Enriching for the Nucleic Acid of Interest before NGS Profiling of Plant and Animal Samples

Next Generation Sequencing (NGS) is the standard method for large-scale nucleic acid profiling. Sequencing whole eukaryotic genomes and transcriptomes is not always necessary and often selection of regions of interest is required to increase sensitivity and reduce sequencing costs.

For example, organelle genome sequencing projects have employed a variety of techniques to obtain sequence data enriched for the organellar DNA of interest. These include laborious centrifugation steps, long-range PCR, and hybrid sequence capture techniques that rely on prior knowledge of the sequence regions to be enriched, which can limit discovery. Here, we present a method that allows partitioning of DNA from different genomic compartments. 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.

For RNA-seq, sequencing the entire transcriptome is inefficient since the majority of sequence reads correspond to a few extremely abundant transcripts. Here, we present a simple method to selectively degrade abundant, unwanted RNAs before library preparation. This method is based on the use of RNaseH to digest unwanted RNA sequences that are hybridized to DNA probes.  The DNA probes are designed to target cytoplasmic and organellar (mitochondrial and chloroplast) ribosomal RNA (rRNA). We assessed the performance of the method via extensive directional RNA sequencing. Efficiency (up to 99% rRNA depletion) was determined through comparison of targeted transcript levels between depleted and untreated samples. Excellent transcript correlation between depleted and non-depleted libraries demonstrated minimal off-target effect.