To determine if multi-mapped reads influenced the break preference at "ACGT", we retained uniquely mapped reads only (899 reads) and counted all of the possible 4-mer sequence of "xxxx▼" in the stitched reads as we had done previously.
Furthermore, the mapping quality score is limited in that it measures the reliability of the mapped reads only, but does not directly identify the problematic repeats in the genome.
After excluding ambiguously mapped reads, only 80,168 of the genomic loci showed transcript coverage above the selected threshold for the statistical analyses (>3 reads for at least two biological replicates, within at least one genotype/time point pair, see Methods).
The -u option was used to report uniquely mapped reads only, whereas -H option was used to breaks ties using Hamming distance when there was more than one best mapping location for a read, which would give the most accurate mapping results with little or no cost to the mapping percentage.
Because the SAGE protocol is designed to produce reads that represent mRNA regions adjecent to the 3'-most NlaIII endonuclease cleavage site, we mapped reads only to 15,169 Ref-Seq mRNA sequences that were computationally found to be longer than 22 bases after restriction enzyme cleavage and were non-redundant in the 23-bp mRNA region, to which the reads were predicted to be aligned.
For expression profiling the incomplete mapping of reads only becomes a problem if genes are differentially affected.
Depending on the references used to map the reads, only about 30%, 23%and25%5% of reads could be mapped to the 5AS, 5BS and 5DS assemblies, respectively.
For mapped read counts, not only we considered uniquely mapped reads but also multimapped reads were included in the mapped reads to provide a rough perspective of alignment sensitivity for each aligner.
After mapping, we selected the reads only mapped to novel exon-exon junctions.
The reads from heterozygous materials could be divided into three groups: reads specifically mapped to Zhenshan 97, reads only mapped to Minghui 63 genome, and reads mapped to both Zhenshan 97 and Minghui 63 at the same position.
