TSS assembly pipeline for At_EPDnew_004

Introduction

This document provides a technical description of the transcription start site assembly pipeline that was used to generate EPDnew version 004 for A. thaliana.

Source Data

Promoter collection:

Name	Genome Assembly	Promoters	Genes	PMID	Access data
TAIR10 Genes	Feb 2011 TAIR10/araTha1	31615	27149	26201819	SOURCE	DOC	DATA

Experimental data:

Name	Type	Samples	Tags	PMID	Access data
Morton et al., 2014	PEAT	1	22,578,668	25035402	SOURCE	DOC	DATA
Cumbie et al., 2015	NanoCAGE-XL	3	226,177,104	26268438	SOURCE	DOC	DATA
Tokizawa et al., 2017	CAGE and OligoCap	2	178,605,048	28214361	SOURCE	DOC	DATA
Ushijima et al., 2017	CAGE	7	552,864,502	29129375	SOURCE	DOC	DATA

Assembly pipeline overview

Description of procedures and intermediate data files

1. Download annotated TSS

Primary annotation data was downloaded from TAIR the 06-02-2015.

Genes annotations downloaded from TAIR did not contain direct links to RefSeq ID. For this reason, RefSeq ID has been parsed from NCBI RefSeq files.

A total of 31615 promoters were selected.

2. TAIR10 TSS collection

The TAIR10 TSS collection is stored as a tab-deliminated text file conforming to the SGA format under the name:

arabidopsisTair10Genes.sga

The six fields contain the following information:

• NCBI/RefSeq chromosome id
• "TSS"
• position
• strand ("+" or "-")
• "1"
• TAIR ID

Note that the second and forth fields are invariant.

3. Import CAGE data

Data was imported from the lab web page or GEO in BAM or SRA file formats. Please refer to the "Source Data" table at the beginning of this document for the links to raw data archives. A detailed guide on how to import, map and convert these samples can be found in the corresponding "MGA doc" files.

5. mRNA 5' tags peak calling

For the 12 samples present, peak calling has been carried out using ChIP-Peak on-line tool with the following parameters:

• Window width = 1
• Vicinity range = 200
• Peak refine = Y
• Count cutoff = 9999999
• Threshold = 5

6. TSS validation and shifting

The 12 samples used were then individually processed in a pipeline aiming at validating transcription start sites with mRNA peaks. A TAIR10 TSS was experimentally confirmed if a CAGE peak lied in a window of 100 bp around it. The validated TSS was then shifted to the nearest base with the higher tag density.

7. Promoter collections

Each sample in the dataset was used to generate a separate promoter collection. Potentially, the same transcript could be validated by multiple samples and it could have different start sites in different samples. To avoid redundancy, the individual collections were used as input for an additional step in the analysis (Assembly pipeline part B).

8. Quality controls

Each promoter collection was then checked for the presence of core promoter elements (TATA-box and Inr) in the expected positions.

9. Merging sample-specific collections and further TSS selection

All sample-specific promoter collections were merged into a unique file and further analyzed. Transcripts validated by multiple samples could potentially have the TSS set on a broader region rather than a single position. To avoid such inconsistency, for each transcript we selected the position that was validated by the largest number of samples as the true TSS.

10. Filtering

TSSs that mapped close to other TSSs that belonged to the same gene (100-bp window) were merged into a unique promoter following the same rule: the promoter that was validated by the highest number of samples was kept.

11. Final EPDnew collection

The 22703 experimentally validated promoters were stored in the EPDnew database, which can be downloaded from our ftp site. Scientists are welcome to use our other tools ChIP-Seq (for correlation analysis) and SSA (for motif analysis around promoters) to analyze the EPDnew database.