Mixed cytomegalovirus genotypes in HIV positive mothers show compartmentalization and distinct patterns of transmission to infants

Pang, Juanita; Slyker, Jennifer A.; Roy, Sunando; Bryant, Josephine M.; Atkinson, Claire; Cudini, Juliana; Farquhar, Carey; Griffiths, Paul; Kiarie, James; Morfopoulou, Sofia; Roxby, Alison C.; Tutill, Helena J.; Williams, Rachel; Gantt, Soren; Goldstein, Richard A.; Breuer, Judith

doi:10.1101/2020.09.17.20196790

Cited by 1 publication

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“…Due to short read lengths of second generation sequencing techniques, determination of individual genomes within mixtures can be challenging. It requires model-based analysis to reconstruct unique haplotypes from short sequencing reads [19, 22, 25, 26] but this might be difficult for genomic segments interspersed by longer conserved sections, low-complexity repetitions and GC-deviant regions [27].…”

Section: Introductionmentioning

confidence: 99%

Long Range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

Brait

Külekci

Görzer

2021

Preprint

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Short read sequencing, which has extensively been used to decipher the genome diversity of human cytomegalovirus (HCMV) strains, often falls short to assess co-linearity of non-adjacent polymorphic sites in mixed HCMV populations. In the present study, we established a long amplicon sequencing workflow to identify number and relative quantities of unique HCMV haplotypes in mixtures. Accordingly, long read PacBio sequencing was applied to amplicons spanning over multiple polymorphic sites. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-free viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Our data show that artificial HCMV DNA mixtures were correctly determined upon long amplicon sequencing down to 1% abundance of the minor DNA source. Total error rate of mapped reads ranged from 0.17 to 0.43 depending on the stringency of quality trimming. PCR products of up to 7.7 kb and a GC content <55% were efficiently generated when DNA was directly isolated from bronchoalveolar lavage samples, yet long range PCR may display a slightly lower sensitivity compared to short amplicons. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Intra-patient haplotype diversity is unevenly distributed across the target site and often interspersed by long identical stretches, thus unable to be linked by short reads. Moreover, diversity at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed populations. Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

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