Complete genome sequence of a KI polyomavirus isolated from an otherwise healthy child with severe lower respiratory tract infection

Abstract

Unbiased, deep sequencing of a nasal specimen from an otherwise healthy 13-month-old boy hospitalized in intensive care revealed high gene expression and the complete genome of a novel isolate of KI polyomavirus (KIPyV). Further investigation detected minimal gene expression of additional viruses, suggesting that KIPyV was potentially the causal agent. Analysis of the complete genome of isolate NMKI001 revealed it is different from all previously reported genomes and contains two amino acid differences as compared to the closest virus isolate, Stockholm 380 (EF127908). J. Med. Virol. 89:926-930, 2017. © 2016 Wiley Periodicals, Inc.

Comprehensive viral enrichment enables sensitive respiratory virus genomic identification and analysis by next generation sequencing

“…Virus-specific probes were designed to span the genome, with tiling probes targeted against a selection of common respiratory viruses ( Supplemental Table S8) as previously described (Dehority et al 2017). Prior to probe design and selection, viral sequences were masked for low-complexity sequences and common repetitive elements in the human transcriptome (e.g., transposable elements).…”

“…Library preparation and target enrichment were performed using a TruSeq RNA Access Library Prep kit (currently, TruSeq RNA Exome kit; Illumina) which contains reagents for both library preparation and hybridization (Dehority et al 2017). Briefly, 10-50 ng of viral NA mixture or up to 5 µL of clinical sample NAs were used for library preparation.…”

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

“…The targeted spike-in values were chosen based on reports in the literature for clinical samples containing each virus to mimic a realistic co-infection scenario [22][23][24][25]. Given that in the literature there is at least one report of clinical samples being probed singly rather than pooled and probed [9] and it is not well known how pooling may affect virus detection levels, in this [13,20] Human Bocavirus 2 5196 ssDNA NC_012042.1 Previously used in [13,20] Human Bocavirus 3 5242 ssDNA NC_012564.1 Previously used in [13,20] experiment we also sought to evaluate whether probing singly or within a pool would affect our ability to identify virus. Therefore, total RNA extracted from these samples was probed singly ("pool of 1") and also pooled in groups of four and 12 with singly-spiked and mock-spiked serum samples consisting of the other components of the pool.…”

“…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…”

“…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]. 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].…”