Analysis and Integration of Big Data from Next-Generation Genomics, Epigenomics, and Transcriptomics

Christopher Benner, PhD

Director, Integrative Genomics and

Bioinformatics Core (IGC)

iDASH Webinar, April 17

2015

Analysis and Integration of Big Data

from Next-Generation Genomics,

Epigenomics, and Transcriptomics

Overview for Webinar:

•

Quick introduction to the wider world of next-generation

sequencing (NGS)

•

Overview of HOMER, our software for NGS analysis

•

Using advanced NGS assays to understand B cell

development and the generation of antibody repertoires

•

Quick teaser on how innovative NGS assays and genetics

can enhance our understanding of transcriptional

mechanisms

Next-Generation Sequencing

•

Large Consortiums

–

1000 Genomes Project

–

TCGA (cancer)

–

many many more…

Illumina sequencing can sequence any

NGS Innovation

(i.e. gene expression) GRO-Seq

(i.e. transcription rates) ChIP-Seq

HOMER

(Hypergeometric Optimization of Motif EnRichment)

http://homer.salk.edu

•

Next-generation Sequencing

Analysis for Quantitative Genomics

– Software suite for UNIX command-line

environment (works downstream of manufacture’s pipeline and mapping to reference genome)

– Quality Control for Experiments

– Basic and advanced analysis, annotation,

and visualization capabilities

– General framework handles data from

different types of quantitative

sequencing (ChIP-Seq/RNA-Seq/GRO-Seq/DNase-Seq/etc.)

– Can work with any organism

•

Regulatory element analysis

– De novo Motif Discovery

– Sort out spatial relationships between

HOMER Functionality

Any organism with a FASTA file

can be analyzed with HOMER

•

Model organisms are

preconfigured with

annotation information:

– Human, mouse, rat,

zebrafish, drosophila, C. elegans, yeast, pombe, arabidopsis

•

Genomes annotated on

the UCSC Genome

Browser are easy to

incorporate, but any

custom genome can be

added with annotation

files (i.e., GTF files)

Best way to develop NGS Analysis

methods: Do it in the context of research!

Biology

NGS Methods Development

Interplay between epigenetics, spatial genome conformation,

and transcription in B-lymphocyte development

Interplay between epigenetics, spatial genome conformation,

and transcription in B-lymphocyte development

Why study transition from

pre-pro-B

to

pro-B

cells?

•

Lineage commitment:

pro-B cells cannot

dedifferentiate back to hematopoietic stem cells.

–

i.e. pre-pro-B cells can be used to reconstitute the whole

immune system

•

Antibody Recombination:

Pro-B cells are paused at the

exact stage when VDJ recombination is set to occur

•

B cell marker expression:

Key cell-surface markers and

transcription factors are induced in pro-B cells,

including CD19, Ebf1 (Early B cell factor), Pax5, and

Foxo1.

Unbiased

Discovery of

Regulatory

Features in

pro-B cells

Relationship between Transcription

Factors and Epigenetic Modifications

Unbiased Discovery of Lineage

Determining Transcription Factors

Hi-C: Mapping 3D interactions in the genome

Hi-C method from Lieberman-Aiden et al., Science 2009

GRO-Seq

Most significant interactions in the genome

occur at epigenetically modified locations

Cell-type specific

interactions often

change their DNA

methylation status

Genome Organization into

topological domains

TAD definition by Dixon et al. 2012

pre-pro-B pro-B

CTCF binding site is directional

CTCF only makes interactions with other CTCF sites in a specific direction along the DNA determined by the orientation of the motif

Clusters of CTCF sites form

‘Super Anchors’

pre-pro-B pro-B

Clusters of CTCF sites form

‘Super Anchors’

Borrowing from Richard Young’s Super Enhancer concept, we can define over 2500 CTCF ‘super anchors’ in the data Only 25% of CTCF sites are found at boundaries. However, nearly 50% of ‘Super Anchors’ are found at the boundaries of

topological domains. Foxo1

Igh Firre

Overview of Immunoglobulin Heavy

Chain Locus

Igh Locus in the Genome (~3 Mb)

Igh Locus in the Genome (~3 Mb)

Top Super Anchor

To generate full repertoires of

Antibodies, each V region needs to find a way to interact with the D regions to recombine

V regions in Igh locus are associated

with CTCF sites

Igh Locus Model

Top Super Anchor (looping backstop) VD recombination target

Summary

•

NGS is a lot more than genome sequencing

•

Integration of different data types empowers

discovery where any given data type alone

falls short

•

The DNA sequence (CTCF motifs and their

orientation) dictates the structure of the

genome to accomplish critical tasks such as

VDJ recombination