Abstract
Illumina's sequencing technology provides high quality reads of DNA fragments with error rates below 1/1000 per base. Runs typically generate millions of reads where the vast majority of the reads has also an average error rate below 1/1000. However, some paired-end sequencing data show the presence of a subpopulation of reads where the second read has lower average qualities. We demonstrate that the fragment length is a major driver of increased error rates in the R2 reads. Fragments above 500 nt tend to yield lower base qualities and higher error rates than shorter fragments. We show the fragment length dependency of the R2 read qualities using publicly available Illumina data generated by various library protocols, in different labs and using different sequencer models. Our finding extends the understanding of the Illumina read quality and has implications on error models for Illumina reads. It also sheds a light on the importance of the fragmentation during library preparation and the resulting fragment length distribution.