Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine

Abstract

We describe strand-specific, base-resolution detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA with single-molecule sensitivity, combining a bioorthogonal, selective chemical labeling method of 5-hmC with single-molecule, real-time (SMRT) DNA sequencing. The chemical labeling not only allows affinity enrichment of 5-hmC–containing DNA fragments but also enhances the kinetic signal of 5-hmC during SMRT sequencing. We applied the approach to sequence 5-hmC in a genomic DNA sample with high confidence.

Dynamics of 5-hydroxymethylcytosine during mouse spermatogenesis

“…To evaluate the reliability of our whole procedure, the correlation of 5hmC levels of the duplicate within 2-kb bins in the genome with 1-kb steps was calculated. The correlation coefficient of each cell type ranged from 0.85-0.98 (Supplementary Table S3), indicating that our 5hmC profiling procedure was highly reliable, as we previously showed 28,47 . Reads from the duplicate of each cell type were pooled to identify clusters of reads or otherwise termed peaks or hMRs (5hmC regions) with the MACS software 30 .…”

The emergence of nanopores in next-generation sequencing

“…Although the devices are very expensive ( figure 1(c)), they are ideal for de novo sequencing of unknown genomes due to their long read lengths of 8 kbp ( figure 2(b)). They even permit the quantification of methylation, DNA damage, and other epigenetic information [11].…”

Current technological advances in mapping new DNA modifications

“…In this context, the recently developed singlemolecule real-time sequencing (SMRT-seq) method allows identification of epigenetic modifications on individual DNA molecules with single base resolution [56,57]. For this thirdgeneration sequencing, a single DNA polymerase molecule is monitored during incorporation of fluorescently labelled nucleotides into newly synthesised DNA.…”

“…In a first attempt to optimize this technique for the detection of DNA modifications, Flusberg et al successfully detected N6-methyladenine, however differential kinetic signatures were missing for 5mC and 5hmC [56]. Coupled with chemical modification using T4-BGT enzyme and UDP-6-N3-glucose, a modified version of SMRT-seq with a 5hmC enrichment procedure has been optimised, increasing the confidence of 5hmC assignments [57]. However, the field is still eagerly awaiting the validation of this method for Aside from the technological challenges, an important question concerns the underlying biological function of 5hmC, 5fC and 5caC, which still remains the subject of intense scientific debate.…”