Hi everyone,

I have designed my own library prep protocol by means of a home brew multiplexed PCR and adapter ligation protocol for targeted sequencing on both Ion Torrent and Illumina machines. Looking at the quality metrics of the Ion Torrent machine, a very large amount of reads (> 40%) is categorized as low quality, indicating that the drop in read quality already starts at the beginning of the sequencing reaction. Also the amount of adapter-dimmer is on the high side. Similar poor performance (overall low Phred scores) is observed for the Illumina machine; the test fragments have good quality.

For sure, this has something to do with my library protocol, but I have difficulties to pinpoint where the problems start. I have read that poor read quality can be attributed to secondary structures in the template, but I cannot understand where this would come from. Can someone indicate how my protocol can be debugged?

Thanks!