For all samples, 24-nt reads represent the highest number of uniquely mapped sequences.> Mapping data was investigated for correlation between sequence size and mapping location in repetitive regions.
Despite that bisulphite sequencing gives highly valuable information about the sequence context of methylated cytosine, it has limitations at the lower concentration range: incomplete bisulphite conversion of unmethylated cytosines and missalignment of sequencing reads in the mapping process in repetitive, telomeric, and GC-rich regions result in false positive rates of ∼0.5% and higher.
Assembly and mapping: once sequencing is accomplished, the initial analysis of base calling is performed by proprietary software on the sequencing platform, followed by aligning the data to a known reference genome if available or by de novo assembly; short read lengths can make mapping in the repetitive regions challenging.
Now we have used Illumina sequencing to map DSBs in repetitive rDNA units in human HEK293T cells.
It has been reported that the longer Roche 454 reads as compared to the Illumina reads, results in an improvement in mapping of repetitive regions [ 32].
Therefore, we sought to reduce ambiguity in mapping repetitive elements by constructing a reference Mtb H37Rv genome with only a single MIRU copy at each locus.
Approximately 60% of small RNAs mapped to the sense strand of protein coding genes in both libraries, whereas small RNAs mapping to repetitive elements mapped in nearly equal frequency to both strands.
As demonstrated by these results, using short paired-end data alone produced a high false-positive rate (FPR) due to ambiguous read mapping in locations that contain repetitive regions.
A total of 308 X-linked probes were removed from analysis as they were located in repetitive elements, mapped to more than one location in the genome and/or were located in the promoters of the cancer-testis family of genes (summarized in Supplementary Figure S1).
