Optimized library preparation for sequencing of whole bacterial genomes and low density microbiota by Illumina-based NGS

Abstract
Next-Generation Sequencing (NGS) technologies have paved the way for rapid and cost-efficient analyses of transcriptomes, microbiomes as well as de novo sequencing and re-sequencing of genomes. Library preparation is a crucial step in the generation of high quality data and may have a major impact on the success of data analysis and interpretability of data. Difficulties and improvements of two different sample preparation procedures are presented.
With the introduction of PCR-free library preparation procedures (LPPs) for de novo genome sequencing major improvements were made in comparison to the initial PCR containing LPPs, as PCR biases are largely reduced. In this study modified versions of the widely used Illumina TruSeq® DNA PCR-free library preparation protocol were developed that enable the generation of sequencing libraries with longer average insert sizes leading to substantial assembly improvements using SPAdes, which is currently one of the best performing assemblers with regard to bacterial de novo genome assembly. Through the introduced modifications, DNA quantitation by qPCR can be omitted and fewer reagents are consumed. Furthermore, the relationships between genomic GC content, average library insert size and sequencing quality were investigated.
For analyzing the biodiversity of microbiota, PCR is an indispensable step, as it is needed to amplify a fragment of the 16S rRNA gene, which is sequenced subsequently. Here, the extraction of DNA and use of adequate PCR conditions are of utmost importance. This is particularly true for microbial communities with low cell counts in difficult matrices. Raw milk microbiota are of high interest, but raw milk contains high fat and protein contents as well as high amounts of accompanying eukaryotic DNA originating from the cow’s somatic cells. We have developed a protocol for DNA extraction minimizing the content of somatic DNA and have performed different PCR strategies such as droplet digital PCR for analysing possible PCR bias. The data obtained show that DNA extraction out of low density communities requires substantial effort, but sequencing of such microbiota is possible.