View Single Post
Old 01-27-2015, 06:04 AM   #8
JBKri
Member
 
Location: Greece

Join Date: Jan 2014
Posts: 66
Default

Quote:
Originally Posted by stinky View Post
Pmiguels you are right, I took this off of the Nextera XT page at illumina....

Smaller amplicon inputs into Nextera XT preps typically yield smaller insert size ranges. To maximize recovery of smaller fragments out of the AMPureXP cleanup we recommend > 300 bp to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50 bp from each distal end of a fragment may be seen. This is because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing this can be easily averted by simply designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.

Are there any protocol differences based on different amplicon lengths?
For amplicons greater than 500bp, Illumina recommends using a 0.6x AMPure XP cleanup (30 μl volume of beads). For amplicons less than 500bp, Illumina recommends using a 1.8x AMPure XP cleanup (90 μl volume of beads) to maximize yield."
Reviving an old thread here....
We have PCR-products without Illumina adapters (~500 bp), and we are thinking to use the Nextera XT kit to add the adapters and indexes.
Am I correct in assuming the resulting reads will be a mix of various lengths (up to 400 bp) and different parts of the insert? Can such data be used for e.g. Qiime processing?

Last edited by JBKri; 01-27-2015 at 06:56 AM.
JBKri is offline   Reply With Quote