The goal of this thesis research was to develop software to be used with RNA-Seq data for transcriptome quantification that was capable of handling multireads and quantifying isoforms on a more global level. Current software available for these purposes uses various forms of parameter alteration in order to work with multireads. Many still analyze isoforms per gene or per researcher determined clusters as well. By doing so, the effects of multireads are diminished or possibly wrongly represented. To address this issue, two programs, GWIE and ChromIE, were developed based on a simple iterative EM-like algorithm with no parameter manipulation. These programs are used to produce accurate isoform expression levels.

Massively parallel whole transcriptome sequencing and its ability to generate full transcriptome data at the single transcript level provides a powerful tool with multiple interrelated applications, including transcriptome reconstruction, gene/isoform expression estimation, also known as transcriptome quantification. As a result, whole transcriptome sequencing has become the technology of choice for performing transcriptome analysis, rapidly replacing array-based technologies. The most commonly used transcriptome sequencing protocol, referred to as RNA-Seq, generates short (single or paired) sequencing tags from the ends of randomly generated cDNA fragments. RNA-Seq protocol reduces the sequencing cost and significantly increases data throughput, but is computationally challenging to reconstruct full-length transcripts and accurately estimate their abundances across all cell types. We focus on two main problems in transcriptome data analysis, namely, transcriptome reconstruction and quantification. Transcriptome reconstruction, also referred to as novel isoform discovery, is the problem of reconstructing the transcript sequences from the sequencing data. Reconstruction can be done de novo or it can be assisted by existing genome and transcriptome annotations. Transcriptome quantification refers to the problem of estimating the expression level of each transcript. We present a genome-guided and annotation-guided transcriptome reconstruction methods as well as methods for transcript and gene expression level estimation. Empirical results on both synthetic and real RNA-seq datasets show that the proposed methods improve transcriptome quantification and reconstruction accuracy compared to previous methods.