DeMott, M. S. | Search bioRxiv

bioRxiv2019

Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

Here we present the Nick-seq platform for quantitative mapping of DNA modifications and damage at single-nucleotide resolution across genomes. Pre-existing breaks are blocked and DNA structures converted to strand-breaks for 3-extension by nick-translation to produce nuclease-resistant oligonucleotides, and 3-capture by terminal transferase tailing. Libraries from both products are subjected to next-generation sequencing. Nick-seq is a generally applicable method illustrated with quantitative profiling of single-strand-breaks, phosphorothioate modifications, and DNA oxidation.

molecular biology↗

bioRxiv2019

Sequencing-based quantitative mapping of the cellular small RNA landscape

Current next-generation RNA sequencing methods cannot provide accurate quantification of the population of small RNAs within a sample due to strong sequence-dependent biases in capture, ligation, and amplification during library preparation. We report the development of an RNA sequencing method - AQRNA-seq - that minimizes biases and enables absolute quantification of all small RNA species in a sample mixture. Validation of AQRNA-seq library preparation and data mining algorithms using a 963-member microRNA reference library, RNA oligonucleotide standards of varying lengths, and northern blots demonstrated a direct, linear correlation between sequencing read count and RNA abundance. Application of AQRNA-seq to bacterial tRNA pools, a traditionally hard-to-sequence class of RNAs, revealed 80-fold variation in tRNA isoacceptor copy numbers, patterns of site-specific tRNA fragmentation caused by stress, and quantitative maps of ribonucleoside modifications, all in a single AQRNA-seq experiment. AQRNA-seq thus provides a means to quantitatively map the small RNA landscape in cells and tissues.

molecular biology↗