TSS assembly pipeline for Sp_EPDnew_002

Introduction

This document provides a technical description of the transcription start site assembly pipeline that was used to generate EPDnew version 002 for S. pombe.

Source Data

Promoter collection:

Name	Genome Assembly	Promoters	Genes	PMID	Access data
RefSeq Genes	Aug 2015 ASM294v2/spo2	5128	5128	22121212	SOURCE	DOC	DATA

Experimental data:

Name	Type	Samples	Tags	PMID	Access data
Li et al., 2015	DeepCAGE	1	17,279,045	25747261	SOURCE	DOC	DATA
Thodberg et al. 2018	CAGE	15	276,570,639	doi:281642	SOURCE	DOC	DATA

Assembly pipeline overview

Description of procedures and intermediate data files

1. Download annotated TSS

Data was downloaded from NCBI RefSeq database the 02-02-2015. Transcrips have been filtered for protein coding gene only, removing pseudogenes from the list.
Gene names were taken from the field "Locus ID". Since the EPD format doesn't allow gene names longer than 18 characters, we checked whether the names repsected this limitation.
A total number of 5128 promoters were selected.

2. SGD TSS collection

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

spo2TssFromRefSeq.sga

The six field contain the following kinds of information:

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

Note that the second and forth fields are invariant.

3. Import CAGE data

Data was imported from ArrayExpress as FASTQ file format. Raw sequence files were mapped to spo2 genome using Bowtie (trimming 1bp from the 5-end). The resulting BAM files were converted to SGA file format using ChIP-Convert.
A step-by-step guide on how to import, map and convert these samples can be found here

5. mRNA 5' tags peak calling

For each individual sample (3), 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 UCSC TSS) was then separately processed in a pipeline aiming at validating transcription start sites with mRNA peaks. A UCSC TSS was experimentally confirmed if an mRNA peak lied in a window of 500 bp around it. The validated TSS was then shifted to the nearest base with the higher tag density.

7. UCSC not-validated TSS

The total number (summing up all samples) of non experimentally validated TSS was around 2000.

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 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 larger number of samples as the true TSS.

10. Filtering

Transcription Start Sites that mapped closed to other TSS that belonged to the same gene (500 bp window) were merged into a unique promoter following the same rule: the promoter that was validated by the higher number of samples was kept.

10. Final EPDnew collection

The 4802 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.