Negative Selection by Spiral Inertial Microfluidics Improves Viral Recovery and Sequencing from Blood

Abstract

In blood samples from patients with viral infection, it is often important to separate viral particles from human cells, for example, to minimize background in performing viral whole genome sequencing. Here, we present a microfluidic device that uses spiral inertial microfluidics with continuous circulation to separate host cells from viral particles and free nucleic acid. We demonstrate that this device effectively reduces white blood cells, red blood cells, and platelets from both whole blood and plasma samples with excellent recovery of viral nucleic acid. Furthermore, microfluidic separation leads to greater viral genome coverage and depth, highlighting an important application of this device in processing clinical samples for viral genome sequencing.

Figure 1.

(A) Schematic of closed-loop separation (“C-sep”) spiral microfluidic device platform with negative selection scheme (B) Photograph of experimental setup (C) Magnified view of spiral microfluidic device (D) Comparison between input (100 times diluted whole blood) and microfluidics (MF)-treated samples

Figure 2.

(A) Total number of cells (calculated from flow cytometry results) in untreated and MF-treated samples for whole blood and plasma samples. Each bar is drawn with data points of 9 samples. (B) Concentration of CMV (viral copies/uL) recovered as output from MF (yaxis) versus input (x-axis).

Figure 3.

(A) Metagenomic classification of sequencing reads by Kraken for samples derived from whole blood spiked with 10⁸ CMV copies/mL. (B) Normalized coverage depth of CMV whole genome assemblies for samples in (A). (C) Comparison of CMV reads before (untreated) and after (MF-treated) microfluidic treatment of whole blood samples spiked with a range of CMV viral loads (10⁴, 10⁶, 10⁸ copies/mL whole blood). (D) Comparison of CMV reads before (untreated) and after (MF-treated) microfluidic treatment of plasma samples (E) Plot showing the depth of coverage (y-axis) across the CMV genome (x-axis) in samples derived from whole blood spiked with 10⁶ CMV copies/mL. Depth of coverage is the number of sequenced reads that aligned to the CMV genome at each base pair position from 450,000 sampled reads.