Technical data

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 4. Prepare samples for multiplexed sequencing

SureSelect

XT2

Target Enrichment System for Illumina 71

concentration of each sample may be accurately determined as described

in “Step 5. Optional: Quantify captured library pools by QPCR” on

page 72.

Sequencing run setup guidelines

If samples will not be further combined in post-capture pools, proceed to

cluster amplification using the Illumina Paired-End Cluster Generation Kit;

refer to the manufacturer’s instructions for this step. The optimal seeding

concentration for cluster amplification from SureSelect

XT2

DNA libraries is

approximately 6 to 8 pM.

For sequencing exome captures, Agilent recommends a cluster density of

approximately 800K to 900K clusters/mm

. If sequencing smaller captures,

with or without pooling, the cluster density can be adjusted appropriately

for improved base quality and amount of coverage desired.

Sequencing runs must be set up to perform an 8-nt index read. For the

HiSeq platform, use the Cycles settings shown in Table 31. Cycle number

settings can be specified on the Run Configuration screen of the

instrument control software interface after choosing Custom from the

index type selection buttons.

For complete index sequence information, see the Reference chapter

starting on page 75.

NOTE

The optimal seeding concentration may vary, depending on the method used for library

quantification and fragment size distribution.

Table 31 HiSeq platform Run Configuration screen Cycle Number settings

* Settings apply to v3.0 SBS chemistry.

Run Segment Cycle Number

Index 1 (i7) 9

Index 2 (i5) 0