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. 2020 Mar 3;21(1):54.
doi: 10.1186/s13059-020-01969-6.

Accurate targeted long-read DNA methylation and hydroxymethylation sequencing with TAPS

Yibin Liu  1   2 , Jingfei Cheng  1   2 , Paulina Siejka-Zielińska  1   2 , Carika Weldon  3 , Hannah Roberts  3 , Maria Lopopolo  3 , Andrea Magri  2 , Valentina D'Arienzo  2 , James M Harris  2 , Jane A McKeating  2 , Chun-Xiao Song  4   5
Affiliations

Accurate targeted long-read DNA methylation and hydroxymethylation sequencing with TAPS

Yibin Liu et al. Genome Biol. .

Abstract

We present long-read Tet-assisted pyridine borane sequencing (lrTAPS) for targeted base-resolution sequencing of DNA methylation and hydroxymethylation in regions up to 10 kb from nanogram-level input. Compatible with both Oxford Nanopore and PacBio Single-Molecule Real-Time (SMRT) sequencing, lrTAPS detects methylation with accuracy comparable to short-read Illumina sequencing but with long-range epigenetic phasing. We applied lrTAPS to sequence difficult-to-map regions in mouse embryonic stem cells and to identify distinct methylation events in the integrated hepatitis B virus genome.

Keywords: 5-Methylcytosine; Bisulfite-free; DNA methylation; Epigenetic phasing; Long-read sequencing.

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Conflict of interest statement

C-XS and YL are named as inventors on pending patent applications filed by the Ludwig Institute for Cancer Research pertaining to one or more aspects of the technologies described here, which have been licensed to Base Genomics.

Figures

Fig. 1
Fig. 1
lrTAPS of 4 kb model DNA. a Schematic of lrTAPS for targeted long-read DNA methylation sequencing. b The upper panel (from top to bottom): methylation of a 4-kb model DNA obtained from bisulfite sequencing (short-read Illumina sequencing), Nano-TAPS, SMRT-TAPS, and native Nanopore methylation sequencing using Nanopolish or Tombo. The lower panel shows examples of individual long-reads from Nano-TAPS and SMRT-TAPS. The red bars indicate methylation. Black and purple bars indicate sequencing errors (deletions and insertions, respectively). c Scatter plots showing all pairwise correlations of all CpG sites among Nano-TAPS, SMRT-TAPS, native Nanopore methylation calling (Nanopolish and Tombo), and Bisulfite-seq, with correlation coefficient showing on top of each plot. d ROC curve and AUC comparing Nano-TAPS, SMRT-TAPS, and native Nanopore methylation sequencing (Nanopolish and Tombo), using DNA methylation from bisulfite sequencing (methylation level > 3% was designated as methylated) as the truth
Fig. 2
Fig. 2
lrTAPS of a previously unmapped region in mESCs and integrated HBV DNA in Huh-1 cells. a Genome browser view of the methylation and coverage detected by Illumina-TAPS, Nano-TAPS, and SMRT-TAPS in Hba-a1 locus. The pink shaded area shows the gap which cannot be mapped with Illumina short-read sequencing. b CpG methylation of integrated HBV DNA in Huh-1 cells detected by Nano-TAPS and SMRT-TAPS. The blue shaded area shows the covered regions with lrTAPS. Regions of methylated CpGs are indicated by the blue/yellow boxes. c Heatmap showing integrated HBV DNA methylation in each SMRT read (34,755 reads were included). Reads were ranked by the average methylation in the first CpG Island. The blue bar indicates the methylated CpG (mCG) while white bar indicates unmethylated CpG (uCG). The number in the bottom indicates the relative position of CpG in the HBV reference genome
See this image and copyright information in PMC

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