Targeted Sequencing of Respiratory Viruses in Clinical Specimens for Pathogen Identification and Genome-Wide Analysis

Abstract

A large number of viruses can individually and concurrently cause various respiratory illnesses. Metagenomic sequencing using next-generation sequencing (NGS) technology is capable of identifying a variety of pathogens. Here, we describe a method using a large panel of oligo probes to enrich sequence targets of 34 respiratory DNA and RNA viruses that reduces non-viral reads in NGS data and achieves high performance of sequencing-based pathogen identification. The approach can be applied to total nucleic acids purified from respiratory swabs stored in viral transport medium. Illumina TruSeq RNA Access Library procedure is used in targeted sequencing of respiratory viruses. The samples are subjected to RNA fragmentation, random reverse transcription, random PCR amplification, and ligation with barcoded library adaptors. The libraries are pooled and subjected to two rounds of enrichments by using a large panel of oligos designed to capture whole genomes of 34 respiratory viruses. The enriched libraries are amplified and sequenced using Illumina MiSeq sequencing system and reagents. This method can achieve viral detection sensitivity comparable with molecular assay and obtain partial to complete genome sequences for each virus to allow accurate genotyping and variant analysis.

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

“…An additional potential protocol difference is the number of samples pooled, which ranges from a single sample to 12 [9,10,15]. The current recommendation by Illumina for viral enrichment is to pool four samples [14].…”

“…A composite panel of 80-mer DNA probes was assembled using a previously described panel for respiratory viruses [13,14,20], a previously described panel for Filoviruses [9][10][11], plus an additional panel of newly designed probes for 41 viruses of biosurveillance and biodefense concern, for a total of 19,077 probes. The methods employed for capture oligo design were…”

“…In this protocol, viral enrichment is coupled with the RNA Access kit, developed by Illumina, Inc. The technical advancements of the RNA Access kit had already enabled the sequencing of previously unsequencable materials such as those of low concentration and formalin-fixed paraffin-embedded (FFPE) tissue [12], and now this protocol has been employed not only for the detection and characterization of EBOV from clinical samples but also for detection and characterization of respiratory viruses in clinical samples [13,14]. In general, hybridization-based viral target enrichment has been successfully employed to characterize viruses found within both contrived samples and clinical samples [3,9,10,13,[15][16][17][18][19][20].…”

“…In general, hybridization-based viral target enrichment has been successfully employed to characterize viruses found within both contrived samples and clinical samples [3,9,10,13,[15][16][17][18][19][20]. The performance of the Respiratory Virus Panel (RVP) version of this method [14], which uses probes for 34 common respiratory viruses in conjunction with the TruSeq RNA Access protocol, was recently investigated and it was demonstrated to work well overall when tested on human clinical samples [13]. Specifically, the authors reported successful enrichment for 30 of 33 human clinical samples tested.…”

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 ].…”