AMplicon Libraries

Our standard approach is to use the Illumina recommended two-step protocol for generating libraries. We are also experienced with a one-step approach that uses the gene region primers as the sequencing primer during sequencing. There are positives and negatives to both approaches. Using a one-step approach allows for slightly longer amplicons because no sequence flows are allocated to the primer, however, unless the entire run is the same amplicon different sequencing primers can interfere with each other and show a reduction in quality. On the other hand, a two-step approach increases the overall read quality of multi primer runs. However, it reduces the fragment size that can be sequenced and stitched together in paired-end sequencing. As there are two PCR steps, there is a higher chance of introducing contamination and bias. We have a number of assays available, a list is found here.

DNA Shotgun Libraries 

We have used Illumina Nextera kits, TruSeq kits and NEB Ultra kits in the past and are willing to use these in the future. Currently, our default kit is the KAPA HyperPlus kit. This is a robust kit, capable of using 6bp single or 8bp dual indexes. It allows for manual or enzymatic fragmentation and is flexible on the amount of starting material.

RNA Shotgun Libraries

As with DNA libraries, there are a number of options for RNA libraries. The standard kit we use is the KAPA Stranded Library kit, with or without RiboErase. This kit (if samples are from certain sample types) allow for some flexibility with the quality of the RNA extract.