An alternative method to multiple displacement amplification for preparing virome DNA in a way adapted for the sequencing of both double-strand and single-strand DNA viruses

Abstract

SummaryUnless some dedicated treatments are applied, virome shotgun sequencing will only reveal the double stranded DNA (dsDNA) content of a given sample. However bacterial virus genomes often consist of a circular single stranded DNA (ssDNA) molecule. Pre-treatment and amplification of DNA with the multiple displacement amplification (MDA) method allows to convert the ssDNA to ds DNA, but it introduces an over-representation of these circular ssDNA genomes. A more recent alternative consists in using an xGen kit, which permits the ligation of adaptators directly to sheared DNA. However, this kit is expensive, and the sonication step might shear ssDNA more efficiently than dsDNA, therefore introducing another bias in virome sequencing. This prompted us to search for an alternative method of DNA preparation for the sequencing of mixed ssDNA and dsDNA viromes. We present here this new method based on the use of the T7 DNA polymerase (T7pol), which is easy to implement and cheap, and compare it to MDA with 30 minutes incubation time (sMDA) and to the direct shearing of DNA without prior conversion of ssDNA to dsDNA. All samples were then prepared with the xGen kit. We found that sMDA overamplifies with 2 to 8.6-fold excess compared to input phage particles, which was less than reported for the full time, 90 minutes MDA treatment. In contrast, the T7pol method led to the under-representation (1.3 to 8.2-fold) of these genomes, and was overall a better reflection of input phage cocktails, compared to the other methods. We conclude a T7pol pre-treatment is a good alternative to MDA for the shotgun sequencing of viromes.