False-Positive Results in Metagenomic Virus Discovery: A Strong Case for Follow-Up Diagnosis

Abstract

A viral metagenomic approach using virion enrichment, random amplification and next-generation sequencing was used to investigate an undiagnosed cluster of dairy cattle presenting with high persistent fever, unresponsive to anti-microbial and anti-inflammatory treatment, diarrhoea and redness of nose and teat. Serum and whole blood samples were taken in the predicted hyperviraemic state of an animal that a few days later presented with these clinical signs. Bioinformatics analysis of the resulting data from the DNA virus identification workflow (a total of 32 757 sequences with average read length 335 bases) initially demonstrated the presence of parvovirus-like sequences in the tested blood sample. Thorough follow-up using specific real-time RT-PCR assays targeting the detected sequence fragments confirmed the presence of these sequences in the original sample as well as in a sample of an additional animal, but a contamination with an identical genetic signature in negative extraction controls was demonstrated. Further investigation using an alternative extraction method identified a contamination of the originally used Qiagen extraction columns with parvovirus-like nucleic acids or virus particles. Although we did not find any relevant virus that could be associated with the disease, these observations clearly illustrate the importance of using a proper control strategy and follow-up diagnostic tests in any viral metagenomic study.

Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

“…Of course molecular identification of viral sequences is only the first step in determining whether or not it is associated with disease. Follow-up studies are needed to prove a link between a candidate infectious agent and disease (Chiu, 2013;Lipkin, 2009;Rosseel et al, 2014). Some recommendations to minimize contamination issues would be (1) alternating use of a large set of barcodes, (2) do not simultaneously make libraries in the same PCR cabinet, (3) perform the recommended maintenance wash steps of your sequencing instrument in between 2 subsequent sequencing runs, (4) avoiding multiplexing samples of different metagenomics experiments on the same sequencing run, and (5) using a different NGS platform with less carryover issues.…”

“…Two pretreatment steps (0.45 m filtration followed by nuclease treatment) were performed on 1 ml of spiked sample as described previously (Rosseel et al, 2014). The nuclease treatment step was performed using 50 U TURBO DNase (2 U/l, Life Technologies), 1× TURBO DNase Buffer and 10 U RiboShredder RNase Blend (1 U/l, Epicentre Technologies) as reported previously (Rosseel et al, 2014 …”

“…The nuclease treatment step was performed using 50 U TURBO DNase (2 U/l, Life Technologies), 1× TURBO DNase Buffer and 10 U RiboShredder RNase Blend (1 U/l, Epicentre Technologies) as reported previously (Rosseel et al, 2014 …”

“…Sequence independent PCR amplification was performed as previously described (Rosseel et al, 2014) on a single replicate of the 2-step virion enriched samples (control & rRNA removal). Random PCR fragments were size selected on a 1% agarose gel, and fragments between 200 and 800 bp were excised and purified using the High Pure PCR Product Purification Kit (Roche).…”

Challenges associated with research on RNA viruses of insects

“…For (+) ssRNA viruses, strand-specific RNA sequencing with good validation [51] may help to detect the negative strand (replicative intermediate); but regular RNA-seq and other NGS techniques cannot. Then researchers should be aware of the limitations of massive sequencing tools and other Àomics approaches to avoid misinterpretation [52][53][54].…”