In April 2018, Nextomics joins hands with research group of Xiaofeng Gu, biotechnology research institute, Chinese academy of agricultural sciences, and research group of Hao Yu, department of biological sciences and Temasek life sciences laboratory, National University of Singapore, to publish a paper titled as “DNA N⁶-Adenine Methylation in Arabidopsis thaliana” in the journal of Development Cell. This research reported that global profiling of 6mA sites at single-nucleotide resolution in the genome of A. thaliana at different developmental stages and ecotypes using single-molecule real-time sequencing.

This is the first research article of distributing eukaryote methylation mapped by PacBio SMRT.

Highlights

• DNA methylation on N⁶-adenine (6mA) widely occurs in the Arabidopsisgenome;
• 6mA is more enriched on gene bodies than intergenic regions;
• 6mA is a dynamic DNA modification during Arabidopsisdevelopment;
• 6mA is associated with actively expressed genes in

Results

6mA widely occurs in the Arabidopsis genome

Dot blot analysis of gDNA extracted from 3- to 21-day-old Col wild-type plants revealed a gradual increase in the 6mA signal intensity from vegetative to reproductive stage (Fig.1).

Fig. 1 6mA Occurs in Arabidopsis Genomic DNA

Genome-Wide Mapping of 6mA in Arabidopsis sequenced by PacBio SMRT

Compare the dynamic and distribution of 6mA in D9 and D21, and combined with the transcriptome data researchers revealed the function of 6mA during the development of Arabidopsis (Fig.2).

Fig.2 Circos plots of 6mA DNA methylation profiles of 9- and 21-day-old Col wild-type Arabidopsis.

6mA is more enriched on gene bodies than intergenic regions

6mA distribution in Col genomic regions divided into gene bodies, promoters, 5’intergenic, 3’intergenic, and other intergenic regions. The results showed that 32% of 6mA sites were located within gene bodies, and protein-coding genes contained more than half of the 6mA sites in all methylated genes (Fig. 3).

Fig. 3 Distribution Pattern of 6mA Methylation in Genomic DNA of 9-Day-Old Col Plants

6mA is a dynamic DNA modification during Arabidopsis development

Genome-wide mapping of 6mA in gDNA from 21-day-old Col wild-type plants at the reproductive stage by SMRT sequencing identified 184,633 6mA sites, among which 9,909 sites were overlapped with those in 9-day-old plants (Fig. 4), which implied that 6mA DNA methylation positively correlates with the transition from vegetative to reproductive growth.

Fig.4 Venn diagram comparison of the numbers of 6mA sites in D9 and D21.

6mA associated with actively expressed genes in Arabidopsis

Analysis of 6mA methylomes and RNA sequencing data demonstrates that 6mA frequency positively correlates with the gene expression level and the transition from vegetative to reproductive growth in Arabidopsis (Fig. 5).

Fig. 5 Correlation between 6mA and Gene Expression in Col Plants.

This research uncover 6mA as a DNA mark associated with actively expressed genes in Arabidopsis, suggesting that 6mA serves as a hitherto unknown epigenetic mark in land plants. Nextomics took part in the program and provided technical support.

Reference

Liang et al., DNA N6-Adenine Methylation in Arabidopsis thaliana, Developmental Cell (2018), https://doi.org/10.1016/j.devcel.2018.03.012

Other eukaryote methylation published papers

[1]Greer, E.L. et al. DNA methylation on N6-adenine in C. elegans. Cell 161, 868–878 (2015).

[2]Wu, T.P. et al. DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature 532, 329–333 (2016).

[3]Mondo, S.J. et al. Widespread adenine N6-methylation of active genes in fungi. Nature Genetics (2017).

The first >2Mb continuous DNA read sequence has been reported by Oxford Nanopore sequencing technology, which is regarded as another giant leap in the development of Nanopore sequencing. The result was published on BioRxiv by a team at the University of Nottingham, led by Alex Payne, etc.^[1]

The output data of NanoporeMinION sequencing is in fast5 format, which can be further converted into fastq format through the base-calling procedure. Previously, the MinKNOW was the conventional choice for MinION base-calling. But Alexander Payne found that the MinKNOW may interrupt long reads by error, while they eliminated this “bug”, the length of reads can achieve over 2Mb.

Why long reads?

Traditional short-read DNA sequencing technologies (also known as the Next-Generation Sequencing, NGS) may provide data about the small fragments of genomic DNA sequences, and it is a great challenge for researchers to assemble these large number of small pieces into a complete genome or dataset.

Nanopore sequencing technology has significant advantages in sequencing read length, including greatly improve the continuity of genome assembly and overcome the problem that caused by complex repeat sequences or structural variation which beyond the capabilities of short-read sequencing. The article recently published on Nature Biotechnology about human Y chromosome centromere sequences achieved by Nanopore sequencing have emphasized the abilities of Nanopore sequencing in solving complex repetitive regions. In addition, the identification of complex tandem rearrangement in the nematode genome and structural variations in the drosophila genome are all outstanding applications of Nanopore long reading sequencing. (For more details, see the “extended reading” at the end of this article)

As the first third-generation sequencing company with the Nanopore sequencing platform in China, NextOmics Biosciences have obtained a great amount of excellent data. Here we present you some results.

Case one: Assembly of an insect’s genome by Nanopore sequencing

The size of the insect genome was estimated to be ~ 330Mb based on k-mer analysis

Fig. 1 K-mer analysis

Qualified sample DNA was extracted, and 30Gb of third-generation data were sequenced on the Oxford Nanopore GridlON X5 platform, with a maximum reading length of 270Kb, and reads N50 length was 26.8kb. Long read length is a prerequisite for more accurate genome assembly.

Fig. 2 Distribution of read length

Genome assembly applying a variety of software and select the optimal scheme. The ultra-long read Nanopore sequencing matching super-computing platform enables genome assembly to be more continuous and faster. In this case, Contig N50 can be >7mb, which has reached the high-quality assembly level as insect model animal fruit flies.

Table1 The assembly results

The assembled genome was compared with the insect genome database by BUSCO to assess the integrity of the assembly of conservative genes, and the entire genome was reflected by indirect measurement f. The results show that after Nanopolish+Pilon (* 2) correction, BUSCO evaluation can reach ~ 98% and genome assembly integrity is good.

Table2 BUSCO assessment

Case 2: Ultra-long sequencing data from an animal

Nanopore ultra-long sequencing can achieve ultra-long reading length. Based on its unique transposase library, DNA sequencing library containing ultra-long fragments, and the ultra-long DNA sequence can be obtained by Nanopore sequencing. Ultra-long sequences will greatly facilitate the de novo assembly of genome and the identification of complex structural variations (SVs) of chromosomes.

Fig. 3 Process of Ultra-long library construction

NextOmics Bioscience conducted ultra-long library construction and sequencing on the blood of a mammal based on Nanopore sequencing platform. The reads N50 of multiple libraries were longer than 70kb, and the longest read length was more than 1Mb.

Fig. 4 Library reads N50 (partial)   

   Fig. 5 Distribution of read-length in a library

[1]Payne, A., Holmes, N., Rakyan, V. & Loose, M. Whale watching with BulkVis: A graphical viewer for Oxford Nanopore bulk fast5 files., doi:10.1101/312256 (2018).

Pacific Biosciences of California, Inc. formally announced a new version of Sequel® Software (V5.1) and a new polymerase on Mar. 7, 2018. Combined, these enhancements increase throughput and the overall performance of Single Molecule, Real-Time (SMRT®) Sequencing for key applications such as de novo assembly, structural variant detection, targeted sequencing, and RNA sequencing (Iso-Seq® method), making genomic research more economical.

With this release the Sequel System can achieve up to 10 Gb per SMRT Cell for genomic libraries, effectively doubling the throughput when using ultra-long inserts (>40 kb) for de novo genome assembly. For targeted and RNA sequencing, customers can achieve up to 20 Gb per SMRT Cell.

For human whole genome sequencing (WGS) studies, the new improvements support sensitive detection of structural variants with as little as 5- to 10-fold coverage per individual. As a result, customers can now complete low-cost WGS studies in thousands of individuals using fewer SMRT Cells.

For long amplicons (>3 kb), the new polymerase increases the number of high-quality sequences per SMRT Cell, reducing costs for HLA sequencing and other targeted applications. Further, software enhancements for multiplexed samples simplify the analytical workflow.

Appendix: Based on PacBio SMRT technology, NectOmics has assembled hundreds of genomes and authored mature process for full-length transcriptome, some published papers as follow:

Classic NextOmics genome-assembling projects

Classic NextOmics Full-length Transcriptome projects based on the Third Generation Sequencing Technology