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Old 10-02-2012, 08:40 AM   #1
Location: St. Catharines

Join Date: Mar 2010
Posts: 11
Default Small insert size Nextera XT library...on purpose

I am looking for information on creating a small insert size Nextera XT library. The goal is 100 bp inserts but Illumina support does not recommend the Nextera kits for this. Before we walk away from and move to the TruSeq kits I wanted to see if anyone has experience on modifiying the Nextera XT workflow to shift the tagmentation to ~ 100 bp peak.

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Old 10-02-2012, 05:19 PM   #2
Location: Sydney, Australia

Join Date: May 2010
Posts: 65

We have accidentally created nextera (though not nextera XT) libraries with small median insert sizes, ~120nt. The Adey et al 2010 paper claims stearic hindrance prevents generating inserts much smaller than 100nt. Small inserts tend to be created with an excess of tnp relative to target gDNA. The Ampure XP cleanup in standard Nextera XT protocol preferentially removes fragments < 200nt or so. It's possible to modulate the fragment cutoff by changing the buffer concentration but 100nt is close enough to the PCR oligo size that bead separation seems unworkable (though I am no expert here). You would probably need another means to get rid of unincorporated PCR oligos. gel purification? pippin? Also with such small fragments the Nextera XT normalization seems unlikely to work as intended. If you can tolerate greater sample-to-sample variation in read counts normalizing the input to library prep across samples can work fairly well if they are all the exact same sample type/dna extraction/quantitation. this is critical.

If you needed to go smaller than 100nt it seems that in principle that longer custom adapter oligos could be used with tnp to get past the stearic hindrance issue. But this is well outside the realm of a kit.
koadman is offline   Reply With Quote

nextera xt, small insert size

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