Much of today's molecular science revolves around next-generation sequencing. Frequently, the first step in analyzing such data is aligning sequencing reads to a reference genome. This step is often taken for granted, but any analysis downstream of the alignment will be affected by the aligner's ability to correctly map sequences. In most cases, for research into chromatin structure and nucleosome positioning, ATAC-seq, ChIP-seq, and MNase-seq experiments use short read lengths. How well aligners manage these reads is critical. Most aligner programs will output mapped reads and unmapped reads. However, from a biological point of view, reads will fall into one of three categories: correctly mapped, incorrectly mapped, and unmapped. While increased sequencing depth can often compensate for unmapped reads, incorrectly and correctly mapped reads appear algorithmically identical but can produce biologically significant alterations in the results. For this reason, we are benchmarking various alignment programs to determine their propensity to incorrectly map short reads. As short-read alignment is an important step in ATAC-seq, ChIP-seq, and MNase-seq experiments, caution should be taken in mapping reads to ensure that the most accurate conclusions can be made from the data generated. Our analysis is intended to help investigators new to the field pick the alignment program best suited for their experimental conditions. In general, the aligners we tested performed well. BWA, Bowtie2, and Chromap were all exceptionally accurate, and we recommend using them. Furthermore, we show that longer read lengths do in fact lead to more accurate mappings.
Keywords: ChIP-seq; NGS; alignment programs.