Universal human papillomavirus typing by whole genome sequencing following target enrichment: Evaluation of assay reproducibility and limit of detection

- eWGS showed high correlation in the number of reads mapped to HPV reference genomes between the two flow-cell lanes within (R 2 = 1) and between experiments (R 2 = 0.99).
- The number of mapped reads was positively correlated to copy number ( β = 13.9, p <.
- The limit of blank (LOB) could be calculated based on mapped reads to HPV types not included in each sample.
- HPV genotyping was reproducible for all 9 types at 625 copies using multiple cut-off criteria but LOD was 25 copies based on number of reads above LOB even when multiple types were present.
- There are more than 200 HPV types in 5 of the 45 genera within Papillomaviridae (Alphapa- pillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus and Nupapillomavirus).
- Conventional typing assays do not capture information on additional viral characteristics, such as integration and variant status within HPV type, therefore, systematic evaluation of the clinical importance of this information is lacking.
- While next generation sequencing (NGS) could address this limitation, current HPV NGS methods rely on sequencing PCR amplicons targeting a limited region of the genome for genotyping [2–7] or for identifying the integration/variant status [8–16] of a restricted number of.
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Given the complexity of the workflow for NGS methods, determination of these important charac- teristics is difficult.
- how- ever, these studies did not give detailed measures of variability at the level of base quality, coverage, and mapped reads [2, 5, 6].
- Moreover, methods evaluating reproducibility and sensitivity of amplicon-based NGS methods targeting a limited region of the genome are not directly applicable to our conceptually different whole-genome enriched NGS method.
- We used overall quality of sequencing reads, number of reads mapped to reference genomes, average depth of coverage, and fraction of genome cov- ered by mapped reads to measure reproducibility and re- sults on samples with decreasing copy number to determine LOD.
- vaccine HPV types (HPV and 58) were received from various sources including ATCC, German Cancer Research Institute (Heidelberg, Germany), Karolinska Institute (Stockholm, Sweden), and Institute Pasteur (Paris, France).
- We used defined samples with known copies of HPV types to evaluate these parameters under con- trolled conditions.
- The first three columns of Table 1 shows the composition for each of the 16 samples used in this study.
- The reproducibility in the number of reads mapped to reference genomes between flow cell lanes and between experiments was evaluated using linear re- gression.
- Reproducibility of HPV type determination was done at L1S1 mapping stringency along with selected cut-off criteria (number of mapped reads ≥ 1000, average coverage ≥20 and fraction of genome covered ≥0.5), ei- ther individually or combined.
- Mean, standard deviation (SD), and coefficient of variation (CV) for number of mapped reads, depth of coverage and fraction of genome covered for each HPV type in each sample were used as additional measures of reproducibility..
- The LOD for each of the 9 HPV types was calculated based on the number of mapped reads using the equa- tion: LOD = limit of blank (LOB.
- 1.64 SD of the lowest.
- Data from all four replicates of the 16 samples was used to calcu- late LOB and LOD.
- LOB, representing signal noise, was defined as mean of blank + 1.64SD of blank where blank is defined as the average number of reads mapped to any HPV type that was not expected (false positive reads).
- Based on this definition, the average number of reads mapped to any unexpected HPV type was 26.4, calculated from a total of 4757 reads in 180 false positive HPV calls from all 16 samples over the four replicates..
- The experimental LOD was defined as the lowest input copy number with number of mapped reads greater than calculated LOD..
- Sequence reads (in fastq format) from the 4 replicates had perfect match in the 8 bp index and passed the default filtering of the Illumina BCL2fastq V1.8.4 software.
- Prior to reference mapping, we evalu- ated the quality of the sequence data in terms of the mean number of reads, mean base quality score and per- cent of bases with quality score ≥ 30 (See Additional file 1:.
- The mean number of reads for each sample with DNA ranged from 9684, 372 to and was highly reproducible among replicates (CV: mean, 14.7%;.
- of the total reads) than samples with DNA, and the number of reads among the water control replicates was highly variable (mean .
- Additional file 2: Figure S2 shows the mean number of reads mapped to HBB, average depth of coverage, and fraction of reference genome cov- ered by mapped reads.
- The mean number of reads mapped to HBB in the 4 replicates for the 15 samples with DNA ranged from 12,155 to 37,414 (mean = 25,596) and was.
- Table 1 Reproducibility of mapping parameters for HPV type determination based on eWGS Sample No.
- HPV types.
- HPV type (eWGS call).
- Mapped reads (Mean ± SD) a.
- CV Fraction of genome covered (Mean ± SD).
- Additional file 2: Figure S2B) and the average fraction of the HBB target region covered by mapped reads (mean CV 0.13%.
- Reproducibility of HPV detection.
- There was a high correlation in number of reads mapped to HPV genomes between the two flow cell lanes (R 2 = 1) in both experiments (Fig.
- Table 1 shows the mean, SD, and CV (based on 4 replicates) for the number of reads mapped to a spe- cific HPV genome, average depth of coverage, and frac- tion of reference genome covered.
- were highly reprodu- cible as measured in number of HPV-specific reads mapped (CV range, 3.4 to 13.4.
- The number of reads mapped to specific HPV genomes in the plasmid pool was correlated to copy numbers, with the number of mapped reads decreasing linearly (β = 13.9, p <.
- reaction in terms of the number of mapped reads (mean CV: 10.08%.
- We evaluated the reproducibility of type-specific HPV detection in each of the 16 samples in the 4 replicate data sets using each cut-off criterion (number of mapped reads ≥1000, average coverage ≥20 and fraction of gen- ome covered ≥0.5) individually or combined (Table 2)..
- As expected, both negative controls (water and placental DNA) and four positive controls were HPV negative and positive, respectively, with each of the HPV type deter- mination criteria when applied individually or combined..
- All 4 replicates with 625 and 125 copies/reaction of the 9 HPV type plasmid pool met the mapped reads criter- ion (>.
- For the depth of coverage (≥20) and fraction of genome mapped criteria (≥0.5), all replicates of the 9 HPV plasmid pool at 625 and 125 copies/reaction de- tected all types with the exception that at 125 copies/re- action depth of coverage was not met for HPV types 6, 16 and 31.
- While HPV detection was not reproducible at copy numbers of 25 and below, it should be noted that when considering only the number of reads greater than 1 read or 5 reads used as cut-off in prior reports by Arroyo et al.
- Evaluation of eWGS for detection bias in multiple infections To evaluate the potential detection bias among HPV types in multiple infections, we compared the number of mapped reads to all 9 HPV types generated from librar- ies prepared in plasmid pools as described earlier (simu- lating multiple infection) with the number of reads generated from libraries prepared with individual Table 1 Reproducibility of mapping parameters for HPV type determination based on eWGS (Continued).
- HPV types (Expected).
- plasmids of the same HPV type (simulating single infec- tion).
- eWGS showed no bias for detection of all 9 HPV types under single or multiple infection (p = 0.16 to 0.99) except for the marginal difference in the number of mapped reads for HPV type 58 (p = 0.042) (Table 3)..
- The mean number of non-specific HPV reads corre- sponding to blank resulting from all samples of the 4 replicates was giving rise to a calculated LOB = 133.3 for each of the 9 specific HPV types in the plasmid pool.
- in terms of number of mapped reads, and the corre- sponding LOD in terms of copy number was determined for each of the 9 HPV types.
- LOD for all 9 types in this study was determined to be 25 copies/reaction since each specific HPV type at 25 copies reproducibly gener- ated specific mapped reads greater (mean .
- range in all 4 replicates of the data set (Table 4)..
- This study provides an in-depth evaluation of the repro- ducibility and LOD for HPV genotyping with our re- cently described eWGS method.
- As each defined sample was a pool of 4 to 5 HPV types with copy number ranging from 625 to 1 (composition shown in Table 1), the limit of detection could be assessed from the replicate results.
- Importantly, the study design considered reproducibility of the enrichment and library preparation steps as well as sequencing.
- We chose to use reproducibility of HPV genotyping using reads mapped under the most stringent L1S1 mapping conditions, while varying cut-off criteria for number of reads, depth of coverage, and fraction of reference gen- ome covered.
- Varying input amounts of HPV genomes (1–625 copies/reaction) and the number of types in the samples provided additional insights into assay robust- ness and LOD..
- The eWGS results from the 4 replicates demonstrates consistency in the number and quality of reads (mean CV: 14.7% for total number of reads.
- The number of reads mapped to HPV genomes were highly correlated between the two flow-cell lanes (R 2 = 1) in both experiments and be- tween the two experiments (R 2 = 0.99).
- Importantly, eWGS results in terms of number of mapped reads (mean CV: 10.1%.
- As expected, we observed a positive correlation be- tween the number of mapped reads and copy number/.
- The positive correlation of mapped reads and fraction of genome covered with target concentra- tion as we observed with bait-based eWGS for HPV agrees with a recently reported study on whole genome sequencing of hepatitis C viral genomes following simi- lar target enrichment [23].
- However, PCR-based methods either targeting whole genome or amplicon se- quencing did not show a copy number dependence with the number of mapped reads [2, 23].
- We found that eWGS method for HPV genotyping outperformed an NGS method based on amplicon sequencing [2] with less variability among the 9 vaccine types in the pooled sample for the number of mapped reads at 25 copies/re- action (CV: eWGS, 29.6%.
- Even at higher input (50 – 500 copies/reaction) of the 9 vaccine types in the pool, variability in the num- ber of HPV reads with amplicon sequencing remained.
- 2 Evaluation of reproducibility in terms of number of reads mapped to expected HPV genomes.
- Amplicon sequencing of replicates of clinical samples for HPV detection also showed high variability in the number of reads between libraries prepared to assess reproducibility (CV: mean 81%.
- We eval- uated this by comparing the number of reads for each HPV type at 625 copies in mixed type plasmid pools (simulating multiple infections) and single plasmid pools.
- The numbers of reads did not vary significantly (p on 8 of the 9 plasmids, suggesting eWGS has minimal bias for HPV typing.
- The data on reprodu- cibility among the replicates (Table 1) with HPV copies down to 25 copies/reaction (CV for number of reads.
- In this study, we used the number of reads mapped to HPV reference genomes as a parameter in relation to the LOB to calculate LOD .
- At an LOD of 25 copies/reaction, eWGS averaged mapped reads for HPV genotype calling, higher than the 1–5 mapped reads used as threshold by some amplicon sequencing methods [2, 5].
- 3 Mapped reads to expected HPV genomes showing the relationship between copy number and (a) mean number of mapped reads, b fraction of reference genome, and (c) mean CV for the 9 HPV plasmids in the number of mapped reads in relation to copy number, and (d) mean CV for the HPV plasmids for the fraction of reference genome covered in relation to HPV copy number.
- of reads ≥ 1000 Depth of coverage ≥ 20 Fraction of genome covered ≥ 0.5 All 3 cut-offs combined.
- a Selected eWGS cut-offs are number of mapped reads ≥ 1000, average depth of coverage ≥ 20, and fraction of reference genome covered ≥ 0.5.
- c Data not shown for 5 and 1 HPV copy/reaction since eWGS did not meet any of the cut-off criteria for type determination.
- Table 3 Comparison of mean number of mapped reads in samples with multiple and single HPV plasmids.
- Cost reductions could be achieved through automating the steps of li- brary production and by increasing the number of sam- ples that are pooled for the sequencing reaction.
- In our continued work (data not shown), we tested a total of 18 HPV types from mixing 4–7 types/reaction, and found that the number of samples/sequencing lane can be in- creased from 16 to 32 while maintaining the level of sen- sitivity (625 copies/reaction) for type determination (based on all three cut-off criteria combined) for all 18 HPV types.
- Additional work is needed to determine the maximum number of samples that can be pooled per se- quencing reaction without compromising sensitivity..
- In summary, we report an in-depth evaluation of the reproducibility of eWGS method for HPV genotyping employing a variety of met- rics that include overall quality of reads, number of reads mapped, depth of coverage, and fraction of refer- ence genome covered.
- Results indicate eWGS is highly reproducible for HPV genotyping at 625 copies /reaction using multiple cut-off criteria, with the possibility of re- ducing LOD to 25 copies/reaction, if HPV detection is based on the widely used number of mapped read cri- teria alone..
- Overall quality of reads from 4 replicates..
- (A) Mean number of reads passing the default filtering of Illumina BCL2fasq V1.8.4.
- Data shown is mean ± SD of 4 replicates/sample for number of reads (A), average depth of coverage (B), and fraction of reference covered (C).
- The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention..
- Table 4 Limit of detection of eWGS assay for different HPV types.
- of mapped reads) LOD (copy number) No.
- of mapped reads at 25 copies of HPV genome (mean ± SD) Concordnace a.
- TL, ERU and MSR contributed to the design of the experiments.
- TL, ERU and MSR participated in drafting and revisions of the manuscript.
- MSR provided oversight to all phases of the study.
- The clinical importance of the nomenclature, evolution and taxonomy of human papillomaviruses.
- Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology.
- Genome-wide profiling of the human papillomavirus DNA integration in cervical intraepithelial neoplasia and normal cervical epithelium by HPV capture technology.
- Methods for the determination of limit of detection and limit of quantitation of the analytical methods.
- Type-specific reproducibility of the Roche linear array HPV genotyping test

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