deSRA

differential expression of SRA: An automated protocol to extract differences in gene expression from public NGS datasets.

Background

The future of biomedical research depends on the ability to rapidly access and analyze Next-Generation Sequencing (NGS) data stored at the NCBI’s Sequence Read Archive (SRA). NGS provides an unprecedented level of resolution, allowing researchers to ask previously unanswerable questions such as how cancer pathogenesis might be mediated by very small changes in gene expression.

Hosting data and performing analyses on the cloud, in a massively parallel fashion, will greatly accelerate the development of new research breakthroughs and innovative pharmaco-therapeutic strategies. Cloud computing would allow researchers to ask computationally intensive questions, such as how liver cells respond to treatment for hepatitis C virus infection (PMID28360091) or how human and mouse gene expression varies across all published datasets (DOI10.1101/189092)

With ~3 million records currently stored in the SRA database, and submissions growing exponentially, the SRA collection represents a treasure-trove of data to be mined by academia and industry. The information contained in a single SRA dataset is equivalent to hundreds of in vitro experiments worth of work, potentially saving thousands of dollars and research hours.

deSRA facilitates the interrogation of SRA datasets for differential gene expression via dockerized pipeline. deSRA makes it easy for biologists to examine the impact of their genes of interest on health and disease in the cloud through a user friendly web-interface.

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Website

(Not fully functional yet i.e. does not accept submissions yet)

Demo for submission:

Demo for result visualization:

Introduction

The differential expression of genes in response to an experimental condition can give valuable information on the pathways involved in the reaction. Unfortunately, it currently takes many steps to compare gene expression levels between Next Generation Sequencing (NGS) data sets. This repository presents code for a simple tool to compare the expression of selected genes between sets of NGS runs, as well as a link to a user friendly web interface.

What’s the problem?

The NCBI Sequence Read Archive (SRA) provides NGS data along with sample and project metadata (NCBI Resource Coordinators 2017). As part of the International Nucleotide Sequence Database Collaboration, the SRA supports access to data from a wide variety of experimental types and sequencing instruments. Unfortunaltely, it can be time-consuming and difficult to access and analyze the data, especially if you want to quickly develop meaningful hypotheses. deSRA bridges this gap between advanced bioinformatic data and users.

Why should we solve it?

The amount of NGS data stored in the Sequence Read Archive (SRA) data-base is growing rapidly. However, many researchers who are interested in this data do not have experience with the tools necessary to analyze it effectively. deSRA increases the utility and return on investment of NGS projects by making the data more accessible to a wider range of individuals.

What is deSRA?

deSRA is a tool to compare two sets of NGS data for differences in gene expression. For example, if the user is interested in how gene expression varies in the liver after treatment for HCV, they may be interested in looking at a BioProject record that links the runs for a relevant experiment, such as https://www.ncbi.nlm.nih.gov/bioproject/328986. From that page, you can select the link for SRA experiments, then view the results in SRA Run Selector, which displays a table including the SRA run accessions and treatment conditions, https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=328986. You will also need at least one Entrez Gene name to search against the SRA datsets.

Build BLAST database
The first step to running the tool is to build BLAST databases for each of the input genes. Building the databases this way allows for rapid querying of only those databases that are relevent to your query, and allows for parallelization, thus increasing the programs speed. The accession, start & stop positions, and gene ID are pulled from ref_GRCh38.p7_top_level.gff3. Based on those positions, a bash script retrieves the sequences in FASTA format and saves each as an individual BLAST database in the active Docker server.

Magic BLAST
MagicBLAST is run iteratively for each gene, once for the treatment runs and once for the experiment runs. The general format for running the magicBLAST command is:
magicblast -sra <accession> -db <database_name> See more at https://ncbi.github.io/magicblast/

Comparison of gene expression
MagicBLAST produces a SAM file, which is processed by separate scripts in Docker encoding samtools commands. The SAM file is converted to a BAM file, which is sorted and indexed, and used to generate a pileup. TPM is calculated for the pileups generated from the experimental runs and the control runs, and a volcano plot is used to display the log2 of the TPM ratios.

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Project team

John Garner
Mingzhang Yang
Eneida Hatcher
Anna-Leigh Brown
Peter Meric
Roberto Vera Alvarez
Ryan Connor
Nora Husain
Aynex Mercado

Installation options:

We provide two options for installing deSRA: Docker or directly from Github.

How to use deSRA

You will need gene names and SRR accessions for two alternative conditions. Launch a website from Docker image!

Installing deSRA from Github

git clone https://github.com/NCBI-Hackathons/deSRA

docker build -t biocontainers/desra deSRA/

Preparing the database

Creates a working directory data

docker run -it -v `pwd`/data:/data biocontainers/desra desra_pre-process.sh -d /data/blastdb

Creating database for a selected number of genes

Create a file into the data directory with the list of genes to process (Gene name per line). Let’s name the file gene_name.txt

Create the database using:

docker run -it -v `pwd`/data:/data biocontainers/desra desra_pre-process.sh -d /data/blastdb -g /data/gene_name.txt

This command will create a folder /data/blastdb/ref_GRCh38.p7_top_level, inside that folder will be a folder per gene including the gene fasta sequence and the blast db.

A gene can have multiple copies in the genome. In this case each copy sequence is included in the database. The database name follows this format: GeneName_GeneID_StarPos_EndPos.fasta

For the gene A1CF these are the files created into the directory /data/blastdb/ref_GRCh38.p7_top_level/A1CF/:

A1CF_29974_50799409_50885681.fasta
A1CF_29974_50799409_50885681.nhr
A1CF_29974_50799409_50885681.nin
A1CF_29974_50799409_50885681.nog
A1CF_29974_50799409_50885681.nsd
A1CF_29974_50799409_50885681.nsi
A1CF_29974_50799409_50885681.nsq

Processing data with the command line options

To align a single gene with a number of SRA accessions. The gene blastdb should be create with the previous step.

docker run -it -v `pwd`/data:/data biocontainers/desra desra_go_mb.sh -d /data/blastdb/ref_GRCh38.p7_top_level -s comma_separated_sra_list -g A1CF -t number_of_threads -c condition1

This command will create two files:

A1CF_29974_50799409_50885681.bam
A1CF_29974_50799409_50885681.bam.bai

Processing a list of gene vs two lists of SRA accessions

We’ve developed a main script desra_main.sh to process a list of genes vs two lists of SRA accessions (condition 1 and condition 2). This script is also designed to process the jobs submitted by the website. Therefore, it required special working folder.

This kind of jobs should be executed in directory inside data/jobs creating a folder with an unique identifier that will be used as job_id. For example, create a folder data/jobs/00001/.

Inside this directory creates three files named: gene_name, sra_cond1 and sra_cond2. Each files should have an id per line.

Run this command to process the data:

docker run -it -v `pwd`/data:/data biocontainers/desra desra_main.sh -d /data/blastdb/ref_GRCh38.p7_top_level -j 00001 -e r78v10a07@gmail.com -t 4

Please, note that -t 4 will run magicblast with 4 threads. Adapt this command to your host resources.

Running the docker image for the website

Execute this command for the website app. It will be available in the host port 8000

docker run -d -p 8000:8000 -v `pwd`/data:/data biocontainers/desra /home/biodocker/bin/docker_entry.sh

The blastdb database needs to be created before start the website app.

Future Development Plans

The front end needs to make a call to get the job IDs. Currently the files are put in the /data folder, but they should go into the /jobid folder inside the /data folder. Write a service that creates the folder and returns the job ID, and start the bash script. Accept gene IDs in addition to gene names
Support organisms other than human

Output with simulated volcano plot

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Actual plot!

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