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

BMC Genomics ◽  
2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Nadja Brait ◽  
Büşra Külekçi ◽  
Irene Goerzer
Keyword(s):  
Gc Content ◽  
Amplicon Sequencing ◽  
Read Length ◽  
Third Generation Sequencing ◽  
Hcmv Strain ◽  
Novel Approach ◽  
Distinct Haplotype ◽  
Pcr Products ◽  
Long Range Pcr ◽  
Sequencing Platforms

Abstract Background 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. 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. Results Total 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. Conclusions 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 by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

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

2021 ◽  
Author(s):  
Nadja Brait ◽  
Busra Kulekci ◽  
Irene Goerzer
Keyword(s):  
Long Range ◽  
Total Error ◽  
Haplotype Diversity ◽  
Gc Content ◽  
Amplicon Sequencing ◽  
Lower Sensitivity ◽  
Total Error Rate ◽  
Hcmv Strain ◽  
Novel Approach ◽  
Long Range Pcr

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.

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

2021 ◽  
Author(s):  
Nadja Brait ◽  
Büsra Kül ◽  
Irene Goerzer
Keyword(s):  
Long Range ◽  
Total Error ◽  
Haplotype Diversity ◽  
Gc Content ◽  
Amplicon Sequencing ◽  
Lower Sensitivity ◽  
Total Error Rate ◽  
Hcmv Strain ◽  
Novel Approach ◽  
Long Range Pcr

Abstract Background 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. Results 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. Conclusions 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.

scholarly journals High efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells

2018 ◽  
Author(s):  
Andrew D. Johnston ◽  
Claudia A. Simões-Pires ◽  
Masako Suzuki ◽  
John M. Greally
Keyword(s):  
High Efficiency ◽  
Single Cells ◽  
Amplicon Sequencing ◽  
Barr Virus ◽  
Functional Variant ◽  
Pcr Products ◽  
Flow Cytometric Sorting ◽  
Epstein Barr ◽  
Long Range Pcr ◽  
Genomic Editing

ABSTRACTWhile lymphoblastoid cell lines (LCLs) represent a valuable resource for population genetic studies, they are usually regarded as difficult for CRISPR-mediated genomic editing. It would be valuable to be able to take the results of their functional variant studies and test them in the same LCLs. We describe a protocol using a single-stranded donor oligonucleotide (ssODN) strategy for ‘scarless’ editing in LCLs. The protocol involves optimized transfection, flow cytometric sorting of transfected cells to single cells in multi-well plates and growth in conditioned, serum-rich medium, followed by characterization of the clones. Amplicon sequencing reveals the relative proportions of alleles with different editing events, with sequencing of DNA from clones showing the frequencies of events in individual cells. We find 12/60 (20%) of clones selected in this manner to have the desired ssODN-mediated recombination event. Long-range PCR of DNA at the edited locus and of RT-PCR products for the gene traversing the edited locus reveals 3/6 characterized clones (50%) to have large structural mutations of the region that are missed by sequencing just the edited site. The protocol does not require the use of lentiviruses or stable transfection, and makes LCLs a realistic cell type for consideration for CRISPR-mediated genomic targeting.

Quality of Third Generation Sequencing

2020 ◽  
Vol 17 (12) ◽  
pp. 5205-5209
Author(s):  
Ali Elbialy ◽  
M. A. El-Dosuky ◽  
Ibrahim M. El-Henawy
Keyword(s):  
Neural Network ◽  
Deep Neural Network ◽  
Gc Content ◽  
Error Rates ◽  
Third Generation ◽  
Third Generation Sequencing ◽  
Long Reads ◽  
Generation Sequencing

Third generation sequencing (TGS) relates to long reads but with relatively high error rates. Quality of TGS is a hot topic, dealing with errors. This paper combines and investigates three quality related metrics. They are basecalling accuracy, Phred Quality Scores, and GC content. For basecalling accuracy, a deep neural network is adopted. The measured loss does not exceed 5.42.

scholarly journals Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing

PLoS ONE ◽  
2011 ◽  
Vol 6 (11) ◽  
pp. e28240 ◽  
Author(s):  
Ellen Knierim ◽  
Barbara Lucke ◽  
Jana Marie Schwarz ◽  
Markus Schuelke ◽  
Dominik Seelow
Keyword(s):  
Next Generation Sequencing ◽  
Long Range ◽  
Next Generation ◽  
Systematic Comparison ◽  
Pcr Products ◽  
Long Range Pcr ◽  
Generation Sequencing

scholarly journals Reflex: intramolecular barcoding of long-range PCR products for sequencing multiple pooled DNAs

2013 ◽  
Vol 41 (10) ◽  
pp. e112-e112 ◽  
Author(s):  
James A. Casbon ◽  
Andrew F. Slatter ◽  
Esther Musgrave-Brown ◽  
Robert J. Osborne ◽  
Conrad P. Lichtenstein ◽  
...  
Keyword(s):  
Long Range ◽  
Pcr Products ◽  
Long Range Pcr

scholarly journals Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples

2021 ◽  
Author(s):  
Samuel Martin ◽  
Darren Heavens ◽  
Yuxuan Lan ◽  
Samuel Horsfield ◽  
Matthew D Clark ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Web Application ◽  
Adaptive Sampling ◽  
Bacterial Species ◽  
Abundant Species ◽  
Read Length ◽  
Multiple Species ◽  
Sequencing Platforms ◽  
The Relationship ◽  
Pore Stability

Background: Adaptive sampling is a method of software-controlled enrichment unique to nanopore sequencing platforms recently implemented in Oxford Nanopore's own control software. By examining the first few hundred bases of a DNA molecule as it passes through a pore, software can determine if the molecule is sufficiently interesting to sequence in its entirety. If not, the molecule is ejected from the pore by reversing the voltage across it, freeing the pore for a new molecule. User supplied sequences define the targets to be sequenced or ejected. Here we explore the potential of using adaptive sampling for enrichment of rarer species within metagenomic samples. Results: We created a synthetic mock community consisting of seven bacterial species at different proportions ranging from 1.2% to 47% and used this as the basis for a series of enrichment and depletion experiments. To investigate the effect of DNA length on adaptive sampling efficiency, we created sequencing libraries with mean read lengths of 1.7 kbp, 4.7 kbp, 10.6 kbp, and 12.8 kbp and enriched or depleted for individual and multiple species over a series of sequencing runs. Across all experiments enrichment ranged from 1.67-fold for the most abundant species with the shortest read length to 13.87-fold for the least abundant species with the longest read length. Factoring in the reduction to sequence output associated with repeatedly rejecting molecules reduces the calculated efficiency of this enrichment to between 0.96-fold and 4.93-fold. We note that reducing ejections due to false negatives (approximately 36%) would significantly increase efficiency. We used the relationship between abundance, molecule length and enrichment factor to produce a mathematical model of enrichment based on molecule length and relative abundance, whose predictions correlated strongly with experimental data. A web application is provided to allow researchers to explore model predictions in advance of performing their own experiments. Conclusions: Our data clearly demonstrates the benefit for enriching low abundant species in adaptive sampling metagenomic experiments, especially with longer molecules, and our mathematical model can be used to determine whether a given experimental DNA sample is suitable for adaptive sampling. Notably, repeated voltage reversals have no effect on pore stability.

scholarly journals Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4925 ◽  
Author(s):  
Jonathan M. Palmer ◽  
Michelle A. Jusino ◽  
Mark T. Banik ◽  
Daniel L. Lindner
Keyword(s):  
Amplicon Sequencing ◽  
Variable Length ◽  
Its Sequences ◽  
Synthetic Control ◽  
Mock Community ◽  
Software Pipeline ◽  
Initial Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Sequencing Platforms ◽  
Mock Communities

High-throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal internal transcribed spacer (ITS) amplicons, we created two ITS spike-in control mock communities composed of cloned DNA in plasmids: a biological mock community, consisting of ITS sequences from fungal taxa, and a synthetic mock community (SynMock), consisting of non-biological ITS-like sequences. Using these spike-in controls we show that: (1) a non-biological synthetic control (e.g., SynMock) is the best solution for parameterizing bioinformatics pipelines, (2) pre-clustering steps for variable length amplicons are critically important, (3) a major source of bias is attributed to the initial polymerase chain reaction (PCR) and thus HTAS read abundances are typically not representative of starting values. We developed AMPtk, a versatile software solution equipped to deal with variable length amplicons and quality filter HTAS data based on spike-in controls. While we describe herein a non-biological SynMock community for ITS sequences, the concept and AMPtk software can be widely applied to any HTAS dataset to improve data quality.

scholarly journals The effect of variant interference on de novo assembly for viral deep sequencing

2019 ◽  
Author(s):  
Christina J. Castro ◽  
Rachel L. Marine ◽  
Edward Ramos ◽  
Terry Fei Fan Ng
Keyword(s):  
Deep Sequencing ◽  
De Novo ◽  
Gc Content ◽  
Read Length ◽  
Viral Genomes ◽  
Minor Variant ◽  
Main Driver ◽  
Next Generation Sequencing Ngs ◽  
Viral Sequences ◽  
Generation Sequencing

AbstractViruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approach has surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. Our results from >15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This “variant interference” (VI) is highly consistent and reproducible by ten most used de novo assemblers, and occurs independent of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the “rescue” of full viral genomes from fragmented contigs. These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing.

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