The virus variant caller pipeline was implemented as a workflow for the workflow management system Watchdog and is available at https://github.com/watchdog-wms/watchdog-wms-workflows/ (workflow VariantCallerPipeline). The workflow takes as input read alignments against the viral genome in BAM format for one or more virus-infected samples. Read sequences in FASTQ format are only required if inserted sequences are to be identified, which is an optional step. We used BWA ²³ for read alignment as it is very fast and requires little memory, but any read alignment program can be used that provides SAM/BAM output, includes read sequences in the output and produces clipped read alignments if only parts of a read can be aligned to the viral genome. Notably, since we are not interested in identifying splicing events, which are rare in viruses, there is no need to use a splicing-aware aligner for RNA-seq data.

The variant caller pipeline is divided into two main parts, which are described in the following: (1) SNP calling and (optionally) strain identification and (2) indel detection and (optionally) identification of inserted sequences.

SNP calling

Figure 2 provides an overview of the steps performed for SNP calling. First, the variant callers bcftools ¹⁶ and VarScan ¹⁷ (after running ‘samtools mpileup’ ²⁴ ) are applied independently to each input BAM file. Both tools provide the identified SNPs in the variant call format (VCF). ²⁵ Next, so-called consistent SNPs are determined that are identified by both bcftools and VarScan. If more than one replicate is available, SNPs are considered consistent if they are detected by both tools in all replicates. Consistent SNPs are then mapped to viral features, e.g., genes, coding sequences, or introns, given a gene annotation in GTF format for the viral genome.

Furthermore, if a set of reference SNPs for different virus strains is provided by the user, the pipeline performs a prediction of the virus strain for each sample. This is useful both for verifying the virus strain used in the experiment and the parental strain from which a particular null mutant was generated. Such reference SNPs can be obtained by identifying consistent SNPs with our pipeline for functional genomics data of various virus strains. An example file with reference SNPs for HSV-1 strains 17, F and KOS 1.1 is included with example input files at https://doi.org/10.5281/zenodo.14266852 and the Watchdog module for strain identification (identifyStrain, available at https://github.com/watchdog-wms/watchdog-wms-modules).

For strain identification, the following distance D is calculated for each reference strain: D = | S 1 ∪ S 2 | − | S 1 ∩ S 2 | , with S 1 the set of consistent SNPs identified for the virus used in the experiment and S 2 the set of reference SNPs for a reference strain. The strain with the smallest distance D is then predicted for the virus. This measure is largely independent of the reference genome sequence used for read alignment. For illustration, consider the following example. Assume a sample S that was derived from strain X but is aligned against the genome sequence of a different strain Y . Furthermore, reference SNPs for strains X , Y and Z were also obtained by aligning functional genomics data for these strains against the genome for strain Y . This will result in a (relatively) large number of consistent SNPs for sample S , no/few reference SNPs for strain Y and (relatively) large numbers of reference SNPs for strains X and Z . Since consistent SNPs for S and reference SNPs for X will be largely the same, the distance will be close to zero. The distance for Y and Z will be larger since consistent SNPs for S are not in the reference set for Y and will differ from reference SNPs for Z .

Indel detection

Insertions and deletions in viral genomes are determined as outlined in Figure 3. First, per-base read coverage, i.e. the number of reads overlapping each genome position, and clipped reads (= reads with unaligned parts) are extracted from each input BAM file using samtools. ²⁴ The results are then used as input for indel calling as described below. Subsequently, identified indels are also mapped to genomic features. In addition, the pipeline can identify inserted sequences by combining the results from insertion detection with de novo read assembly obtained with rnaSPAdes ²¹ if raw read sequences in FASTQ format are provided.

Candidate deletion detection

The pipeline first detects potential deletions by identifying regions of the genome with very low read coverage compared to (i) the complete genome using a global threshold and (ii) the surrounding genomic regions using a local threshold. For this purpose, a global z-score is calculated for each position, comparing the logarithm of the read coverage (= log read coverage) for this position to the mean and standard deviation of the log read coverage for the complete genome. If this is below a stringent global threshold, the position is labelled as a potential deletion. If it passes only a less stringent global threshold, a local z-score is calculated comparing the log read coverage at this position to the mean and standard deviation of the previous n nucleotides (nt) before the current position (by default n = 500 ). If the local z-score is below the local threshold, the position will also be labelled as a potential deletion.

The local z-score is used as read coverage can vary massively between positions in functional genomics data. This is exemplified for an RNA-seq sample in Figure 1. However, calculating local z-cores for every position is very costly as it requires calculating the mean and standard deviation over the preceding n nt for every genomic position. Thus, the stringent global z-score threshold is first employed to identify clear-cut cases of potential deletions. Local z-scores are only calculated for less clear-cut cases. Optionally, a user-defined length threshold can also be used to exclude very short deletions.

Deletion verification

Candidate deletions are subsequently verified using clipped read alignments. As depicted in Figure 4, reads crossing a deletion in the genome can only be aligned with gaps to the reference genome. If the alignment is performed using a non-splice-aware read aligner, such as BWA, this will result in clipped read alignments where only parts of the read are aligned to the genome. Notably, this often also occurs with splice-aware read aligners as the start and end nucleotides of the deletion generally do not match canonical splicing signals expected by many splice-aware read aligners.

Deletions should exhibit a peak of right-clipped reads ending at the deletion start and a peak of left-clipped reads beginning at the deletion end. Such peaks of clipped reads are again identified using both a global and local z-score, both of which are calculated separately for peaks of right-clipped and left-clipped reads. For the global z-score at each position, the number of clipped reads is compared against the mean and standard deviation for the same type of clipped reads across the whole genome. For the local z-score, the number of clipped reads is compared against the mean and standard deviation for a window starting x nt upstream of the candidate peak and ending x nt downstream of the candidate peak (by default x = 20 ), excluding the peak position itself. If both the global and the local z-scores pass a global (default: 10) and local (default: 50) threshold, respectively, the position is considered a peak. In addition, a minimum number of reads is required for a peak (default: 10 reads).

To verify deletions, the pipeline identifies pairs of right-clipped and subsequent left-clipped peaks (i.e. the clipping pattern of deletions) and determines whether the positions of the two peaks overlap with a candidate deletion detected based on the per-base read coverage. Subsequently, the clipped sequences of the corresponding clipped read alignments (i.e. the unaligned part of the read in this alignment) are extracted from the BAM file and position-weight-matrices (PWMs) are computed from the sequence profiles of the clipped sequence parts. As can be seen in Figure 4, the PWMs of the clipped sequence parts on either side of the deletion should match the reference sequence on the opposite side of the deletion.

To test this, the best match of each PWM is determined in a window around the opposite deletion end. The score of a potential match is calculated as the sum of log-odds scores over all positions comparing the value of the PWM for the nucleotide at this position against the background probability of that nucleotide in the complete genome sequence. The best match for a PWM is the match with the highest score. If the best matches for both PWMs have a score >0 or at least one has a score >1, the deletion is accepted. If neither match is good enough, the deletion is flagged as a potential deletion that may contain an insertion. This special case was observed for one of the data sets analysed in the results section. In this case, the potential insertion sequence is determined as described in the next section and can be further analysed.

It should be noted that our approach for predicting deletions may also identify splicing events in RNA-seq data. However, splicing is rare in viruses and the few cases detected can easily be excluded after mapping the deletions to the genome annotation. For instance, even a very thorough re-annotation of the HSV-1 genome, a relatively large viral genome of ~152 kb, based on short- and long-read RNA-seq identified only 15 splicing events. ²⁶ Most of those had only low abundance compared to the corresponding unspliced transcripts.

Insertion detection

Our pipeline also uses clipped read alignments to determine insertions since reads containing part of an inserted sequence cannot be completely aligned to the genome (see Figure 5). Originally, we expected that the resulting clipping pattern should consist of a peak of right-clipped reads at a reference genome position n preceding the insertion in the genome followed by a peak of left-clipped reads at position n + 1 . However, the examples of null mutants created by insertions that we investigated showed a different pattern, consisting of a peak of left-clipped reads at position n and a peak of right-peaked reads at position n + 1 (see Figure 5). This results from the first and last position of inserted sequences matching the genome on the other side of the insertion and is likely a consequence of the use of homologous recombination for inserting sequences. ²⁷

To allow for such matches between the insertion start and/or end to the surrounding genomic regions, we introduced a parameter ϕ determining the maximum number of such matches that are allowed. Thus, any pair of positions for a left-clipped peak p l and a right-clipped peak p r is used to predict an insertion if p r − p l + 1 ≤ ϕ . In the example in Figure 5, p r − p l + 1 = 2 . For each identified insertion, we extract the non-aligned parts of clipped reads to calculate consensus sequences for the insertion start and end, respectively. These consensus sequences are commonly 30-40 nt long.

To identify the remaining central part of the inserted sequences, the pipeline optionally performs a de novo sequence assembly using rnaSPAdes, ²¹ a modification of the genome assembler SPAdes ²⁸ for application to RNA-seq data. Assembly is performed for all reads, which also includes reads from non-viral sequences, in particular the inserted sequences. Following this, the consensus sequences for the insertion start and end are aligned to the resulting assembled contigs using BWA. If a match for both consensus sequences is found, the assembled sequence starting with the consensus of the insertion start and ending with the consensus of the insertion end is extracted. Insertion sequences containing only one of the consensus sequences are also extracted but are flagged for special attention. The origin of the inserted sequences can then be confirmed using BLAST. ²⁹

We also investigated whether de novo assembly alone was sufficient for detection of both insertions and deletions by aligning the assembled contigs to the viral reference genome (see results). However, this either resulted in too few or too many indels depending on parameters, thus we did not pursue this approach for the pipeline.

Watchdog and the VariantCallerPipeline can be run on Linux and MacOS systems. Running Watchdog requires Java 11 or higher. The deployment of required software during the VariantCallerPipeline run is performed with conda ( https://conda.io, using conda-forge and bioconda channels) using the deployment functionality of Watchdog. Watchdog also supports easy parallelization of workflow runs on computing clusters and monitoring of workflow execution, which can be used when running our pipeline. Example input files can be found at https://doi.org/10.5281/zenodo.14266852. A detailed README on installing and running the pipeline can be found at https://github.com/watchdog-wms/watchdog-wms-workflows/ in the VariantCallerPipeline directory.

We applied our pipeline to previously published 4sU-seq data for infection with null mutants for multiple HSV-1 proteins. ¹⁴ 4sU-seq is a variant of RNA-seq based on sequencing newly transcribed RNA obtained by RNA labelling with 4-thiouridine (4sU). ³⁰ 4sU-seq was performed for null mutant viruses of the following HSV-1 proteins: •

ICP4, null mutant created from HSV-1 strain 17 by a SNP, which resulted in a temperature sensitive mutant (TsK) ^{3, 5}

The precise genomic location for these null mutants have not been described and most of these were created before the first HSV-1 genome sequence (for strain 17) was completed in 1988. ³¹ Two replicates were available for all null mutant viruses, except for the ICP4 knockout by insertion (ΔICP4), for which only one replicate was performed.

In addition, we analysed RNA-seq data for human brain organoids ²² infected with an HSV-1 strain 17 virus engineered to express GFP. ¹² Here, RNA-seq data was available for brain organoids from two genetically distinct induced pluripotent stem cell lines, each infected for 3 and 6 days (2 replicates each, resulting in 8 samples).

All 4sU-seq and RNA-seq samples were aligned against the HSV-1 strain 17 genome (GenBank accession: JN555585) using BWA and then fed into the pipeline. The HSV-1 genome contains two repeat regions at each end of the genome that are repeated internally in the genome. Since read alignment cannot distinguish between the two repeats, one occurrence of each repeat (i.e. the ones at the genome ends) was replaced by N’s for read alignment.

The performance of the pipeline was evaluated by comparing the results with the descriptions of the original publications. The insertion sequences that were extracted by the pipeline from the sequence assembly were investigated with the NCBI BLAST webserver to identify their origin.

Our pipeline identified 28 consistent SNPs in the TsK mutant, three of which were in the ICP4 gene. One of these was consistent with the sequence change identified by Davison et al. ⁵ as responsible for the mutant phenotype: a replacement of a C:G base pair by a T:A base pair that changed the 475th codon of the ICP4 gene from an alanine codon to a valine codon. Our pipeline matched this missense mutation to a SNP at nucleotide 129,708. It furthermore showed that the TsK mutant differed from its parental strain 17 by an additional 27 SNPs, whose effects remain unclear. In particular, one of the other two SNPs identified in the ICP4 gene leads to a second amino acid change in ICP4 from serine to asparagine.

To detect deletions, our pipeline was run with a stringent global z-score cut-off of -2.5, a less stringent global cut-off of 0.0 and a local z-score cut-off of -6.0. No minimum length was required for the deletions. This resulted in the identification of the deletions shown in Table 1. We identified a deletion each in the ICP0 null mutant (ΔICP0) and the ICP22 null mutant (ΔICP22), respectively, that matched the target gene and approximate length described in the corresponding articles. ^{4, 6, 7} Furthermore, the sequences found directly up- and downstream of the predicted deletions matched the target sequences of the restriction enzymes used in the corresponding experiments to create the deletions (XhoI & SalI for ΔICP0; PvuII & BstEII/Eco91I for ΔdICP22). Thus, we could recover the exact locations of the introduced deletions.

A further deletion in the 5’ UTR of ICP22 identified in ΔICP22 infection corresponded to a genome deletion in the parental strain F from which the ΔICP22 virus was derived. Similarly, a deletion identified in the ΔICP27 virus is already present in its parental strain KOS 1.1. In addition, a deletion was identified for the ICP27 null mutant (ΔICP27) and the vhs null mutant (Δvhs) that fell into a known intron in the ICP22 gene. Although this intron is spliced in all samples, it was not detected in ΔICP0, ΔICP4 and TsK infection. For ΔICP4 and TsK infection this was likely due to the fact that read coverage on the whole viral genome was relatively low as ICP4 is necessary for optimal expression of other HSV-1 genes. ³² For ΔICP0 infection the opposite applied as both replicates had by far the highest read coverage on the viral genome of any of the samples. As a consequence, sufficient numbers of reads from unspliced ICP22 transcripts were detected for the intron not to be identified as a deletion.

Insertions were also predicted using default values. Local z-scores were calculated for the 40 nt around each peak position, at least 10 clipped reads were required for each peak and a maximum overlap ϕ of 10 nt was allowed for the insertion ends and the surrounding genome regions. Furthermore, the consensus sequences obtained from the clipped parts of the read had to be at least 10 nt long. The identified insertions are listed in Table 2.

All but one of the identified insertions matched the description in the corresponding publications on how the null mutants were created. ^{8– 11} In particular, we could confirm the insertion of lacZ genes in both the ΔICP27 and Δvhs virus by BLASTing the predicted insertion sequences obtained from the assembly. For the ΔICP4 mutant, the insertion of a small 16 nt sequence could be directly confirmed from the consensus sequences of the insertion start and end as these overlapped. This insertion sequence contained the 3 stop codons, one for each frame, and a recognition site of the HpaI restriction enzyme described in the original publication. The additional insertion identified in the ΔICP27 virus represented a known insertion in the parental strain KOS 1.1.

Interestingly, we found that the position for the insertion in the vhs coding sequence (251st codon) described in the corresponding publication for the Δvhs mutant ¹⁰ may have been calculated based on a wrong strand assignment. The vhs gene is located on the negative strand, with the coding sequence ranging from positions 91,167 (stop codon) to 92,636 (start codon). Accordingly, the insertion position identified by our pipeline (91,923) is in the 238th codon. However, if the codon position is erroneously calculated from the positive strand, the insertion would be after the first position of the 252nd codon (excluding the stop codon), which is closer to the original publication. Since the insertion site was described to be in the unique recognition site of the NruI restriction enzyme in the vhs gene ¹⁰ and the centre of this NruI recognition site is at the insertion position identified by our pipeline, this is indeed the correct position.

According to the original publication describing this virus, ¹² an enhanced GFP (EGFP) gene with a mouse cytomegalovirus promoter was inserted between the open reading frames (ORFs) UL55 and UL56. In addition, a LoxP site (= a 34 nt DNA sequence recognized by the Cre recombinase enzyme) was inserted downstream of the UL23 ORF. Two insertion sites at positions 46,665 and 116,147 were identified in 8 and 7 of the samples, respectively, located downstream of the UL23 coding sequence and between UL55 and UL56, respectively. The insertion sequence for the first insertion indeed contained a LoxP site and BLAST analysis of the insertion sequence for the second insertion site showed that it matched several cloning vectors containing the GFP gene. Thus, we correctly identified the precise genome positions of both insertions.

It should be noted that the insertion at position 116,147 was actually identified as a deletion between positions 116,147 and 116,154 into which an insertion was placed. This special case is predicted by the pipeline if the PWMs obtained from the clipped reads cannot be matched to the opposite end of the deletion and an insertion sequence can be identified from the assembly. Unfortunately, the description in the original publication on how the sequence was inserted is not sufficiently detailed to explain how this small deletion was generated during the insertion process, but it is most likely a consequence of the experimental approach used.

Additional insertions were identified at positions 62,143, 106,984, and 119,496 in 4-8 of the samples. However, no insertion sequences could be extracted from the assembly for insertions at positions 62,143 and 106,984 based on the consensus sequences from the clipped reads, while the insertion sequence for 119,496 matched the genome downstream of the predicted insertion site. Based on these results and inspection of the genome at these positions, we concluded that these represented artefacts from repetitive sequences. This highlights how the combination of consensus sequences from the clipped parts of reads and the assembly can be used to filter out incorrectly identified insertions.

For comparison, we also investigated whether deletions and insertions could be identified directly from the contigs assembled by rnaSPAdes instead of performing the analysis of read coverage and clipped read alignments performed by our pipeline. For this purpose, contigs assembled for the 4sU-seq data of HSV-1 mutant infections were aligned against the reference genome using minimap2. ³³ However, this showed that assembled contigs often contained small indels (~1-50bp) compared to the reference genome, which would result in a large number of predicted indels if we included all of them. Thus, we evaluated different minimum length thresholds on identified indels. Furthermore, we observed that for some insertions the inserted sequence was only partially assembled and thus located at the start or end of assembled contigs. This resulted in a clipped alignment of these contigs to the genome. We thus also evaluated the option to include such clipped alignments to identify the position of the insertion and at least the start or end of the inserted sequence.

Figure 6 shows an evaluation of different thresholds on the indel length with and without inclusion of clipped contig alignments for insertion detection. This showed that a relatively small minimum indel length of 16 nt had to be used to identify all indels and clipped contig alignments had to be included. Higher minimum indel lengths excluded the 16 nt insertion in the ΔICP4 mutant, while the lacZ gene insertion in the ΔICP27 mutant would be missed without allowing clipped contig alignments. However, this parameter combination resulted in large numbers of predicted insertions for ΔICP0, ΔICP22, ΔICP27, and Δvhs mutants, making it difficult to distinguish the correct indels in these mutants.