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Updated: Oct 15, 2025
Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing
Published on: August 25, 2018
Measurement Biases Distort Cell-Free DNA Fragmentation Profiles and Define the Sensitivity of Metagenomic Cell-Free
Adrienne Chang1, Omary Mzava1, Joan S Lenz1
1Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
Background:
Metagenomic sequencing of microbial cell-free DNA (cfDNA) in blood and urine is increasingly used as a tool for unbiased infection screening. The sensitivity of metagenomic cfDNA sequencing assays is determined by the efficiency by which the assay recovers microbial cfDNA vs host-specific cfDNA. We hypothesized that the choice of methods used for DNA isolation, DNA sequencing library preparation, and sequencing would affect the sensitivity of metagenomic cfDNA sequencing.
Methods:
We characterized the fragment length biases inherent to select DNA isolation and library preparation procedures and developed a model to correct for these biases. We analyzed 305 cfDNA sequencing data sets, including publicly available data sets and 124 newly generated data sets, to evaluate the dependence of the sensitivity of metagenomic cfDNA sequencing on pre-analytical variables.
Results:
Length bias correction of fragment length distributions measured from different experimental procedures revealed the ultrashort (<100 bp) nature of microbial-, mitochondrial-, and host-specific urinary cfDNA. The sensitivity of metagenomic sequencing assays to detect the clinically reported microorganism differed by more than 5-fold depending on the combination of DNA isolation and library preparation used.
Conclusions:
Substantial gains in the sensitivity of microbial and other short fragment recovery can be achieved by easy-to-implement changes in the sample preparation protocol, which highlights the need for standardization in the liquid biopsy field.
