Direct transposition of native DNA for sensitive multimodal single-molecule sequencing.

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90-99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000-50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA.

Bisulfite sequencing (BS-seq) to detect 5-methylcytosine (5mC) is limited by lengthy reaction times, severe DNA damage, overestimation of 5mC level and incomplete C-to-U conversion of certain DNA sequences. We present ultrafast BS-seq (UBS-seq), which uses highly concentrated bisulfite reagents and high reaction temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in reduced DNA damage and lower background noise. UBS-seq allows library construction from small amounts of purified genomic DNA, such as from cell-free DNA or directly from 1 to 100 mouse embryonic stem cells, with less overestimation of 5mC level and higher genome coverage than conventional BS-seq. Additionally, UBS-seq quantitatively maps RNA 5-methylcytosine (m5C) from low inputs of mRNA and allows the detection of m5C stoichiometry in highly structured RNA sequences. Our UBS-seq results identify NSUN2 as the major 'writer' protein responsible for the deposition of ~90% of m5C sites in HeLa mRNA and reveal enriched m5C sites in 5'-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation.

Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.