Short-read and Long-read PCR-Free Sequencing of Bacteriophages Using Ultra-Low Starting DNA Input.

Abstract

Preparing phage DNA in sufficient quantities for sequencing is often a challenging task, especially when a sensitive bacterial host is not available for phage propagation.¹ This limitation poses a significant obstacle in phage research as the availability of adequate phage DNA is often considered crucial for various analyses, including genome sequencing, functional studies, and therapeutic developments. Also, because DNA extraction from phage samples (e.g., from bacterial induction) can yield low amounts of genomic DNA, many studies utilize tagmentation for amplification-free quantitative sequencing. However, this technique has the drawback of losing phage genome ends (termini) and creating biases in genome coverage.²,³ Polymerase chain reaction (PCR)-free sequencing is often recommended or even necessary to obtain an unbiased characterization of phage genomes or communities. However, sequencing very low quantities of DNA without PCR amplification is challenging, and sequencing service providers, as well as library kit manufacturers, will only guarantee products and results with relatively high DNA inputs. In this study, we aimed to assess the feasibility of sequencing phage genomic DNA with very low DNA starting material and to determine the impact of decreasing DNA input on sequencing quality using both Illumina short-read and Nanopore long-read technologies. We analyzed the quantity and quality of output sequences (and their impact on genome assemblies) for different ranges of input DNA concentrations, starting at the recommended DNA inputs for each technology. We concluded that it is achievable to perform sequencing of high quality with DNA inputs that are lower (i.e., 1000-fold lower) than manufacturers' recommendations or requirements. In this study, we successfully sequenced phage genomic DNA (without PCR amplification) using as little as 1 ng of total input DNA (or 0.02 ng/uL in 50 uL eluted volume) for short-read sequencing with Illumina technology and 0.4 ng (or 0,036 ng/uL in 11 uL eluted volume) for long-read sequencing with Nanopore technology.