Enrichment post-library preparation enhances the sensitivity of high-throughput sequencing-based detection and characterization of viruses from complex samples

Abstract

Background Sequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids. Hybridization-based target enrichment is one potential method for increasing the sensitivity of viral detection via high-throughput sequencing. Results This study expands upon two previously developed panels of virus enrichment probes (for filoviruses and for respiratory viruses) to include other viruses of biodefense and/or biosurveillance concern to the U.S. Department of Defense and various international public health agencies. The newly expanded and combined panel is tested using carefully constructed synthetic metagenomic samples that contain clinically relevant amounts of viral genetic material. Target enrichment results in a dramatic increase in sensitivity for virus detection as compared to shotgun sequencing, yielding full, deeply covered viral genomes from materials with Ct values suggesting that amplicon sequencing would be likely to fail. Increased pooling to improve cost- and time-effectiveness does not negatively affect the ability to obtain full-length viral genomes, even in the case of co-infections, although as expected, it does decrease depth of coverage. Conclusions Hybridization-based target enrichment is an effective solution to obtain full-length viral genomes for samples from which virus detection would fail via unbiased, shotgun sequencing or even via amplicon sequencing. As the development and testing of probe sets for viral target enrichment expands and continues, the application of this technique, in conjunction with deeper pooling strategies, could make high-throughput sequencing more economical for routine use in biosurveillance, biodefense and outbreak investigations. Electronic supplementary material The online version of this article (10.1186/s12864-019-5543-2) contains supplementary material, which is available to authorized users.

Unveiling Viruses Associated with Gastroenteritis Using a Metagenomics Approach

“…A target enrichment approach (TES) was performed in one pool (F1-3A) to evaluate its usefulness for the detection of gastroenteritis viral agents compared with untargeted metagenomics (UVM) applied in this study. The application of a target enrichment probe-based capture method, prior to sequencing, has been demonstrated to overcome the low sensitivity or the lack of sequencing depth in complex samples with large viral diversity [ 12 , 101 ].…”

Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection

“…HTS would need to be compared with qPCR if it is to become routinely used. The sensitivity of HTS is influenced by genomic size 22 , 23 , as well as genomic structure, and for RNA viruses, the relative efficiencies in reverse transcription and cDNA synthesis 22 . Nevertheless, HTS is virus-sequence independent and can detect all types of virus genomes, including single-stranded/ double-stranded RNA and DNA.…”

“…HTS may also be more sensitive than quantitative PCR (qPCR) 19 , 20 ; however the method may be overly sensitive to the detection of background and cross-contaminating viral nucleic acids originating from the laboratory environment or from other sources 21 . Viral genome size can also influence the sensitivity of HTS 22 , 23 as sensitivity is expected to be proportional to the mass ratio of nucleic acids in a given matrix. Although HTS results for adventitious virus testing may differ between laboratories 24 , the development of well-characterized model virus stocks would support standardization and validation of the different HTS platforms 22 .…”

Comparison of Nucleic Acid Extraction Methods for a Viral Metagenomics Analysis of Respiratory Viruses

“…The development of metagenomics next-generation sequencing (mNGS) has enabled the exploration of whole viral nucleic acids within a clinical sample (human virome) in order to detect pathogens not targeted by conventional PCR and to identify emerging viruses [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. Several studies have used various mNGS protocols to explore the human virome in diverse clinical samples, including human stools [ 9 , 10 ], blood [ 11 , 12 ], cerebrospinal fluid [ 13 , 14 ], human tissues [ 15 , 16 ], and respiratory tract samples [ 17 , 18 , 19 , 20 , 21 ].…”