Detection of Viral Pathogens With Multiplex Nanopore MinION Sequencing: Be Careful With Cross-Talk

Abstract

Metagenomic sequencing with the Oxford Nanopore MinION sequencer offers potential for point-of-care testing of infectious diseases in clinical settings. To improve cost-effectiveness, multiplexing of several, barcoded samples upon a single flow cell will be required during sequencing. We generated a unique sequencing dataset to assess the extent and source of cross barcode contamination caused by multiplex MinION sequencing. Sequencing libraries for three different viruses, including influenza A, dengue, and chikungunya, were prepared separately and sequenced on individual flow cells. We also pooled the respective libraries and performed multiplex sequencing. We identified 0.056% of total reads in the multiplex sequencing data that were assigned to incorrect barcodes. Chimeric reads were the predominant source of this error. Our findings highlight the need for careful filtering of multiplex sequencing data before downstream analysis, and the trade-off between sensitivity and specificity that applies to the barcode demultiplexing methods.

Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples

“…Despite time reductions in wet-laboratory processing, this method requires further modification to simplify and accelerate the protocol if it is to become viable as a near-to-patient test. High error rates are a recognized concern in Nanopore sequence data, and cross-barcode contamination can create challenges when low-and high-titer samples are batched (21). To avoid these problems, we batched samples according to C T value and applied stringent barcode demultiplexing criteria; however, this reduces the total data available for analysis, typically by ϳ50% but with variation between sequencing runs (21).…”

“…High error rates are a recognized concern in Nanopore sequence data, and cross-barcode contamination can create challenges when low-and high-titer samples are batched (21). To avoid these problems, we batched samples according to C T value and applied stringent barcode demultiplexing criteria; however, this reduces the total data available for analysis, typically by ϳ50% but with variation between sequencing runs (21). For future primary diagnostic use, it would be preferable to sequence samples individually using a lower-throughput flow cell, e.g., ONT Flongle (each paired with a negative-extraction-control sample, for which a prior spike with Hazara virus, using the same methods described here, would remain appropriate).…”

“…Data yield varied between flow cells (range, 2.5 ϫ 10 6 to 13.2 ϫ 10 6 reads from up to 12 multiplexed samples). Within flow cells, barcode performance was inconsistent when using a stringent, dual-barcode, demultiplexing method (21). From each clinical sample, the range of total reads generated was 1.0 ϫ 10 5 to 2.4 ϫ 10 6 (median, 3.8 ϫ 10 5 reads) (Table S1).…”

“…Irrespective of the test used, most clinical diagnostic facilities report a nonquantitative (binary) diagnostic result, and the data routinely generated for influenza diagnosis have limited capacity to inform insights into epidemiological linkage, vaccine efficacy, or antiviral susceptibility. On these grounds, there is an aspiration to generate new diagnostic tests that combine speed (incorporating the potential for POCT [18,19]), sensitivity, detection of coinfection (20,21), and generation of quantitative or semiquantitative data that can be used to identify drug resistance and reconstruct phylogeny to inform surveillance, public health strategy, and vaccine design.…”

Current Trends in Diagnostics of Viral Infections of Unknown Etiology

“…This poses a problem that is particularly important for the research of drug resistance, the substrate for which is often a few single-nucleotide polymorphisms, particularly in RNA-viruses [132]. Xu et al suggest that incorrect sample differentiation and chimeric reads that emerge during sequencing with MinION might significantly alter the experiment outcome [197].…”