Analysis of, and software development for, ChIP‐Seq and RNA‐Seq data by Yu‐Hsuan Lin A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Bioinformatics) in The University of Michigan 2012 Doctoral Committee: Professor James Douglas Engel, Co‐Chair Assistant Professor Maureen A. Sartor, Co‐Chair Professor Kerby A Shedden Assistant Professor Ivan Patrick Maillard Research Assistant Professor Fan Meng © Yu‐Hsuan Lin 2012 To my grand parents and parents ii Acknowledgements I would like to thank Dr. Sakie Hosoya‐Ohmura for her work in writing our manuscript on the Gata3 enhancer, and Dr. Henriette O'Geen for the initial version of the manuscript relating to chapter 3. I would also like to thank Dr. Lihong Shi for developing the ex vivo CD34 culture system in our laboratory and for performing the RNA‐Seq experiments that provide the basis for the bioinformatics analyses presented in Chapter 4. I would also thank Yanxiao Zhang for revising the ChIP‐Seq analysis pipeline to make it more efficient and to incorporate additional functionality. Finally, I greatly appreciate the advice of Dr. Engel and Dr. Sartor for their great contribution to this thesis. iii Table of Contents Dedication ..................................................................................................................................................... ii Acknowledgements .................................................................................................................................. iii List of Figures ............................................................................................................................................ vii List of Tables ............................................................................................................................................... ix Chapter I. Introduction .................................................................................................................... 1 A. Overview ............................................................................................................ 1 B. Thesis chapter summary ............................................................................ 3 i. Chapter II summary ....................................................................... 3 ii. Chapter III summary ...................................................................... 5 iii. Chapter IV summary ...................................................................... 6 iv. Chapter V summary ........................................................................ 8 C. Thesis contribution ....................................................................................... 9 II. An NK and T Cell Enhancer Lies 280 Kilobase Pairs 3’ to the Gata3 Structural Gene ............................................................................................................ 11 A. Introduction ................................................................................................... 11 B. Materials and methods .............................................................................. 14 i. Mice ..................................................................................................... 14 ii. BAC recombineering .................................................................... 14 iii. eGFP reporter BAC recombinants .......................................... 15 iv. Deletion of TCE‐7.1 from BAC 43G17 ................................... 15 v. Construction of an eGFP reporter plasmid containing TCE‐7.1 ................................................................................................. 16 vi. Flow cytometry .............................................................................. 16 vii. In vitro CD4+ T cell differentiation assay ............................. 18 viii. In vivo and ex vivo imaging ........................................................ 19 ix. Bioinformatics ................................................................................ 19 x. ESTs ..................................................................................................... 19 C. Results .............................................................................................................. 20 i. Neither the Gata3‐1a nor ‐1b promoter confers T cell autonomous expression in vivo .............................................. 20 ii. A potent T cell element located far 3′ to Gata3 ................. 21 iii. A 7.1‐kbp genomic fragment directs Gata3 activity at multiple T cell stages ................................................................... 26 iv. TCE‐7.1 bears Gata3 T and NK cell‐specific regulatory information ...................................................................................... 35 D. Discussion ....................................................................................................... 39 iv III. Genome‐Wide Binding of the Orphan Nuclear Receptor TR4 Suggests Its General Role in Fundamental Biological Processes .............................. 44 A. Introduction ................................................................................................... 44 B. Results and discussion ............................................................................... 47 i. Identification of genome‐wide TR4 binding sites ........... 47 ii. TR4 target genes are involved in fundamental biological processes ........................................................................................... 53 iii. Motif analysis suggests the importance of ETS family members in TR4 action ............................................................... 59 iv. ETS transcription factor ELK4 co‐occupies TR4 target sites ..................................................................................................... 63 C. Conclusions ..................................................................................................... 69 D. Methods ............................................................................................................ 71 i. Cell culture and crosslinking .................................................... 71 ii. Chromatin immunoprecipitation (ChIP) assay and library preparation ....................................................................... 71 iii. Sequencing and data analysis .................................................. 72 iv. Motif analysis .................................................................................. 73 v. RNA preparation and Illumina expression arrays .......... 73 vi. ChIP assay and quantitative PCR (qPCR) ............................ 74 IV. RNA‐Seq Analysis of Differentiating CD34+ Cells Suggests Novel Isoforms of Erythroid Regulators ........................................................................ 75 A. Introduction ................................................................................................... 75 B. Methods ............................................................................................................ 77 i. RNA sequencing ............................................................................. 77 ii. Alignments of sequence reads and evaluation of data quality ................................................................................................. 78 iii. Differential expression analysis .............................................. 78 iv. Functional analysis ....................................................................... 79 C. Results and discussion ............................................................................... 79 i. Transcriptome dynamics during CD34+ progenitor cell differentiation ................................................................................. 79 ii. Novel isoforms of known erythroid regulators................ 96 iii. Potential novel intergenic/intronic transcripts ............ 103 D. Conclusions .................................................................................................. 104 V. PePr: A ChIP‐Seq Peak Prioritization Pipeline for Testing Replicated ChIP‐Seq Data and Integrating External Annotations ............................. 106 A. Introduction ................................................................................................ 106 B. Methods ......................................................................................................... 109 i. Datasets .......................................................................................... 109 a. ATF4 .................................................................................. 109 b. H3K27 trimethylation ................................................ 110 ii. PePr input formats ..................................................................... 111 iii. PePr preprocessing ................................................................... 112 a. Removal of duplicates ................................................ 112 b. Shift size calculation ................................................... 112 v c. Window size calculation ........................................... 112 d. Normalization ............................................................... 113 iv. PePr peak detection .................................................................. 113 a. Dispersion factor calculation .................................. 113 b. P‐value and FDR calculation ................................... 114 v. Incorporating peak location relative to gene structure (PePr version 2