TSS assembly pipeline for Am_EPDnew_001

Introduction

This document provides a technical description of the transcription start site assembly pipeline that was used to generate EPDnew version 001 for A. mellifera.

Source Data

Promoter collection:

Name	Genome Assembly	Promoters	Genes	PMID	Access data
RefSeq Genes	Apr 2011 Amel_4.5/amel5	17735	10727	22121212	SOURCE	DOC	DATA

Experimental data:

Name	Type	Samples	Tags	PMID	Access data
Khamis et al., 2015	CAGEscan	16	70,802,351	26073445	SOURCE	DOC	DATA

Assembly pipeline overview

Description of procedures and intermediate data files

1. Download annotated TSS

Data was downloaded from RefSeq the 20-07-2016. Transcripts have been filtered according to the following rules:

1. Transcripts of protein coding genes only
2. Transcripts have a non-empty description field

Gene names were taken from the field "Locus ID". Since the EPD format does not allow gene names longer than 18 characters, we checked whether the names respected this limitation.
A total number of 17735 promoters were selected.

2. RefSeq TSS collection

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

amel5TssFromRefSeq.sga

The six fields contain the following information:

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

Note that the second and forth fields are invariant.

3. Import CAGE data

Data was imported from GEO as SRA file format. Raw sequence files were mapped to amel_4.5 genome using Bowtie. The resulting BAM files were converted to SGA file format using ChIP-Convert.

5. mRNA 5' tags peak calling

For each individual sample (16), peak calling for the merged file has been carried out using ChIP-Peak on-line tool with the following parameters:

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

6. TSS validation and shifting

Each sample in the collection (mRNA peaks and RefSeq TSS) was then separately processed in a pipeline aiming at validating transcription start sites with mRNA peaks. A RefSeq TSS was experimentally confirmed if an mRNA peak lied in a window of 300 bp around it. The validated TSS was then shifted to the nearest base with the higher tag density.

7. RefSeq not-validated TSS

The total number (summing up all samples) of non experimentally validated TSS was around 10000.

8. Promoter collection for each sample

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).

9. Merging collections and second TSS selection

The 16 promoter collections were merged into a unique file and further analysed. Transcripts validated by multiple samples could potentially have the TSS set on a broader region and not to single position. To avoid such inconsistency, for each transcript we selected the position that was validated by the larger number of samples as the true TSS.
Different TSSs that belong to the same gene were classified according to their global expression level. The primary TSS of a gene (marked with an '_1' at the end of the ID) always has the highest expression level, followed by all the others in decreasing order of expression (marked with '_2', '_3', etc.).

10. Filtering

TSSs that mapped closed to other TSSs belonging to the same gene (500-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.

10. Final EPDnew collection

The 6493 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.