DI-tector: defective interfering viral genomes' detector for next-generation sequencing data

Abstract

Defective interfering (DI) genomes, or defective viral genomes (DVGs), are truncated viral genomes generated during replication of most viruses, including live viral vaccines. Among these, "panhandle" or copy-back (cb) and "hairpin" or snap-back (sb) DI genomes are generated during RNA virus replication. 5' cb/sb DI genomes are highly relevant for viral pathogenesis since they harbor immunostimulatory properties that increase virus recognition by the innate immune system of the host. We have developed DI-tector, a user-friendly and freely available program that identifies and characterizes cb/sb genomes from next-generation sequencing (NGS) data. DI-tector confirmed the presence of 5' cb genomes in cells infected with measles virus (MV). DI-tector also identified a novel 5' cb genome, as well as a variety of 3' cb/sb genomes whose existence had not previously been detected by conventional approaches in MV-infected cells. The presence of these novel cb/sb genomes was confirmed by RT-qPCR and RT-PCR, validating the ability of DI-tector to reveal the landscape of DI genome population in infected cell samples. Performance assessment using different experimental and simulated data sets revealed the robust specificity and sensitivity of DI-tector. We propose DI-tector as a universal tool for the unbiased detection of DI viral genomes, including 5' cb/sb DI genomes, in NGS data.

Keywords: NGS; copy-back; defective interfering viral genome; panhandle; snap-back; viral replication.

FIGURE 1.

Schematic representation of the main classes of DI genomes that may arise from (A) a full-length negative-sense viral RNA genome and antigenome: (B) DI genomes with deletion, (C) mosaic genomes, (D) copy-back and (E) snap-back DI genomes. C′ represents C complementary region. ΔB and ΔB′ represent shorter B sequence and its complementary region. D represents sequence from other origin. (BP) Breakpoint site, (RI) reinitiation site.

FIGURE 2.

Schematic representation of the DI-tector workflow, which includes four main steps: (i) alignment against host and viral genomes to collect unmatched reads, (ii) segmentation, (iii) second alignment against viral genomes, and (iv) analyses of the matched segmented reads. Dashed lines represent optional steps.

FIGURE 3.

Schematic representation of DI-tector DI genome group clustering on a positive sense viral genome reference.

FIGURE 4.

Characterization of DI genomes identified by DI-tector in RNA samples generated from rMV-ΔV infected cells. (A) Counts of reads overlapping junction for DI_336 and DI_1236 in several rMV-ΔV samples. Values are followed by a star when reads only align on one strand. Data sets were generated from RNA samples obtained from ST-Cherry or ST-RIG-I rMV-ΔV infected cells before (Total) or after purification (Beads) (# 1 to 3 represent three biological triplicates). (B) Example of results’ representation of 5′ cb DI genomes of rMV-ΔV samples after RIG-I affinity purification (# 1 to 3 represent three biological triplicates). Red and gray lines represent, respectively, cb DI genome stem and loop.

FIGURE 5.

Validation of the presence of a 3′ cb DI genome (DI_2202) by RT-PCR in MV infected cells. A RT-PCR was performed on total RNA extracted from HEK293T cells infected with MV. A 3′ cb DI genome of 2202 bp was detected. The amplicon of the expected size of 1219 bp was sequenced.

FIGURE 6.

Impact of RiboZero treatment on SeV DI genomes’ detection. Example of results’ representation of 5′ cb DI genomes of SeV with and without RiboZero treatment (“--Min_Segment” = 15). Red and gray lines represent, respectively, cb DI genome stem and loop.

FIGURE 7.

Predictive performance assessment of DI-tector. Sensitivity and selectivity values using different “--Min_Segment” and “--Min_MAPQ”.

FIGURE 8.

Example of graphical outputs available using DI-tector online tool with rMV-ΔV sample (replicate #1) data (www.di-tector.cyame.eu). (A) Scatter-plot, (B) chord diagram, or (C) sequence logo for 5′ cb DI and DI genomes with deletion.