DNA-spiking in viral metagenome sequencing: A new method with low bias

• Published in: PeerJ preprints
• Main Authors: Cremers, Geert, Gambelli, Lavinia, VanAlen, Theo, VanNiftrik, Laura, Huub Op den Camp
• Format: Journal Article
• Language: English
• Published: San Diego PeerJ, Inc 04.04.2017
• Subjects: Bias; Deoxyribonucleic acid; DNA; DNA sequencing; Gene mapping; Genetic testing; Genomes; rRNA 16S
• ISSN: 2167-9843
• DOI: 10.7287/peerj.preprints.2912v1

With the emergence of Next Generation Sequencing, major advances were made with regard to identifying viruses in natural environments. However, bioinformatical research on viruses is still limited because of the low amounts of viral DNA that can be obtained for analysis. To overcome this limitation, DNA is often amplified with multiple displacement amplification (MDA), which causes an unavoidable bias. Here, we describe a DNA-spiking method to avoid the bias that is created when using amplification of DNA before metagenome sequencing. To obtain sufficient DNA for sequencing, a bacterial 16S rRNA gene was amplified and the obtained DNA was spiked to a DNA sample containing DNA from a bacteriophage population before sequencing using Ion Torrent technology. After sequencing, the 16S rRNA gene reads DNA was removed by mapping to the Silva database. The new DNA-spiking method was compared with the MDA technique. When MDA was applied, the overall GC content of the reads showed a bias towards lower GC%, indicating a change in composition of the DNA sample. Assemblies using all available reads from both MDA and the DNA-spiked samples resulted in six viral genomes. All six genomes could be almost completely retrieved (97.9%-100%) when mapping the reads from the DNA-spiked sample to those six genomes . In contrast, 6.3%-77.7% of three viral genomes were covered by reads obtained using the MDA amplification method and only three were nearly fully covered (97.4%-100%). The new method provides a simple and inexpensive protocol with very low bias in sequencing of metagenomes for which low amounts of DNA are available.