The Origin of Biased Sequence Depth in Sequence-Independent Nucleic Acid Amplification and Optimization for Efficient Massive Parallel Sequencing

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.

Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

“…We observed that this random PCR amplification negatively impacted upon viral identification power. The observed biases in sequence distributing confirms our previous findings (Rosseel et al, 2013). Moreover, rPCR amplification increased the proportion of reads for which megablast could not find similar sequences in the NCBI nt database, suggesting the creation of amplification artefacts.…”

“…Several attempts have been performed to reduce amplification introduced bias (Motley et al, 2014;Rosseel et al, 2013). Although the sequencing library preparation kit (Illumina Nextera XT) used in this study is not completely PCR-free, it supports ultra-low DNA input of only a single nanogram which eliminates the need for extra sequence independent genome amplification step.…”

Diversity and Ecology of Viruses in Hyperarid Desert Soils

“…Desert soils frequently produce suboptimal (Յ10 ng/l) viral DNA yields (66), forcing the inclusion of a random PCR amplification step for NGS library construction. The use of whole-genome amplification (WGA) by multiple-displacement amplification (MDA) or random-priming, sequence-independent, single-primer amplification (RP-SISPA) (67) almost certainly results in biased amplification of certain virus groups (68)(69)(70)(71) and prevents the accurate determination of viral abundances and diversity. While viral amplification is widely accepted as a necessity in metaviromic studies (72), we argue that amplification of virus metagenomic DNA should be avoided where possible.…”

Measurements of Intrahost Viral Diversity Are Extremely Sensitive to Systematic Errors in Variant Calling

“…The method has been used in conjunction with statistical algorithms to control for accuracy in studies of intrahost influenza virus diversity (5,22,23). However, the bar-coding reaction used in SISPA can be biased in unpredictable ways, resulting in uneven coverage and sensitivity across the genome (24).…”