Novel Solutions for NGS Library Preparation for Challenging Samples, from Picograms to Microgram Input Amounts

As the use of Next Generation Sequencing (NGS) is becoming more widespread, the use of more challenging samples is increasing. These include samples for which only small amounts are available, samples that are heavily degraded or samples where the DNA or RNA of interest is overwhelmed by other nucleic acid. At the same time, demand is increasing for faster protocols that perform reliably and do not compromise the quality of the libraries produced. To address these challenges we have developed novel reagents and streamlined workflows.

The new NEBNext Ultra II library preparation solution has been optimized to enable high-yield library preparation from a broad range of inputs (from picograms to microgram of DNA input) while retaining library quality (high yield and evenness of coverage regardless of GC content).

Sequencing whole eukaryotic genomes and transcriptomes is not always necessary and often selection of regions of interest is required to increase sensitivity and to reduce sequencing costs. We have developed a method that allows partitioning of DNA from different genomic compartments into enriched fractions. We illustrate the use of human methyl-CpG-binding domain (MBD2) to fractionate angiosperm DNA into eukaryotic nuclear (methyl-CpG-rich) vs. organellar and prokaryotic (methyl-CpG-poor) elements for genomic and metagenomic sequencing projects. To enrich for RNA of interest, we have optimized a method based on hybridization of probes to the unwanted RNA, followed by RNAseH enzymatic degradation of targeted RNAs. We applied this method to remove cytoplasmic and mitochondrial ribosomal RNA from different eukaryotic total RNA samples (human and plant RNA).