Engineering annotation- agnostic tools for differential expression analysis Alyssa Frazee Johns Hopkins University @acfrazee 

Why annotation-agnostic? 

Why annotation-agnostic? (1) isoform-level analysis 

scientifically important   cell differentiation (Trapnell 2010) organism development (Graveley 2010) cancer (Govindan 2012) 

24376000 24378000 24380000 24382000 24384000 genomic position 332 333 334 335 336 337 338 but read-counting is challenging   

Why annotation-agnostic? (2) allows for new discoveries 

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One solution: assembly genome some possible assemblies 

Approach 1: avoid assembly altogether 

DER Finder PMID 24398039 idea: scan genome base-by- base, highlight segments showing differential expression signal 

DER Finder 0 2 4 6 8 log2(count+1) chr22: 17684448−17684670 normal tumor −1 0 1 2 3 4 5 t statistic states genomic position DE signal read coverage 

DER Finder 0 2 4 6 8 log2(count+1) chr22: 17684448−17684670 normal tumor −1 0 1 2 3 4 5 t statistic states genomic position DE signal read coverage 

find signal at each nucleotide samples indexed by i locations indexed by l j confounders indexed by k expression confounders covariate of interest 

samples indexed by i locations indexed by l j confounders indexed by k expression confounders covariate of interest  v   find signal at each nucleotide 

DE DE not DE segment genome into groups of nucleotides with similar signal t 1 t 2 t 3 t 4 t 5 DE not DE 

DE DE not DE segment genome into groups of nucleotides with similar signal t 1 t 2 t 3 t 4 t 5 DE not DE Hidden Markov Model 

0 2 4 6 8 log2(count+1) chr22: 17684448−17684670 normal tumor −1 0 1 2 3 4 5 t statistic xaxinds exons states 17684041 17684451 17684551 17684651 17684754 genomic position linear models HMM (candidate DERs) 

0 2 4 6 8 log2(count+1) chr22: 17684448−17684670 normal tumor −1 0 1 2 3 4 5 t statistic xaxinds exons states 17684041 17684451 17684551 17684651 17684754 genomic position linear models HMM permutation tests for statistical significance 

0 2 4 6 8 log2(count+1) chr22: 17684448−17684670 normal tumor −1 0 1 2 3 4 5 t statistic xaxinds exons states 17684041 17684451 17684551 17684651 17684754 genomic position match to annotation if desired: CECR1, “may play a role in regulating cell proliferation” 

engineering challenges •  creating and handling nucleotide-by- sample matrix •  efficient linear model fitting (solution: lmFit) •  efficient segmentation with HMM •  efficient p-value calculations Initial solution: https://github.com/alyssafrazee/derfinder 

Approach 2: improve the current software infrastructure for analysis of transcriptome assemblies 

Ballgown biorXiv preprint: http://biorxiv.org/content/early/2014/03/30/003665, Bioconductor package “ballgown” Align Reads! (e.g. TopHat)! Assemble Transcripts! (e.g. Cufflinks)! Estimate Expression! (Cufflinks via Tablemaker, RSEM)! Differential Expression Tests! (default Ballgown models, limma, EdgeR, DESeq,…)! paired-end RNA-seq reads! Transcriptome Assembly! Pipelines! R/Bioconductor Pipelines! Ballgown as connecting framework! transcriptome assembly pipelines R/Bioconductor DE analysis 

S4 class for transcript assemblies ballgown object data structure indexes exon intron transcript exon intron transcript e2t i2t t2g pData bamfiles expr matrices GRanges data frames 

easy exploration and DE analysis 24376000 24378000 24380000 24382000 24384000 genomic position 332 333 334 335 336 337 338 plotting functions 

stat_results = stattest(my_assembly, feature='transcript', meas='FPKM', covariate='group’) head(stat_results) ## feature id pval qval ## transcript 10 0.01381576 0.105212332 ## transcript 25 0.26773622 0.791149753 ## transcript 35 0.01085070 0.089518254 ## transcript 41 0.47108019 0.902537475 ## transcript 45 0.08402948 0.489348136 ## transcript 67 0.27317385 0.79114975 easy exploration and DE analysis statistical tests (drop-in replacement for Cuffdiff) 

ballgown object data structure indexes exon intron transcript exon intron transcript e2t i2t t2g pData bamfiles expr easy exploration and DE analysis statistical tests easily connects to existing DE packages 

easy exploration and DE analysis annotation functions 24615000 24620000 24625000 24630000 24635000 24640000 genomic position Assembled and Annotated Transcripts annotated assembled get corresponding gene names, match assembled and annotated transcripts, plot assembly alongside annotation 

highly flexible 

freely available! Bioconductor (devel): source(“http://bioconductor.org/biocLite.R”) biocLite(“ballgown”) GitHub:   https://github.com/alyssafrazee/ballgown Cufflinks users will also need Tablemaker: https://github.com/alyssafrazee/tablemaker 

Thanks Collaborators: Jeff Leek (advisor), Steven Salzberg, Ben Langmead, Andrew Jaffe, Rafa Irizarry, Sarven Sabunciyan, Kasper Hansen, Geo Pertea, Leonardo Collado Torres Contact: alyssafrazee.com, [email protected], @acfrazee (Twitter) 

differential expression model for each transcript, compare the fits of the following models using an F-test. Null hypothesis is that the fits of model (a) and model (b) are equally good; alternative is that (a) fits better. (a) (b) BRIEF ARTICLE THE AUTHOR expressioni = ↵ + 0groupi + P X p=1 pconfounderip + noiseip expressioni = ↵⇤ + P X p=1 ⇤ pconfounderip + noise ⇤ ip expressioni = ↵ + K X k=1 ksplinek(timei) + P X p=1 pconfounderip + noiseip P