Technical data

55
SureSelect
XT2
Target Enrichment System for Illumina Multiplexed
Sequencing Protocol
Agilent Technologies
4
Hybridization
Step 1. Pool indexed DNA samples for hybridization 56
Step 2. Hybridize gDNA library pools to the SureSelect Capture Library 57
Step 3. Prepare streptavidin-coated magnetic beads 59
Step 4. Capture the hybridized DNA using streptavidin beads 60
This chapter describes the steps to pool indexed gDNA libraries and then
hybridize the pooled gDNA libraries with a SureSelect
XT2
Capture Library.
Pools of 8 or 16 indexed samples are hybridized to the appropriate
SureSelect
XT2
Capture Library and the targeted molecules are captured for
sequencing.
The size of your SureSelect
XT2
Capture Library determines the number of
indexes that may be combined for hybridization. See Table 23 for the
recommended number of indexes per gDNA library pool.
R
CAUTION
The ratio of SureSelect Capture Library to indexed gDNA library is critical for
successful capture.
CAUTION
You must avoid evaporation from the small volumes of the capture during the 24 hour
or greater incubation.
If you want to use a duration of hybridization >24 hours, first test the conditions.
Incubate 60 µL of SureSelect XT2 Hybridization Buffer (without DNA) at 65°C for
24 hours (or longer, if applicable) as a test. Include buffer in each well that you might
use, including those in the center and those on the edges. Check that you do not get
extensive evaporation. Evaporation should not exceed 8 µL.