The orientation of reads was calculated from the SAM bit flag data.
The parameters collected of the 378 genes included orientation, length and exon-number, distance to neighboring genes and their orientation as well as the number and orientation of reads.
Low-coverage regions or mis-orientation of read pairs suggests mis-assemblies, while aberrant insert sizes indicate small insertions or deletions.
For paired-end reads, O a = 1 if the two reads come from different strands, point to each other, and, in the case of directional RNA-Seq, the orientation of first read matches the known isoform orientation.
Additionally, the orientation of the read at the fragment end is important and defined by the protocol (Fig. 2b).
In part, this is achievable only because the RNA-Seq protocols used here preserved 'strandedness' information, allowing comparisons to be performed in the knowledge of the 5'-3' orientation of each read.
The output of "Bowtie" includes the names of the reads that aligned, the orientation of the reads in the alignments, the names of the sequence to which the reads aligned, and the offsets into the reference sequences of the alignments.
For example, when dealing with single-end reads that do not originate from a known strand of DNA or RNA, a given read r and its reverse complement rc (r ) are often equivalent for the purpose of most analyses the same is also true of non-strand-specific paired-end reads, though the relative orientation of these reads should be preserved (Section 2.2).
These would be read-pairs that deviate from the expected mate-pair orientation of both reads occurring in the same direction or read-pairs that are outside the expected 1.5 kb insert size.
After adapter trimming and adjustment of the orientation of all reads to correspond to that of the original RNA sequence, reads were collapsed into unique reads prior to further analysis.
