The fragmentation of genomic DNA according to the Agilent SureSelectXT protocol and subsequent downstream processing produces a Gaussian distribution of DNA fragments with different lengths. A small percentage of the resulting library fragments have an insert size below 250 bp. Sequencing those library fragments with 150 bp paired-end reads will generate partially overlapping reads that cover the same bases and hence create an artificial doubling of coverage at those positions. To assure accurate analyses, those bases are excluded from further downstream analyses, such as single-nucleotide variant and insertion and deletion detection. Furthermore, Eurofins Genomics is continuously optimising its protocols to minimise the number of overlapping bases.