Ordering Primers

Ordering primers

Once you've created indexing primers using GIL, you can simply upload the automatically generated primer order sheets to order from IDT, Sigma, Thermo, or Eurofins. Below are some considerations for ordering primers generated by GIL.

Phosphorothioate Modification

Most commercial NGS primers contain a phosphorothioate modification between the last two nucleotides on the 3' end, which protects against primer degradation by the 3'-5' exonuclease activity of high-fidelity polymerases. By default, primers in the order sheet contain an asterisk to specify that a phosphorothioate modification should be added (this code is used by IDT, Sigma, and Eurofins). Thermo's code is different (a different letter code for each base), but you can also generate primers for ordering from Thermo by using the --company Thermo argument. This costs ~$4 per primer with IDT, and can be removed with the --no-mod option.

Oligo Quality

We ordered our indexing primers as standard 100 nmol oligos from IDT, with only standard desalting for purification. Higher quality oligos, such as IDT Ultramers, will likely result in better quality sequencing data, but come at a much higher cost. We observed ~0.2% of reads with deletions in the index region for our 8 bp dual index primers, while the higher quality PhiX spike-in had only ~0.02% deletions.

Preparing Primer Plates

We ordered our oligos in TE buffer normalized to 100 μM concentration in multiple replicate plates. We created "master plates" by diluting the i5 and i7 indexes into a single 96-well plate (polypropylene is preferable to polystyrene due to lower DNA binding) to a final concentration of 10 μM each primer. We then aliquoted each master plate into multiple single-use plates to prevent index contamination between wells.