ArticlePloS one2013
The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient massive parallel sequencing.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 31 papers.
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Who cites it
31 citing papers in PubMed.
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- Genetic diversity and molecular characterization of avian paramyxoviruses from wild birds in South Korea between 2020 and 2024.Frontiers in microbiology · 2026Article
- IBDV-SSA, a novel molecular approach for the recovery of infectious bursal disease virus whole genomes from FTA cards.Microbiology spectrum · 2025Article
- Assessing Bias and Reproducibility of Viral Metagenomics Methods for the Combined Detection of Faecal RNA and DNA Viruses.Viruses · 2025Article
- An interlaboratory proficiency test using metagenomic sequencing as a diagnostic tool for the detection of RNA viruses in swine fecal material.Microbiology spectrum · 2024Article
- Iterative epigenomic analyses in the same single cell.Genome research · 2021Article
- Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results.Viruses · 2021Article
- Enrichment approach for unbiased sequencing of respiratory syncytial virus directly from clinical samples.Wellcome open research · 2021Article
- Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses.Journal of virological methods · 2020Article
- Laboratory Methods in Molecular Epidemiology: Viral Infections.Microbiology spectrum · 2018Review
- A Versatile Sample Processing Workflow for Metagenomic Pathogen Detection.Scientific reports · 2018Article
- Evaluation of bias induced by viral enrichment and random amplification protocols in metagenomic surveys of saliva DNA viruses.Microbiome · 2018Article
- An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.PloS one · 2018Article
- Towards a Universal Molecular Microbiological Test.Journal of clinical microbiology · 2017Review
- Article
- Characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics.PloS one · 2017Article
- Article
- Measurements of Intrahost Viral Diversity Are Extremely Sensitive to Systematic Errors in Variant Calling.Journal of virology · 2016Article
- Development and evaluation of a non-ribosomal random PCR and next-generation sequencing based assay for detection and sequencing of hand, foot and mouth disease pathogens.Virology journal · 2016Article
- Diversity and Ecology of Viruses in Hyperarid Desert Soils.Applied and environmental microbiology · 2016Review
Corrections and comments
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Authors and funding
7 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Sequence Independent Single Primer Amplification is one of the most widely used random amplification approaches in virology for sequencing template preparation. This technique relies on oligonucleotides consisting of a 3' random part used to prime complementary DNA synthesis and a 5' defined tag sequence for subsequent amplification. Recently, this amplification method was combined with next generation sequencing to obtain viral sequences. However, these studies showed a biased distribution of the resulting sequence reads over the analyzed genomes. The aim of this study was to elucidate the mechanisms that lead to biased sequence depth when using random amplification. Avian paramyxovirus type 8 was used as a model RNA virus to investigate these mechanisms. We showed, based on in silico analysis of the sequence depth in relation to GC-content, predicted RNA secondary structure and sequence complementarity to the 3' part of the tag sequence, that the tag sequence has the main contribution to the observed bias in sequence depth. We confirmed this finding experimentally using both fragmented and non-fragmented viral RNAs as well as primers differing in random oligomer length (6 or 12 nucleotides) and in the sequence of the amplification tag. The observed oligonucleotide annealing bias can be reduced by extending the random oligomer sequence and by in silico combining sequence data from SISPA experiments using different 5' defined tag sequences. These findings contribute to the optimization of random nucleic acid amplification protocols that are currently required for downstream applications such as viral metagenomics and microarray analysis.
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