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These faults are most apparent when combining multiple lanes of data in the post-processing phase.
With Illumina pair-end sequencing technology, a total of 8 "lanes" of data was produced.
Illumina data were separated by tag using FastX Barcode Splitter and concatenating the two lanes of data for each tag respectively.
Figure 4 shows the result for 14 lanes of data, consisting of 3 replicate lanes for each of the 2 "control" rats and 4 replicate lanes for each of the 2 "SNL" rats.
Each size selected mRNA-seq library was loaded in one lane of flow cell and paired-end 2 × 120 nt sequencing was conducted on Illumina Genome Analyzer IIx, totaling three lanes of data per pooled transcriptome.
All lanes of data were required to show low overall error rate (< 1.5%), low inter-base phasing (< 1.0), and all reads passed the Illumina GA quality filters (PF = Y).
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Each lane of data was demultiplexed by index sequence and reads were combined from both lanes for each sample.
Each lane of data was first evaluated with TopHat [ 20] to create a set of read-supported junctions.
We used only one lane of data for each strain for copy number analysis (NCBI Sequence Read Archive accessions SRX014130 and SRX014132).
Although the maximum CV is smaller for the data combined across 5 lanes, than for a single lane of data, the trend is still apparent.
The numbers of reads attributable to each index/sample are calculated as a percentage of the total number of sequence reads per lane of data.
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