TSS assembly pipeline for Rm_EPDnew_001

Introduction

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

Source Data

Promoter collection:

Name	Genome Assembly	Promoters	Genes	PMID	Access data
ENSEMBL92 genes	Nov 2015 BCM Mmul_8.0.1/rheMac8	36903	20593	29092050	SOURCE	DOC	DATA

Experimental data:

Name	Type	Samples	Tags	PMID	Access data
francescatto17	CAGE	15	217,883,048	29087374	SOURCE	DOC	DATA

Assembly pipeline overview

Description of procedures and intermediate data files

1. Ensembl Download

Data was downloaded from ENSEMBL in July 2018. Transcripts have been filtered according to the following rules:

1. Transcripts of protein coding genes only
2. Transcripts on full chromosomes
3. Genes must be annotated [Gene Name present]
4. Genes' and transcripts' status known

Gene names were taken from the field "Gene Name". Since the EPD format does not allow gene names longer than 18 characters, we checked whether the names respected this limitation. Transcripts with the same TSS position were merged under a common ID, leaving 36903 uniquely mapped promoters in the input list, from a total of 44758 transcripts originally present in the Ensembl database.

2. Ensembl TSS collection

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

Rm_ensembl92_tss_rheMac8.sga

The six fields contain the following information:

• NCBI/RefSeq chromosome id
• "TSS"
• position
• strand ("+" or "-")
• "1"
• ENSEMBLGeneID .. geneName

Note that the second and forth fields are invariant.

3. Data import from FANTOM5

BAM files for high quality CAGE samples (hCAGE) were downloaded from FANTOM5 ftp site (link above). Files were then converted into SGA format using in-house software. There are a total number of 15 samples in this collection. Individual SGA files can be downloaded from our ftp website (link above).

5. mRNA 5' tags peak calling

For each individual sample, peak calling for the merged file 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

Each sample in the collection (mRNA peaks and Ensembl TSS) was then separately processed in a pipeline aiming at validating transcription start sites with mRNA peaks. An Ensembl TSS was experimentally confirmed if an mRNA peak lied in a window of 200 bp around it or if it mapped in the 5' UTR region. The validated TSS was then shifted to the nearest base with the highest tag density.

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 15 promoter collections were merged into a unique file and further analyzed. The promoter of a transcript was kept in the list only if validated by at least two samples. Transcripts validated by multiple samples could potentially have the TSS set on a broader region rather than a single position. To avoid such inconsistency, we selected for each transcript 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 (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 9575 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.