PCR Amplicons

When starting with PCR amplicons, the PCR amplicon must be > 300 bp. The standard clean-up protocol depletes libraries < 500 bp. Therefore, Illumina recommends that amplicons < 500 bp undergo a 1.8 x Illumina Purification Beads volume normal ratio to supernatant during Clean Up Libraries. Shorter amplicons can otherwise be lost during the library cleanup step.

Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end.