carrier DNA to ultra-low input metagenomic samples did not significantly improve library preparation. While it enables sequencing of extremely low DNA amounts, carrier DNA can negatively impact mock community distributions and reduce sequencing output.

Keywords:

DNA library preparation next-generation sequencing shotgun metagenome sequencing

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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.