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

Nadja Brait,Büşra Külekçi,Irene Goerzer

doi:10.1186/s12864-021-08272-z

Nadja Brait, Büşra Külekçi + Show 1 more

Open Access

https://doi.org/10.1186/s12864-021-08272-z

Copy DOI

Abstract

BackgroundShort read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections.ResultsTotal substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations.ConclusionsQuantitative 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 by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Highlights

Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations
Widely used in-depth short read sequencing of the whole genome comprehensively assessed genome-wide intra- and inter-host diversity at the nucleotide level, and intrahost variability over time [18, 19, 21,22,23,24,25]. These studies revealed that recombination between and within polymorphic regions of different HCMV strains have been common and single nucleotide variants that may emerge during replication periods will further contribute to the overall strain diversity
Establishment of long range PCR for highly polymorphic UL regions First, a long range PCR approach was established by targeting two genomic regions, spanning from UL55 to UL76 (30 kb), and from UL139 to UL146 (6.7 kb), respectively (Fig. 1). These two UL regions were primarily chosen based on high interstrain polymorphism and well-characterised genotype assignments of the respective polymorphic genes [19]

Summary

Introduction

Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. We established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Widely used in-depth short read sequencing of the whole genome comprehensively assessed genome-wide intra- and inter-host diversity at the nucleotide level, and intrahost variability over time [18, 19, 21,22,23,24,25] These studies revealed that recombination between and within polymorphic regions of different HCMV strains have been common and single nucleotide variants that may emerge during replication periods will further contribute to the overall strain diversity. It appears that a huge variety of different HCMV strains circulate in the human population and the frequency of occurrence of completely identical “strain genotypes” among HCMV-infected persons seems to be very rare [19]

Objectives

Methods

Results

Discussion

Conclusion