ChIP- based nuclear DNA isolation for genome sequencing in Pyropia to remove bacterial and cytosol DNA contamination

Abstract

Abstract Contamination from epiphytic bacteria and cytosolic DNA (plastid and mitochondrion) is challenging the accuracy of genome-wide analysis of nori-producing marine seaweed Pyropia yezoensis. Unlike bacteria and organellar DNA, Pyropia nuclear DNA is tightly associated with histone proteins. In this study, we applied Chromatin Immuno-precipitation (ChIP) of histone H3 to isolate nuclear DNA followed by high-throughput sequencing. More than 99.5% of ChIP-sequencing data are successfully aligned to the reference nuclear genome, remarkably higher than the ones from direct-extraction and nuclei-extraction data in which 40%-50% are from plastid. The proportion of data that mapped to the bacterial database when using ChIP extraction was very low. Additionally, ChIP-data can cover up to 89% of the nuclear genome, higher than direct-extraction data at equal data size and comparable to the latter at equal sequencing depth. The uncovered regions from the three methods are mostly overlapping, suggesting that incomplete sequencing accounts for the missing data, rather than failed chromatin-antibody binding in ChIP-extraction method. This ChIP-extraction method can successfully separate nuclear DNA from cytosolic DNA and bacterial DNA, thus overwhelmingly reducing the sequencing cost in genome resequencing project and provides a strictly purified reference data for genome assembly. The applicability to other macroalgae would makes it a valuable contribution to the algal research community.