Adapters are short DNA sequences that are added to the ends of DNA fragments during library preparation. These adapters contain necessary sequences for downstream processes, such as priming sites for amplification or binding sites for sequencing platforms.
In common short read sequencing, the DNA insert (original molecule to be sequenced) is downstream from the read primer, meaning that the 5' adapters will not appear in the sequenced read. But, if the fragment is shorter than the number of bases sequenced, one will sequence into the 3' adapter. To make it clear: In Illumina sequencing, adapter sequences will only occur at the 3' end of the read and only if the DNA insert is shorter than the number of sequencing cycles (see picture below)!
In ChIP-seq (Chromatin Immunoprecipitation sequencing), mapping refers to the process of aligning or mapping the sequencing reads obtained from the ChIP-seq experiment to a reference genome. The purpose of mapping is to determine the genomic locations of the DNA fragments that were enriched through the immunoprecipitation step.
View & Sort
View is to filter those good quality mapping results
Sort is to reorder data by location
Remove Duplication
Duplicated reads refer to sequencing reads that are identical or nearly identical, indicating potential PCR amplification artifacts or technical biases during library preparation.
PCR amplification artifacts: During library preparation, PCR amplification is often employed to generate enough DNA fragments for sequencing. However, PCR amplification can introduce biases, leading to the over-amplification of certain fragments and the creation of duplicate reads. Removing duplicate reads helps mitigate the impact of PCR biases and provides a more accurate representation of the actual DNA fragments in the sample.
Peak calling is commonly used in ChIP-seq (Chromatin Immunoprecipitation sequencing) and other sequencing-based assays to identify genomic regions where DNA-binding proteins, histone modifications, or other chromatin features are localized.