S1.1 mRNA-Seq data Sequencing reads from both MAQC-2 and MAQC-3 experiments have been deposited to the short-read archive under the accession number, SRA010153.1 The calibration method used in Bustard for quality-scoring of base-calls is highly relevant in terms of experimental design. In the auto-calibration method, base-calls are scored in a manner that is similar to the phred base-caller . An alternative, recommended by Illumina, is to reserve one control lane per flow-cell for sequencing DNA, typically bacteriophage phi X genomic DNA . Bustard also provides a variety of read quality measures. For a given cluster, the chastity ck at cycle k is defined as the highest of the four fluorescence intensities divided by the sum of the highest two intensities. The purity filter (PF) discards any read for which the chastity at any of the first 12 sequencing cycles is less than 60%, i.e., min1≤k≤12 ck < 0.60 [15, Supplementary Information, p. 6]. For the MAQC-2 and MAQC-3 datasets, the percentage of reads passing the purity filter (out of the total number of clusters) varies between 50 % and 76 % per lane. Summaries of the Genome Analyzer output are provided in Tables S1 and S2. We used Bowtie , Version 0.10.1, to align reads to the genome (H. sapiens, NCBI 37.1 assembly). W
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