Ab initio assembly of transcripts from RNA-seq data

We mapped each replicate of directional paired-end RNA-seq data to the human reference genome (hg19/GRCh37) using TopHat v2.0.10 [59, 60] before assembling transcripts using both Cufflinks [61] and Scripture [62]. The TopHat settings were as follows:

tophat -p 8 --library-type fr-firststrand --mate-inner-dist 50 --mate-std-dev 50 --microexon-search --GTF genes.gtf -o < output-folder > <index of reference genome > Reads_end1.fastq Reads_end2.fastq

The reference genes in GTF file format (genes.gtf) were downloaded from the University of California, Santa Cruz (UCSC) genome browser [63]. We then assembled transcripts through the following settings of Cufflinks using TopHat output bam file as input:

cufflinks -p 8 --max-bundle-frags 100000000 --library-type fr-firststrand --frag-bias-correct --multi-read-correct -o < output_folder > <tophat_output_bam_file>

Max-bundle-frags was set to 100,000,000 such that highly expressed genes would be included in the output.

For analysis in Scripture, we used the following TopHat settings:

tophat -p 4 --microexon-search --GTF genes.gtf -o < output_folder > <index_of_reference_genome > <Reads_one_end.fastq>

We used Scripture (beta2 version) to assemble transcripts by following the protocol for transcript assembly (http://www.broadinstitute.org/software/scripture/). All transcripts assembled in Cufflinks and/or Scripture were then merged into one list through Cuffmerge [61].