ATAC-Seq of mus musculus:NTD brain
1 Clean Reads Filtering Reads obtained from the sequencing machines included raw reads containing adapters or low quality bases which would affect the following assembly and analysis. Raw reads would be processed to get high quality clean reads according to three stringent filtering standards: i Removing reads containing adapters; ii Removing reads containing more than 10% of unknown nucleotides (N); iii Removing low quality reads containing more than 50% of low quality (Q-value20) bases. 2 Reads Alignment BWA[1] (version 0.7.12) using default parameters was used to align the clean reads from each sample against the reference genome, and only the alignments within 2 mismatches were considered in peak scanning. 3 Strand Cross Correlation Strand cross-correlation of tag density providing a quick assessment of IP-seq data set quality and binding characteristics. It is based on the fact that a high quality IP-seq experiment produces significant clustering of enriched DNA sequence tags at locations bound by the protein of interest, and that the sequence tag density accumulates on forward and reverse strands centered around the binding site. SPP R package was used to calculate the correlation between forward and reverse strands, which can reflect whether the chromatin immunoprecipitation effect is optimal. 4 Peak Detection MACS[2] (version: macs14)( -p 1e-3 -m 10,30) is a computational method that was designed to identify read-enriched regions from ChIP-seq data. Dynamic Possion Distribution was used to calculated p-value of the specific region based on the unique mapped reads. The region would be defined as a peak when p-value<1e-5. Then, the distribute of chromosome distribution, peak width, fold enrichment, significant level and peak summit number were displayed. 5 Peak Related Genes Annotation According to the genomic location information and gene annotation information of peak, peak related genes can be confirmed. Besides, the distribution of peak on different genome regions, such as intergenic, introns, downstream2k, upstream2k and exons) was performed.