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Introduction

m⁶ 🅐 -Atlas

N⁶-methyladenosine (m⁶A) is currently the most prevalent RNA modification on mRNAs and lncRNAs, and plays a pivotal role during various biological processes and disease pathogenesis. Here, we developed m⁶A-Atlas, a comprehensive knowledgebase for unraveling the m⁶A epitranscriptome. (1) 442162 high-confidence m⁶A sites identified from seven base-resolution technologies were collected, and the quantitative (rather than binary) epitranscriptome profiles were estimated from 1363 high-throughput sequencing samples. (2) The conservation of m⁶A sites among seven vertebrate species, including human, mouse and chimp, and the 10 viruses’ m⁶A epitranscriptomes, including HIV, KSHV and DENV. (3) The putative biological functions of individual m⁶A sites predicted from epitranscriptome data, and the potential pathogenesis of m⁶A sites inferred from disease-associated genetic mutations that can directly destroy m⁶A directing sequence motifs. (4) Annotation of m⁶A epitranscriptome sites with post-transcriptional machinery (RBP-binding, microRNA interaction and splicing sites), and the landscape of multiple RNA modifications, together provided users to browse and do further studies.

Pipeline Construction

① High-confidence collection of m⁶A sites

High-confidence m⁶A sites were collected from 67 datasets generated by seven different base-resolution technologies, covering seven species, including human, mouse, rat, zebrafish, fly, Arabidopsis and yeast. The positional information was directly obtained from the original papers or relevant NCBI GEO datasets.

② Quantification of m⁶A methylation levels

The raw m⁶A-seq datasets were directly downloaded from NCBI GEO and Genome Sequence Archive in BIG Data Center. Adaptors and low quality nucleotides were removed by Trim Galore, and the processed reads were aligned to the reference genome by Tophat2.

③ Viral and host epitranscriptomes

A total of 278 m⁶A-seq samples profiling the epitranscriptomes during virus infection were collected and the data covered different infection stages of a total 10 virus species. The m⁶A sites located on viral transcripts were detected by MACS2 from m⁶A-seq data, and are thus not at base-resolution as previously explained.

④ Conservation of m⁶A sites in vertebrate

By integrating and comparing a large number of m⁶A sites across different species, we provided the first comprehensive profile of the conserved epitranscriptome in vertebrates at single-base resolution. During the cross-species comparison, the m⁶A sites of one species were mapped to the homologous coordinates of a target organism using the LiftOver tool from the UCSC genome browser.

⑤ Putative functions of individual m⁶A sites

The putative biological functions of individual m⁶A sites were predicted for the conserved between human and mouse according to guilt-by-association principle and this method relied on the associated methylation patterns (or co-methylation) among functionally related m⁶A sites inferred from the collected quantitative epitranscriptome data.

⑥ Basic annotation for m⁶A sites

The splicing sites, miRNA target sites, RBP-binding sites and subcellular location were integrated to help understand the regulatory roles of m⁶A. The splicing sites and subcellular location were obtained from UCSC database and RNALocate, respectively. The miRNA target sites and RBP-binding sites in human were obtained from the starBase and POSTAR. Transcriptional regulation data (ChIP-seq) were obtained from GTRD database.

⑦ Landscape of m⁶A and other RNA modifications

Here we provide the landscape of multiple RNA modifications on the same gene so as to provide a more comprehensive overview. To do so, a total of 96 datasets generated from base-resolution technologies were collected, covering six other RNA modifications in human, including 5-Methylcytosine (m⁵C), Pseudouridine (Ψ), N6,2-dimethyladenosine (m⁶Am), N1-methyladenine (m¹A), N7-methylguanosine (m⁷G) and 2’-methylation (Nm). For mouse, we integrated m⁵C, m¹A and Ψ with m⁶A modification; and we integrated m¹A and Ψ for yeast epitranscriptomes

Contents of m⁶A-Atlas

Models	Human	Mouse	Rat	Zebrafish	Fly	Arabido-psis	Yeast
Reliable  m⁶A Sites
Quantitative Profiles
Basic Annotation
Conservation in Vertebrates
Putative Functions
Landscape of Modifications
Disease Association

User guide

Getting Started

We present here m6🅐-Atlas, a centralized online platform for a high-confidence collection of unique m⁶A sites. The m⁶A-Atlas consists of the following two major components, including: eukaryote and virus m6 A site databases. On the Home Page, the Quick search feature allows users to select seven species and find m⁶A sites using a search query (i.e. Gene, Region or Disease-Associationenomic location). Quick search is available by using the search bar; The Sidenav Buttons allow users to directly go to the database with a particular species; The following two box buttons allow users to the eukaryote and virus m6 A site databases by clicking, respectively.

Eukaryote

1. Eukaryote Table

m⁶A-Atlas provides users a webpage for a high-confidence collection of reliable m⁶A sites identified from seven base-resolution technologies covering seven eukaryotes. The detail list of m⁶A sites, corresponding cell lines and tissues, relative technologies and the statistical results were summarized and presented, according to the needed filter selection of species, RNA modification, cell line, and technique (e.g. Human).

2.Basic Info

Users can click “Site ID” to see more information. On Basic Info section, users click on button, to link to Jbrowser showing m⁶A-sites, gene, RNA Binding region, miRNA target and other RNA modfication sites.

3. Methylation Level

m⁶A-Atlas provides users an interactive drawing to show the differential methylation levels under various conditions. The first choice is to select the methylation level on normal cell line; the second choice is to display the methylation level after virus infection. Users can select functions of several choices to query the various cell lines, techniques or viruses which you are interested in.

4. Gene Expression Level

Similarly, m⁶A-Atlas also provides users an interactive drawing to show the differential expression gene levels where m⁶A site(s) located under various conditions. The first choice is to select the gene expression level on normal cell line; the second choice is to display the gene expression level after virus infection. Users can select functions of several choices to query the various cell lines, techniques or viruses which you are interested in.

5. Conservation

Here, m⁶A-Atlas also offers conservation analysis was performed among human, mouse, rat, zebrafish, pig, monkey and chimpanzee to identify the conserved m6A sites between two vertebrate species.

6. Landscape

m⁶A-Atlas provides users an interactive RNA modfication landscape, where gene regions and different RNA modification sites distribution plots are demonstrated and it also show the annotation information involving in m6A sites shown at the top. Users can view the summary table for the number of m⁶A sites and their closer RNA modification sites by clicking the Table button and downloading this table.

7. Annotation

m⁶A-Atlas provides the basic annotation for m⁶A sites, including the splicing sites, miRNA target sites, RBP-binding sites and subcellular location integrated to help understand the regulatory roles of m⁶A. Moreover, the potential pathogenesis of human m⁶A sites inferred from disease-associated genetic mutations that can destroy m⁶A forming sequence motif are also demonstrated.

8. GO prediction

m⁶A-Atlas makes the putative biological functions of individual m⁶A site were predicted for the conserved between human and mouse according to guilt-by-association principle, as previously described. On the GO predication section, the two tabular listings show neighbor sites of target m⁶A site and related predict GO functions. Click to see Network Graph of network ralation between the neighbor m⁶A site and GO functions.

Virus

1.Virus Table

Try to scroll this section and look at the navigation bar while scrolling!

Tool

1. m⁶A-conservation-finder

m⁶A-Atlas provides users a convenient Web Server to do a batch query. Among the m⁶A-conservation-finder is used to identify the conserved m⁶A sites among a user-provided list of candidate sites. According to inputted data or uploaded file (required bed format),and the corresponding samples are demonstrated; Users can choose different species (assemblies) including Human/hg19, Mouse/mm10, Rat/rn6 and Zebrafish/danRer10. It is noted that users are required to enter their E-mail address to receive a notification after their job is completed. After submitting , each job will be assigned a unique ID to the users. The job can be retrieved by waiting on the pop-up results webpage directly.

After the work is finished, the detail list of query species with predicted conservation sites in different species as well as related information are presented. Click on Download in the upper right corner, users can get their predicted results. Under the table, statistic analysis of predicted results is also illustrated.

2. differential-m⁶A-finder

m⁶A-Atlas provides users a convenient Web Server to do a batch query. Fot the differential-m⁶A-finder, it returns the differentially methylated m⁶A sites between two experimental conditions. Users can have several choices including the species and two different conditions, i.e. Homo sapiens (Human), GSE99017_brain_cortex_Ctrl and GSE128443_HeLa_Ctrl. Users can also look up the following table to select their interested conditions. It is noted that users are required to enter their E-mail address to receive a notification after their job is completed. After submitting, each job will be assigned a unique ID to the users. The job can be retrieved by waiting on the pop-up results webpage directly.

After the work is finished, the detail list of query specie with the differentially methylated m⁶A sites between two experimental conditions as well as related information are presented. Click on Download in the upper right corner, users can get their predicted result.

3. Retrieve Results

When users leave the job page or lose it by accident, the job ID can be used to retrieve their job.

Download

The data used on this website from m⁶A-Atlas online data and Quantification of m⁶A methylation levels is available and the statistic results were summarized and presented at bottom.