Next-generation sequencing data processing: Analysis of unmapped reads and extremely high mapped peaks

Next-generation sequencing (NGS) and its applications are widely used in studying gene regulation and epigenetic mechanisms due to its decreasing cost and high throughput. Here we used MNase-seq technology to determine the nucleosome positions in human erythroleukemia k562 cells by direct sequencing of nucleosome ends with the SOLiD high-throughput sequencing technique. However, during the reads mapping and data pre-analysis steps, only 40% of the sequenced reads can be mapped to the reference genome hg19 and there are some extremely high peaks (EHPs) in the profiles of mapped reads on the reference genome. Mathematical models were developed to analyze the unmapped reads and nearly 25.3% of the unmapped reads were found due to genome variants, base-calling errors and gaps of the reference genome. We also investigated EHPs and proposed methods to deal with the EHPs for the downstream data analysis.